Biochemical Investigation into the HNH Motif of HK97 Gp74

by

Batool Zafar Hyder

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Graduate Department of Cell & Systems Biology

University of Toronto

© Copyright by Batool Z Hyder 2014

ii

Biochemical Investigation into the HNH motif of HK97 Gp74

Batool Zafar Hyder

Master of Science

Graduate Department of Cell & Systems Biology

University of Toronto

2014

Abstract

Bacteriophages are viruses that infect bacteria. This thesis describes studies of gp74 from

the bacteriophage HK97, which functions as an HNH endonuclease. HNH endonucleases are

DNA digestion proteins characterized by two highly conserved His residues and an Asn residue.

Like other HNH endonucleases, the activity of gp74 is dependent on binding of divalent metal

ions to the HNH motif.

Current work focused on confirming the identity of conserved HNH motif residues of

gp74. We hypothesized the catalytic His residue is H43, the structural Asn residue is N73, and

that H82 is involved in metal–binding. Additional residues in the fold, such as D42, may

also bind the metal. Our bound metal analysis and the sequence of gp74 also suggest the

presence of a Zn2+

–finger motif. Mutations of D42 and H82 decrease the activity of gp74,

without affecting the structure. These studies advance our understanding of the gp74 activity.

iii

Acknowledgments

It has been a great pleasure researching under the supervision of Professor Voula Kanelis.

I thank you for always encouraging, guiding, and supporting me from the very first day to my

last in the laboratory. Without your encouragement, guidance, and support this thesis would not

have been possible. I thank you for your enthusiasm that also kept me interested and increased

my understanding of biochemical research. I would like to thank my laboratory members

Marijana, Jorge, Elvin, Claudia, Sasha, Richard, Yasmine, and Alex for their help, friendship,

and a healthy laboratory experience.

I would like to pay my gratitude to my parents, siblings, my husband, and my best friends

for always supporting me throughout my graduate studies. To my parents, I thank you for

understanding when I could not attend any family gatherings and for always being supportive of

the choices I made. To my siblings, I appreciate your patience when I was on the verge on losing

my sanity and for always trying to keep me sane and happy. To my husband, I am grateful for

understanding when I could not make it to the Skype calls due to unplanned experiments on the

weekends. I thank you for understanding that long–distance relationship and graduate studies can

be a tough choice especially when we are in different time–zones. To my best friends, I will

never be able to thank you enough for always being there for me. I thank you for always picking

me up and relieving me of my frustrations when things were not the way I wanted them to be.

I would like to thank my collaborators Karen L. Maxwell for sharing her results and

guidance. I am grateful to my supervisory committee members Professor Bryan Stewart and

Professor Deborah Zamble for their counsel and support. I would also like to thank Professor

George Espie, Professor Jumi Shin, Professor Scott Prosser, and Professor Guojan Yang for the

use of their laboratory equipments.

iv

Table of Contents

Abstract ........................................................................................................................................... ii

Acknowledgments.......................................................................................................................... iii

Table of Contents ........................................................................................................................... iv

List of Figures ................................................................................................................................ vi

List of Tables ............................................................................................................................... viii

List of Abbreviations ..................................................................................................................... ix

List of Units ................................................................................................................................... xi

1. Introduction ................................................................................................................................. 1

1. 1. Bacteriophages .................................................................................................................... 1

1. 2. The Biological Relevance of Bacteriophages ..................................................................... 4

1. 2. 1. Phage Therapy………………………………………………………………..………4

1. 3. The Caudovirales Order ...................................................................................................... 5

1. 4. Bacteriophage HK97 ........................................................................................................... 8

1. 5. HNH Endonucleases ......................................................................................................... 11

1. 5. 1. HK97 gp74………………………………………………………………………….15

2. Hypothesis and Objectives ........................................................................................................ 17

3. Materials and Methods .............................................................................................................. 17

3. 1. Materials ............................................................................................................................ 17

3. 2. Bioinformatics on Gp74 .................................................................................................... 18

3. 3. Expression of HK97 gp74 ................................................................................................. 19

3. 4. Purification of HK97 gp74 ................................................................................................ 21

3. 5. Fluorescence Temperature Melting Studies ...................................................................... 24

3. 6. CD Spectroscopy ............................................................................................................... 24

3. 6. 1. Secondary Structure Determination ........................................................................... 24

v

3. 6. 2. Thermal Stability Measurements by CD ................................................................... 24

3. 7. HNH Endonuclease DNA Cleavage Assay ....................................................................... 25

3. 8. Rate of DNA Digestion ..................................................................................................... 26

3. 9. Expression and Purification of TEV Protease ................................................................... 27

4. Results and Discussion ............................................................................................................. 27

4. 1. Structure–Based Sequence Alignment .............................................................................. 27

4. 2. Expression and Purification of HK97 gp74 ...................................................................... 31

4. 3. Total Protein Content and Purity Assessment via Mass Spectrometry ............................. 38

4. 4. Metal dependence of gp74 expression .............................................................................. 39

4. 5. Gp74 and Proteolysis ........................................................................................................ 41

4. 6. Gp74 and Storage .............................................................................................................. 42

4. 7. ICP Analysis ...................................................................................................................... 43

4. 8. Fluorescence Studies ......................................................................................................... 44

4. 9. Circular Dichroism Spectroscopy Analysis ...................................................................... 46

4. 9. 1. Melting Temperature of HK97 gp74 ......................................................................... 48

4. 10. HNH Endonuclease Assays by HK97 gp74 .................................................................... 48

4. 10. 1. Gp74 and Mutations……………………………………………………………….48

4. 10. 2. HindIII Digested λ DNA as a Substrate for Cleavage ............................................. 51

4. 11. Rate of DNA Digestion by HK97 gp74 .......................................................................... 52

5. Conclusion ................................................................................................................................ 54

6. Future Work .............................................................................................................................. 55

7. References ................................................................................................................................. 58

vi

List of Figures

Figure 1. A schematic drawing of a bacteriophage structure. ......................................................... 1

Figure 2. The replication cycle of the typical temperate phage coliphage–λ ................................. 3

Figure 3. Electron micrographs and schematic diagrams in the Caudovirales bacteriophages ...... 6

Figure 4: A schematic drawing of HK97 bacteriophage with its structural components ............... 9

Figure 5. Genome map of bacteriophage HK97. .......................................................................... 10

Figure 6. Crystal structure of I–HmuI of homing endonuclease bound to its target DNA. .......... 13

Figure 7. A 3–D ribbon diagram of an HNH motif of colicin E9 DNase. .................................... 13

Figure 8. Proposed mechanisms for DNA cleavage by I–HmuI and colicin E9. .......................... 14

Figure 9. A BLASTp search alignment of HK97 gp74. ............................................................... 16

Figure 10. Structure–based sequence alignment of HK97 gp74 based on GS–15. ...................... 30

Figure 11. A homology model of HK97 gp74 based on G. metallireducins GS–15 protein........ 30

Figure 12.Purification of HK97 gp74. .......................................................................................... 32

Figure 13. Further purification of gp74–WT via SEC and cation exchange chromatography. .... 34

Figure 14. Gp74 mediated digestion of λ DNA in the presence of Ni2+

ions. .............................. 35

Figure 15. HNH endonuclease activity of gp74 affected by altering Ni2+

ions concentrations.

…36

Figure 16. Purification of HK97 gp74–H82A. ............................................................................. 37

Figure 17. A 17% SDS gel for gp74–WT with reducing and non–reducing conditions .............. 38

Figure 18. SDS–PAGE is showing pre– and post–IPTG inductions of gp74–WT ...................... 40

vii

Figure 19. SDS–PAGE showing degraded vs. normal gp74–WT. ............................................... 42

Figure 20. Gp7–WT denaturation studies monitored by intrinsic Trp fluorescence. ................... 45

Figure 21. CD spectra of gp74 proteins. ....................................................................................... 47

Figure 22. The effect of the H82A mutation on gp74 activity. ..................................................... 50

Figure 23. The effect of the D42A mutation on gp74 activity. ..................................................... 50

Figure 24. HindIII digested λ DNA vs. λ DNA as a substrate for endonuclease digestion .......... 51

Figure 25. Rate of DNA digestion by gp74 using densitometry. .................................................. 53

Figure 26. CD spectra of natively folded gp74 vs. gp74 refolded with and without Zn2+

ions. ... 57

Figure 27. Limited Proteolysis of gp74–WT by trypsin in the absence and/or presence of Ni2+

or

Zn2+

ions. ....................................................................................................................................... 57

viii

List of Tables

Table 1: HK97 genome’s relation to lambda phage and function….……………………………11

Table 2: Primers for generating HK97 gp74 mutant proteins………………………………........20

Table 3: Gp74 proteins concentrations were determined using absorbance at 280 nm …………39

Table 4: Purity assessment of gp74 proteins via mass spectrometry………………………..…...39

Table 5: Determination of percent metal–bound for gp74 proteins via ICP–AEOS …….……...44

ix

List of Abbreviations

3D Three–dimensional

ATP Adenosine triphosphate

BLAST Basic local alignment search tool

CD Circular dichroism

DNA Deoxyribonucleic acid

dsDNA Double–stranded deoxyribonucleic acid

DTT Dithiothreitol

EDTA Ethylenediaminetetraacetic acid

G. metallireducins Geobacter metallireducins

GdmHCl Guanidinium chloride

gp74 Gene product 74

gp74–WT Wild type gp74 protein

HEPES 4–(2–hydroxyethyl)–1–piperazineethanesulfonic acid

HK97 Hong Kong 97th strain

HMM Hidden Markov model

HNH Endonuclease with a conserved histidine–asparagine–histidine motif

ICP Inductively coupled plasma

ICP–AEOS Inductively coupled plasma–atomic emission optical spectroscopy

IPTG Isopropyl β–D–1–thiogalactopyranoside

Kd Dissociation constant

λ Lambda

LB Lennox Broth

NESG Northeast Structural Genomics Consortium

NMR Nuclear magnetic resonance

NR SDS Non–reducing SDS buffer

o/n Overnight

PDB Protein Database Bank

Phyre2 Protein Homology/analogY Recognition Engine

PMSF Phenylmethanesulfonylfluoride

PONDR Predictor of Naturally Disordered Regions

SDS Sodium Dodecyl Sulfate

SDS–PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

x

SEC Size exclusion chromatography

SS Pred Secondary structure Prediction

Std Standard

TEMED Tetramethylethylenediamine

TEV Tobacco etch virus

Tm Melting temperature

TMP Tape measure protein

Tris Tris(hydroxymethyl)aminomethane

UV Ultraviolet

v/v Volume by volume

w/v Weight by Volume

xi

List of Units

% Percentage

Å Angstrom

bp Base pair

cm Centimeter

ΔA Change in absorbance values

G Gram

g/L Gram per litre

g/mol Gram per mole

kB Kilobases

kDa Kilodaltons

L Litre

M Molarity

mAU Milliabsorbance units

mg/L Milligram per litre

mg/mL Milligram per milliliter

mL Millilitre

mL/min Millilitre per minute

mM Millimolar

MW Molecular weight

N Normality

Ng Nanogram

Nm Nanometer

OD600 Optical density measured at 600 nm

pM Picomolar (10–12

M)

Rpm Rotations per minute

V Volt

μg/mL Microgram per milliliter

μL Microlitre

μM Micromolar (μmol/L)

µm Micrometer

Page 1 of 74

1. Introduction

1. 1. Bacteriophages

Bacteriophages (or phages) were first discovered in 1915 by Frederick W. Twort as

agents that infect and kill bacteria. In 1917, Félix d’Hérelle gave these agents the name

bacteriophages or bacteria–eaters [1]. Bacteriophages, like other virus particles, cannot carry out

any metabolic processes because they are unable to generate ATP [2] and thus they must make

use of a host cell for their survival. Phage particles are much smaller than the bacterial host cell

they infect, usually between 20–200 nm in size [3]. Bacteriophages can also be considered to be

intracellular parasites because bacteriophages infect, grow, and multiply within bacteria by

utilizing their host biosynthetic machinery [4, 5].

The bacteriophage structure consists of a head, collar, sheath, tail fibers, and base plate

(Figure 1). All of these structural components are made up of proteins. The head is connected to

a tail by a collar protein and an adaptor protein. The head contains the viral genome, which

encodes for all of the structural proteins and proteins necessary for the phage replication cycle.

Figure 1. A schematic drawing of a bacteriophage structure.

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A phage particle, such as the coliphage–λ, has a simple replication cycle consisting of the

following steps (Figure 2) [6]: (1) Adsorption or attachment where the phage particle encounters

and attaches to the cell surface by the tip of the phage tail. (2) Phage DNA enters the cell leaving

an empty protein shell attached to the outside of the cell. Injection of the phage genome into a

bacterial host is followed by transcription, translation, and replication of the phage DNA. The

phage has entered into a lytic cycle. The phage can undergo lytic cycle only when activated

under stressful conditions. (3) The replicated linear double–stranded DNA (dsDNA) is packaged

into preformed protein shells or capsid heads and tails are added to these heads. This process is

known as virion assembly and packaging. (4) Once the phage particles are assembled, the cell

lyses and liberates the phage progeny [3, 7]. (5) Lysogeny occurs when the expression of the

phage genome is repressed, the phage development is repressed, and the phage DNA integrates

into the bacterial chromosome [8, 9]. However, before lysogeny occurs, circularization of the

linear phage DNA takes place where the ends of the linear DNA molecule join to form a circle

and the point of closure is called the cohesive site (cos) [6]. The resulting lysogenic cell can

replicate indefinitely, but the phage can be induced to adopt a lytic cycle with the excision of

phage DNA from the chromosome and subsequent cell lysis [6]. The phage particles that only

undergo the lytic cycle are known as virulent. For example, T2 phage is a virulent phage that

terminates any host–controlled cellular machinery after its penetration resulting in cellular lysis

[10]. Phages, such as the lambda (λ) phage, that can undergo the switch between their replication

cycles or only allow for lysogeny are referred to as temperate phages [11].

Although discovered many years ago, interest in bacteriophages and phage proteins

continues for many reasons. Many genes in bacterial genomes originally thought to be of

bacterial origin have been now identified to be of phage origin [12]. These incorporated phage

genes can remain dormant in bacteria until expression is induced by a stress–related response

Page 3 of 74

resulting in cell lysis or can be constitutively transcribed along with the bacterial genes [5].

Many phages have developed a mutual relationship with their bacterial host cells via lysogeny

[13]. Bacterial genomes have been shown to consist of 3–10 % of prophage–encoded genes [6, 8,

13], which are thought to be the major contribution to the vast diversity among the bacterial

strains. For example, the E. coli 0157:H7 bacteria strain has 16% of prophage genes consisting of

18 prophage elements in its genome [14]. Further, toxin phage genes have been widely spread

out among bacterial strains [15]. For example, the β–phage infects Corynebacterium diphtheria

bacteria and causes this bacterium to produce toxin [11]. When the host conditions begin to

deteriorate, there is a switch from the lysogenic to lytic cycle, with transcription and translation

of all phage genes [13].

Figure 2. The replication cycle of the typical temperate phage coliphage–λ [6]. Temperate phage can

undergo the lytic and/or lysogenic cycle, as explained in the text. This figure is extracted from reference 6.

Page 4 of 74

1. 2. The Biological Relevance of Bacteriophages

In almost all well–studied ecosystems, there are about ten bacteriophages for every

microbial cell present in the system [16]. This makes bacteriophages the most abundant

biological entities on the planet [16, 17]. Phages have colonized all ecosystems such as water,

soil, and gastrointestinal cavities [18]. Thus, phages have a huge impact in biology, such as in

species distribution, nutrient cycling, and controlling the population density of bacteria in

different ecosystems. For example, coastal areas have a higher population of phages due to the

complexity in coastal bio–diversity when compared to the deep sea [18–20]. Phages are also

found in industrial settings. A study used to identify Salmonella–specific phages isolated a total

of 232 phages from 26 sampling sites that included broiler farms, poultry abattoirs, and

wastewater plants [21]. Phages are also present in a wide variety of foods consumed every day.

Therefore, it can be safe to say that many are non–toxic to humans [22]. However, there are

some phages that may indirectly influence humans by first directly affecting the bacteria. For

example, bacteriophages that use E. coli as their host are commonly present in sewage, hospital

waste water, polluted rivers and fecal samples [23], and some of those E. coli are toxic to

humans. Further, E. coli phages have been recovered from fresh chicken, pork, ground beef,

mushrooms, lettuce and other raw vegetables [24]. However, E. coli cells existing in the

gastrointestinal cavities of humans and mice have not been infected by phages [24, 25]. This is

probably because E. coli found in living bodies, such as those found in mice, live in niches not

easily accessible to environmental phages [24, 25].

1. 2. 1. Phage Therapy

Initial research that indicated that phage DNA is incorporated into many bacterial

genomes gave rise to phage therapy [26]. For example, in one case study patients having

purulent disease of the lungs and pleura were treated using phages of Staphylococcus aureus

Page 5 of 74

[26]. Although first investigated in the 1920s, the interest in the potential usage of the phage

therapy was lost due to the discovery of antibiotics in 1941 [27, 28]. However due to the

escalating cases of antibiotic resistance, the ability of phage therapy to eliminate bacterial

infections has been renewed.

Phages can be modified such that they are harmless to their secondary host, such as

humans, and the beneficial bacteria in the gastrointestinal tract [29]. Phage therapy has been

successful against some bacterial infections because phages can disrupt the polysaccharide layer

(biofilm) that antibiotics are unable to penetrate [30]. The potential use of phages to eliminate

Campylobacter in raw food [31], Listeria in fresh food [30], and to reduce food spoilage bacteria

has also been investigated [3]. Further, the agricultural industry has been using phages to

eradicate Campylobacter, Escherichia, and Salmonella in farm animals, Lactococcus and Vibrio

pathogens in aquaculture fish, and Erwinia and Xanthomonas in plants [3]. Phage therapy,

however, was first performed on humans [3]. Phage therapy has been used to treat diarrheal

infections caused by E. coli, Shigella, or Vibrio bacteria and skin pathogens such as

Staphylococcus and Streptococcus [3]. Continued human phage therapy is prescribed in the

Republic of Georgia. Here, phage therapy is currently being used to treat bacterial infections,

such as E. coli, Staphylococcus, Streptococcus, and Enterococcus, that are resistant to standard

or advanced antibiotics [32–34]. The Republic of Georgia has also established therapies for

several acute disorders, such as acne, and chronic diseases, such as cystic fibrosis and the

resulting bacterial infections [34]. Recently in 2008, the usage of phages to deliver antitumor

agents in vivo has also been investigated for cells in tissue culture [35].

1. 3. The Caudovirales Order

About 96% of all bacteriophages belong to the Caudovirales order [12]. Caudovirales

phages are characterized by a hollow, helix–shaped protein tail that is used to attach the phage

Page 6 of 74

onto the host bacterial cell membrane and to inject DNA into the host cell [36]. The

Caudovirales phages are also referred to as the tailed viruses, and are further sub–divided into

three families based on their morphology: (1) Myoviridae (bacteriophages with long contractile

tails); (2) Siphoviridae (bacteriophages with long non–contractile tails); and (3) Podoviridae

(bacteriophages with short non–contractile tails) [3, 37] (Figure 3) [3]. Myoviridae phages have a

different mode of DNA injection due to their contractile tails than phages of the other two

Siphoviridae and Podoviridae families.

Figure 3. Electron micrographs (upper panel) and schematic diagrams (lower panels) of examples of

bacteriophages in the Caudovirales order [3].

The widths of electron micrographs are of sizes 50, 50, and 100 nm for the examples in panels A, B,

and C, respectively. A. The Myoviridae family T4 E. coli phage has a prolate head and a long contractile

tail. B. The Siphoviridae family TP901–1 L. lactis temperate phage has an isometric head and a long non–

contractile. C. The Podoviridae family KSY1 L. lactis virulent phage has a rare morphology characterized

by elongated head and a short non–contractile tail. This figure is adapted from reference 3.

One of the interesting features of the long tailed viruses is the presence of a tape measure

protein (TMP) in their tails. The size of the TMP gene determines the length of the phage tail

[38], where shortening or lengthening the gene would shorten or lengthen the phage tail

accordingly. Further, preliminary studies of TMP have also demonstrated its role in degradation

of the bacterial biofilm (polysaccharide layer) during phage DNA injection [39].

A B C

Page 7 of 74

Different Caudovirales families have different pathways for phage generation [5]. For

example, for phages of the Myoviridae and Siphoviridae families long tails are first completely

assembled and then grafted on to a completed head. In contrast, for phages of the Podoviridae

family short tails are sequentially assembled onto completed heads [5]. Some Caudovirales

species also have tails that are accessorized with fibers for an increased binding to the host cell.

Another distinguishing attribute of the Caudovirales order is the icosahedral–shaped head

or capsid [40, 41]. This capsid is composed of repeating units of a specific monomeric protein

(or capsomere). Individual monomeric capsomere protein molecules associate to form a shell or

capsid [42]. The capsid serves to protect the viral genome [40, 41]. The tail and the capsid are

then connected by a collar protein and an adaptor protein [5] (Figure 1).

Caudovirales genomes consist of a linear double stranded DNA that adopts a circular

form after it is injected into its host [37, 43]. The genome in Caudovirale phages can range from

18,000 base pairs to 500,000 base pairs in length [36, 41], and thus code for approximately 3–5

average–sized gene products (about 150–500 amino acid residues in length) in simple phages

and 100 gene products in complex phages [3, 44].

One more feature that sets Caudovirales phages apart from other phages is that genes that

are related by function and structure are clustered together [37]. For example, the genes encoding

the capsomere are located in the same region as the scaffolding protein that is required for DNA

packaging. These genes are located also close to the genes encoding the portal protein, which

forms a hole that allows the DNA to be inserted into the capsid during DNA packaging [45]. The

portal protein also forms the junction between the phage head and tail protein to allow for phage

genome injection into a host bacterial cell [45].

Page 8 of 74

1. 4. Bacteriophage HK97

The bacteriophage HK97 is named so because it was the ninety seventh strain isolated in

Hong Kong, among other related viral strains, in 1980 [44, 46]. HK97 is a temperate phage of E.

coli bacteria and HK97 belongs to the class of Siphoviridae of the Caudovirales order of

bacteriophages (Figure 3b). HK97 is very closely related to bacteriophage lambda (λ) with

respect to virion morphology and thus, it is also classified as a lambdoid phage [19]. The

lambdoid phages propagate only within the E.coli cells as their host and are therefore known as

the coliphages [8]. Like other phages of the Siphoviridae family, HK97 phage has a capsid, a

long non–contractile tail, and an adaptor that connects the two together. The capsid is composed

of repeating units of the monomeric protein gp5 to create an icosahedral head [47, 48]. Repeating

units of the portal protein are associated as a ring on the bottom of the capsid (Figure 4). After

DNA is packaged into the head, gp15 is assembled as a ring onto the portal protein. Another

protein ring, composed of gp16, is attached onto the gp15 ring to form an interface for tail

attachment [49]. The head and tail assembly (consisting of the capsid and the portal gp15 and

gp16 rings) are connected by a connector protein known as gp6 that forms the collar and an

adaptor protein gp7 [49].

Like other lambda– and lambdoid–like phages, HK97 phage has a linear double stranded

DNA–based genome. The HK97 phage has a genome size of 39.7 kb consisting 61 protein

coding genes [45]. Like other phages related to the lambda phage, structurally and functionally

related genes are clustered together in the HK97 genome (Figure 5 [45, 53], Table 1). Although

the structure and function of several HK97 gene products have yet to be determined, the genome

of HK97 has several genes that are demonstrated to be similar to that of lambda–like phage

particles [45]. Genes encoding the structural components of the HK97 phage, such as the capsid,

Page 9 of 74

Figure 4: A schematic drawing of HK97 bacteriophage with its structural components

tail, collar, and adapter proteins are clustered together. These genes are followed by genes that

encode for DNA packaging proteins, such as the integrase, excisionase, and recombinase genes.

Genes responsible for transcription of the phage proteins within the host cell are found right after

the structural and phage genome integration genes [45, 50]. The integration genes include genes

for transcription activator protein cII and the protein cIII, which binds to cII protein to protect it

from degradation. Following, cII and cIII genes are transcription inhibiting genes, such as cro

and cI [45]. Next, are genes responsible for DNA replication followed by lytic cycle repressor

proteins genes [45, 50]. Genes responsible for DNA repair mechanisms and the lysis of host

bacterial cells are located right after lytic cycle repressor genes. The gene for the RusA resolvase

(which ligates Holliday junctions as a DNA repair mechanism [45, 51]) is followed by the genes

responsible for lysis. The R gene that encodes for the R endolysin protein, which degrades the

bacterial peptoglycan wall and allows for bacterial cell lysis, is found right after the S gene [52].

The S gene encodes for the holin protein, which generates pores through which endolysin protein

Page 10 of 74

is released [52]. Finally, gene product 74, or gp74, is the very last gene in the HK97 genome.

The structure and cellular function of gp74 is yet to be determined. However, previous work in

our laboratory indicated that the biochemical function of gp74 is an HNH endonuclease [54].

After circularization of the phage genome, the location of gp74 in the HK97 genome is similar to

that of gp74 homologues or other lambdoid–like phages and is adjacent to the terminase and

structural proteins [5]. For instance, the putative HNH endonuclease genes in the Lactococcus

phage p2 and Geobacillus phage E2 are located very close to the terminase subunits, structural

and portal proteins implying that the HK97 gp74 HNH endonuclease functions alongside these

proteins in DNA packaging [54].

Figure 5. Genome map of bacteriophage HK97 [45]. Adapted from references 45 and 53.

Genes are represented by red outlined arrows. The gene size and location is represented by the length and

position of the arrow and is indicated in kilobase pairs on a ruler below the gene. The known genes for

HK97 are indicated by their gene number and letter, which are named after homologous lambda phage

genes. Unknown genes are only identified by their order number in the genome and their putative

function is identified in blue script above the genes [45, 53].

Page 11 of 74

Table 1: HK97 genome’s relation to lambda phage genome and function.

Gene Relation to lambda phage Function

1–16 Homologous Structural components

17–21 Homologous DNA packaging

22, 23 Unknown Unknown

24–39, 41 Homologous Phage genome integration

40, 42, 45, 46, 48,

49, 53 Unknown Unknown

43, 44, 47, 52 Homologous Transcriptional activation

50, 51 Homologous Transcriptional inhibition

54, 55 Homologous Lytic cycle repression

56–60, 62–64,

66–68 Unknown Unknown

61 Homologous DNA replication

65 Homologous DNA repair mechanism

69–72 RusA Homolog Host cell lysis

73 Unknown DNA binding

74 Unknown HNH endonuclease

Citations for information about gene products with known function are given in the text.

1. 5. HNH Endonucleases HNH endonucleases are small DNA binding and DNA digestion proteins that include,

but are not limited to the colicins, restriction enzymes, and homing endonucleases [55, 56]. The

HNH motif is approximately 30 residues long and contains two highly conserved His residues

and a conserved Asn residue [57]. The first His residue of the HNH motif is the catalytic residue,

the Asn residue is responsible for secondary structure stabilization, and the second His residue is

involved in divalent metal ion binding [58]. The consensus HNH motif sequence adopts a ββα or

ββα–metal fold. The HNH family of proteins can be divided into eight sub–families based on the

presence of specific residues in the HNH core [55, 59, 60].

As described previously, the HNH motif containing nucleases have a metal–binding site

in their ββα–metal binding motif with similar position and stabilization of the active site complex

between protein, DNA backbone, and a divalent metal ion [61, 62]. However, the chemical

identities, the coordination, and the interaction of each metal with the active site and the DNA

backbone differ considerably with each class of HNH containing nucleases [63]. HNH

Page 12 of 74

endonucleases bind a wide variety of divalent metal ions, such as Ni2+

, Zn2+

, Mg2+

, Ca2+

, Co2+

,

Mn2+

, Sr2+

[64]. Each metal prefers a different coordination scheme. For example, Zn2+

acquires

tetrahedral coordination whereas Ni2+

adopts on octahedral coordination geometry [60].

Consequently, there will be a different mode of interaction between the metal ion and the

residues of the protein and perhaps the DNA backbone.

One well–studied HNH motif containing protein is I–HmuI. I–HmuI contains an HNN

sequence, rather than an HNH motif, meaning that the canonical divalent metal–binding His is

replaced with an Asn. The X–ray structure of I–HmuI (Figure 6 [65]) shows Mn2+

ion

coordination involving the non–bridging oxygen from the scissile phosphate group of the DNA,

the conserved metal–binding Asn (N96) residue, a nearby metal–binding Asp (D74) residue, and

a water molecule employing a tetrahedral geometry [57, 58]. The X–ray structure of I–HmuI

bound to DNA also shows multiple interactions between the protein and DNA. These additional

interactions are necessary for recognition of I–HmuI for its target DNA. Further, I–HmuI is

known to be a homing endonuclease but it cleaves only a single strand of the target DNA

sequence [57, 61]. However, there are some HNH homing endonucleases that cleave double–

stranded DNA, such as the T4 phage enzyme I–TevIII generates a double–stranded break [66].

In contrast to I–HmuI, the bacterial colicins (E7 and E9) possess non–specific DNA

digestion activity. Unlike I–HmuI, the colicin structures consist of mainly an HNH motif (Figure

7 [62]). In the E9 colicin, the catalytic H103 residue interacts with the DNA minor groove at the

phosphate backbone, whereas the metal–binding His residues (H102 and H127) come together to

coordinate a divalent metal ion (Zn2+

or Mg2+

) to cleave the phosphate group at the 3’ end, and

the N118 is involved in secondary structure stabilization [58, 61].

Page 13 of 74

Figure 6. Crystal structure of I–HmuI of homing endonuclease bound to its target DNA sequence [65].

The enzyme I–HmuI, displayed with blue β–sheets and gold α–helices, has an extended monomeric

structure. The ββα–metal–binding motif (consensus HNH core sequence)of I–HmuI (red box) expands to

about 25 base pairs of the DNA minor groove (black) and interacts with the DNA phosphate and 3’

hydroxyl group of the scissile phosphate for a non–specific DNA digestion. Green circle represents

divalent metal ion. This figure is adapted from reference 65.

The DNA cleavage mechanism described below is now accepted for most HNH

endonucleases (Figure 8). Once the target DNA sequence is bound to the active site of the HNH

motif, transition states are produced while the digested DNA is removed. The conserved Asn of

(N83 in I–HmuI or N118 in E9) polarizes the active site His residue (H75 in I–HmuI or H103 in

E9) by removing a proton from the imidazole side chain [58]. This nucleophilic attack causes a

Figure 7. A segment of primary and secondary

sequence of an HNH motif (top) and a 3–D ribbon

diagram (bottom) of an HNH motif of colicin E9

DNase, PDB 1FSJ [62].

The HNH motif is comprised of two β–strands

followed by an α–helix. One of the β–strands

contains the first His residue. The Asn residue is

found in the loop, and the α–helix contains the

second His residue. Structure of the His–rich E9

HNH motif is denoted by H103 (general base),

N118 (secondary structure stabilizer), and H127

(metal ion ligand), respectively. Green circle

represents a divalent metal ion. This figure is

adapted from reference 62.

Page 14 of 74

resonance shift within the aromatic ring of the catalytic His (H75 in I–HmuI or H103 in E9) that

allows a nitrogen atom of this conserved catalytic His residue to attack a nearby water molecule

present in the active site, resulting in a hydroxyl anion formation. This hydroxyl ion in return

attacks the DNA backbone forming a phospho–anion transition state. This phospho–anion

transition state along with the 3’ hydroxyl end of the DNA molecule as the leaving group is

stabilized by metal ion coordination within the active site by metal–binding residues (N96 and

D74 in I–HmuI or H102 and H127 in E9). The digested DNA is then removed from the cleavage

site of the enzyme.

Figure 8. Proposed mechanisms for DNA cleavage by I–HmuI and colicin E9. Adapted from reference 58.

HNH motif residues present in the active site of I–HmuI and colicin E9 are shown. The conserved

structural Asn residue (N83 in I–HmuI or N118 in colicin E9) polarizes the catalytic His residue (H75 and

H103 in I–HmuI and E9, respectively), as shown by the dotted line. The polarized His in turn attacks

water to form a hydroxyl ion in the active site [57]. The newly formed hydroxyl ion then attacks the

phosphate ion on the DNA backbone, forming a phospho–anion intermediate. The binding of a divalent

metal ion (red circle) stabilizes the protein and shields the negative charge on the phosphate oxygen. The

divalent metal ion is bound by N96 and D74 in I–HmuI and H102 and H127 in colicin E9.

Page 15 of 74

1. 5. 1. HK97 gp74

A BLASTp search using a primary sequence of HK97 gp74 as a query indicated that

gp74 possesses significant sequence similarity with the HNH endonuclease family of enzymes

(Figure 9) [54]. BLAST generates sequence alignments based on local information in the

following manner. First, BLAST splits the query sequence into overlapping mini–sequences or

short “words”. Next, BLAST selects “neighbourhood words” that are similar to the mini–

sequences and scores them. Neighbourhood words above a specific threshold value are selected

to search the database. All high–scoring neighbourhood words derived from the query sequence

are used to search the database. Once matches are made between the neighbourhood words and

sequences in the database (i.e. seeding), the alignment is extended in both reverse and forward

directions and scored for the expected value (E–value). If the E–value is higher than the

threshold value for a particular sequence, it will be included in the alignment. Therefore, by

attempting to look for the best matches using information from the local elements, BLAST was

able to suggest that HK97 gp74 is closely related to the HNH endonuclease family of enzymes.

Our sequence alignment suggested that the catalytic residue in gp74 is H43, the

conserved Asn residue is N73, and the metal–binding His residue is H82. However, there have

been no biochemical or structural studies on the HNH proteins shown in the alignment and the

HNH proteins for which there is biochemical and structural data, such as I–HmuI and the

colicins, do not share significant sequence similarity with gp74. Thus, confirmation of the

identity of the HNH motif residues in gp74 is necessary. Further, as seen for other HNH proteins,

additional residues are likely to be involved in HNH activity of gp74. One such residue in gp74

is D42, which may also participate in divalent metal ion binding.

In addition to the HNH motif, gp74 also possesses a CXXC sequence and a CXXH

sequence. The CXXC motif is found outside of the residues that form ββα–fold, approximately

Page 16 of 74

Figure 9. A BLASTp search alignment of HK97 gp74 with closely related HNH–endonuclease family of enzymes [54].

The conserved amino acid residues are boxed in red, red letters represent acidic residues, dark blue letters represent basic residues, pink letters

represent His residue, brown letters represent Cys residues, orange letters represent Gly residue, letters with several shades of blue represent polar

uncharged residues, letters with several shades of green represent hydrophobic residues. Grey cylinders indicate α–helices and grey arrows indicate β–

strands. The catalytic His residue is indicated by an arrow, the structural Asn residue is indicated by a diamond, and the metal–binding residues are

indicated by closed circles. This figure was taken from reference 54.

Page 17 of 74

13 amino acid residues N–terminal to the catalytic His, H43 in gp74. The CXXH motif is found

within the sequence of the ββα–fold, just N–terminal to the metal–binding residue H82. The

CXXC/CXXH motifs have been shown to form a Zn2+

finger [67] and bind to Zn2+

in other

HNH–endonucleases, such as KpnI [68, 69] and PacI [70]. Currently, there is no data

demonstrating the role of the CXXC/CXXH motifs in gp74. Further, as mentioned above, as the

sequence of gp74 is divergent from the sequences of the known HNH endonucleases, residues of

HNH motif and any role for the CXXC/CXXH motifs need to be confirmed.

2. Hypothesis and Objectives

The objective of this research is to confirm the identity of each putative HNH residue in

HK97 gp74. Much of this research involved biophysical and biochemical studies of wild type

gp74 (gp74–WT) and mutant gp74 proteins. The following mutants were studied gp74–H43A,

gp74–N73A, gp74–H82A, gp74–H43A/H82A and gp74–D42A. ICP–AEOS analysis, CD and

fluorescence thermal denaturation studies, and endonuclease assays were carried out on the gp74

mutant proteins and compared to gp74–WT.

3. Materials and Methods

3. 1. Materials

Agar, agarose, Lennox broth, D–glucose, sodium monobasic phosphate, sodium dibasic

phosphate, potassium monobasic phosphate, ammonium chloride, ammonium persulfate, biotin,

thiamine–HCl, magnesium sulfate hexahydrate, calcium chloride, sodium chloride, zinc sulfate

heptahydrate, ampicillin, IPTG, Tris base, HEPES, DTT, EDTA, SDS, SDS as a 20% (w/v)

solution, benzamidine, 6–aminocaproic acid, PMSF, lysozyme as a lyophilized powder, β–

mercaptoethanol, acrylamide/bis–acryl 40% solution, glycine, and TEMED were all purchased

from BioShop. Glacial acetic acid and 12 N hydrochloric acid solutions were obtained from

Caledon Laboratory Chemicals. A 10 N sodium hydroxide solution was purchased from VWR.

Page 18 of 74

Imidazole and Chelex® 100 sodium form resin were purchased from Sigma Aldrich. GeneRuler

High Range DNA Ladder (ready–to–use), GeneRuler 1 kb DNA Ladder, 6x DNA Loading Dye,

and 8.0 M ultra pure guanidinium HCl were purchased from Thermo Scientific. Nickel sulfate

hexahydrate and 3,500 MW cut–off dialysis tubing were bought from Fisher Scientific.

SeeBlue® Plus pre–stained protein standard was purchased from Invitrogen. Lambda phage

DNA and HindIII digested lambda phage DNA were purchased from New England BioLabs Inc.

Nickel resin, 5 mL SP Sepharose High Performance cation exchange column, and a 23.562 mL

Superdex 75 column were purchased from GE Healthcare. Primers were synthesized by

Integrated DNA Technologies, Inc. The E. coli BL21 (DE3) STAR and BL21 (DE3) cell lines

were purchased from Novagen, whereas the DH5α cell line was purchased from Life

Technologies.

3. 2. Bioinformatics on Gp74

Currently, there is no information about the tertiary structure of gp74. While we assume

that the HNH motif residues adopt a ββα–metal fold, information regarding the structure of the

rest of the protein is lacking. Phyre2 (Protein Homology/AnalogY Recognition Engine) [71] was

used to predict the structure of gp74. One of the top hits for the structure–based alignment and

model template from Phyre2 was the GS–15 protein from Geobacter metallireducens (PDB

2QGP). However, the secondary structure of the ββα–fold, which is a crucial structural property

of HNH endonucleases, in the homology model generated by Phyre2 is not well defined. Thus,

we sought to generate a homology model of gp74 using another program, MODELLER.

First, we used ClustalW [72] to generate a structure–based sequence alignment of HK97

gp74 and G. metallireducins GS–15. The ClustalW results were verified with PONDR (Predictor

of Naturally Disordered Regions), which predicts disordered residues in proteins based on

Page 19 of 74

primary sequence [73]. The structure–based sequence alignment from ClustalW and the GS–15

structure were used as inputs into MODELLER [74] to generate a homology model of gp74.

3. 3. Expression of HK97 gp74

A plasmid expressing gp74 with an N–terminal 6x His tag, pET15b–6x His–HK97 gp74, was

obtained from Karen L. Maxwell (CCBR, Toronto). The plasmid also encodes a tobacco etch

virus (TEV) protease site between the 6x His tag and gp74 sequence for removal of the tag.

QuikChange mutagenesis was used to obtain mutant gp74 proteins. All 5’3’ primers used to

generate the mutations are shown in Table 2, with the desired mutation shown in bold letters and

underlined.

The 6x His–gp74 proteins wild type and mutants were expressed in E. coli BL21 (DE3)

STAR cells using the following protocol [54]. Single colonies from freshly transformed E. coli

BL21(DE3) STAR cells were inoculated into 3 mL LB media with 100 µg/ml ampicillin and

incubated at 37 ˚C at 250 rpm for 4 hours or until OD600 of 1.0 was obtained. A total of 300 μL

of the culture was used to inoculate in 300 mL of M9 minimal media to give a starting OD600 of

0.001. The M9 minimal media contained 6 g/L Na2HPO4 7H2O, 3 g/L KH2PO4 and 0.5 g/L NaCl

at a pH of 7.4, 3 g/L glucose, 100 µg/mL ampicillin, 10 µM D–biotin, 10 µM thiamine, 1 g/L

NH4Cl, 0.1 mM CaCl2, 1 mM MgSO4, and 10 µM ZnSO4. The pH of the media was 7.4. The

culture was incubated overnight at 30 ˚C with shaking at 250 rpm. The next day, the 300 mL

culture was harvested by centrifugation at 4000 rpm for 20 min at room temperature. The cells

were resuspended and inoculated into 6x 1L of fresh M9 media. The 6 1L cultures were

incubated at 37 ˚C with shaking at 250 rpm until OD600 was approximately 0.5 at which point the

temperature was reduced successively to a final temperature of 18 ˚C when OD600 reached 0.7–

0.8. Following 30 minutes of incubation at 18 ˚C, protein expression was induced with 1 mM

IPTG and the cultures were incubated overnight at 18 ˚C. The next day (after 16–18 hours of

Page 20 of 74

induction), the cells were harvested and stored at –20 ˚C until purification. The HK97 gp74 wild

type and mutant proteins were also expressed in 2 L of LB containing 100 µg/mL ampicillin,

Table 2: Primers for generating HK97 gp74 mutant proteins

Mutation Primer 5’ 3’ Template Tm

(°C) % GC

MW of

gp74

proteins

(g/mol)

pI of

gp4

H43A ACA CCA GCA ACG GTG GTC

GAC GCT ATC GTG CCC CAC

AAA CTG AAA

gp74–WT1 84.8 55.6 12959 9.36

H82A CAG CCA CTG TGC AAA GCG

CAT GCT GAC TCA ACG AAA

CAG AGA ATG

gp74–WT 83.0 51.1 12959 9.36

N73A TTC TGG AGT AAA GAG GCC

TGG CAG CCA CTG TGC gp74–WT 78.0 57.0 12982 9.36

D42A CA ACG GTG GTC GCC CAT

ATC GTG CCC CA gp74–WT 80.0 64.3 12981 9.51

H23A CGT TTT CTC CAG CAG GCC

CCA CTG TGT GTG ATG gp74–WT 78.0 58.0 12959 9.36

H47A GTC GAC CAT ATC GTG CCC

GCC AAA CTG AAA GAT GCG

CTT

gp74–WT 81.1 53.8 12959 9.36

H43A/H82A H43A Primer gp74–H82A 84.8 55.6 12893 9.36

H43A/N73A/

H82A N73A Primer

gp74–

H43A/H82A 78.0 57.0 12850 9.36

C26A/C29A G CAC CCA CTG GCT GTG ATG

GCC GAG CAG CAG GGG C gp74–WT 80.0 71.4 12961 9.60

C78A/H81A G AAC TGG CAG CCA CTG GCC

AAA GCG GCT CAT GAC TCA

ACG AAA C

gp74–WT 80.4 56.8 12927 9.47

C26A CAG CAG CAC CCA CTG GCT

GTG ATG TGC GAG CAG gp74–WT 81.0 63.6 12993 9.47

C29A CA CTG TGT GTG ATG GCC

GAG CAG CAG GGG CG gp74–WT 81.0 67.7 12993 9.47

C78A AAC TGG CAG CCA CTG GCC

AAA GCG CAT CAT GAC gp74–WT 79.0 57.6 12993 9.47

H81A G CCA CTG TGC AAA GCG GCT

CAT GAC TCA ACG AAA C gp74–WT 79.0 54.3 12959 9.36

N101A2

GTA ATC GGT TGT GAT GCC

GCC GGC TAC CCG CTC GAT

CCT

gp74–WT 89.4 61.5 12982 9.36

H110A2

TAC CCG CTC GAT CCT GCG

TCT GCC TGG AGC ACG TAA

TGA AAG ACC

gp74–WT 85.7 57.8 12959 9.36

1 = gp74–WT: original plasmid obtained from Karen L. Maxwell (CCBR, Toronto) 2 = Designed by Serisha Moodley, 2011

Page 21 of 74

following a similar protocol. In this case, the 2 L of LB cultures were inoculated directly with the

small 3 mL cultures and thus, there was no need for 300 mL overnight growth.

3. 4. Purification of HK97 gp74

Wild type and mutant gp74 proteins were purified using a previously established protocol

[54]. All steps were performed at 4 ˚C. The cellular pellets were resuspended in 15 mL of lysis

buffer (20 mM Tris HCl, pH 7.9, 150 mM NaCl, 5 mM imidazole, 5 mM benzamidine, 5 mM

amino–caproic acid, 2 mM β–mercaptoethanol, 1 mM PMSF, and 2 mg/mL deoxycholic acid).

The cells were lysed by sonication followed by separation of the lysate from the cellular debris

by centrifugation at 13,000 rpm in a JA20 rotor (Beckmann) for 40 minutes at 4 ˚C. The lysate

was filtered with 0.45 µm syringe filter and then applied to a 5 mL Fast Flow Ni2+

column (GE

Healthcare) that was pre–equilibrated with buffer A (20 mM Tris HCl, pH 7.9, 500 mM NaCl,

2 mM β–mercaptoethanol, and 20 mM imidazole). The column was run at 1 mL/min throughout

the purification. The column was then washed with 10 column volumes (50 mL) of buffer A to

remove any non–specific residues bound to the Ni2+

resin. The 6x His–gp74 protein was eluted

using buffer B (20 mM Tris HCl, pH 7.9, 500 mM NaCl, 2 mM β–mercaptoethanol, and

400 mM imidazole). Additional reductant, DTT, was pre–added to the elution fraction collector

tubes so that the final concentration would be 5 mM. The N–terminal 6x His tag was removed

from the 6x His–gp74 protein using TEV protease. Gp74 and TEV protease, in a 40:1 ratio, were

combined in presence of 5 mM EDTA and dialyzed against 50 mM sodium phosphate, pH 7.0,

50 mM NaCl and 5 mM β–mercaptoethanol at 4 ˚C.

In order to improve the purification protocol previously designed in our laboratory [54],

we included an additional step. The cleaved gp74 was loaded on a 5 mL HiTrap SP Sepharose

FF cation exchange column pre–equilibrated with 20 mM sodium phosphate buffer, pH 7.0. A

cation exchange column was used because the pI of gp74 is 9.3 and thus, the protein is positively

Page 22 of 74

charged at pH 7.0. The column was run at a flow rate of 2 mL/min. For the removal of any non–

specific bound proteins, the column was washed with three column volume (15 mL) of 20 mM

sodium phosphate buffer, pH 7.0. Proteins were eluted from the cation exchange column using a

NaCl gradient of 100 mL from 0 M salt to 1 M salt in 20 mM sodium phosphate buffer, pH 7.0.

We expect that the weak positively charged molecules (proteins) would be eluted in low salt and

strong positively charged proteins would be eluted in high salt. The column was then washed

with the 1 M NaCl high salt buffer to remove all proteins bound to the column. Elution fractions

of 5 mL were collected and protein eluted was monitored by absorbance at 280 nm. Fractions

containing gp74 were identified by SDS–PAGE, pooled, and concentrated. Purification of gp74

using cation exchange chromatography was only done initially as it did not enhance the purity of

the protein.

Regardless of whether a cation exchange step was used in the purification, gp74 was

purified to homogeneity using a 23.562 mL Superdex 75 column (GE Healthcare) as a final step.

As done previously [54], the column was run at a flow rate of 0.5 mL/min in 50 mM HEPES

buffer, pH 7.0, 150 mM NaCl, 5 mM EDTA and 5 mM β–mercaptoethanol. Fractions containing

gp74 were verified via SDS–PAGE, pooled, and dialyzed against 20 mM HEPES, pH 7.0, 5 mM

β–mercaptoethanol overnight at 4 ˚C. Because the studies for this thesis rely on knowing the

metal–bound state of the protein, the gp74 purification now includes a step to remove trace

metals. In order to remove any bound metals, 0.5 g Chelex® 100 sodium form resin per 1 L of

buffer was added to the dialysis buffer. The addition of the 0.5 g/L of the resin is based on

preliminary ICP analysis performed on 15 μM of the gp74–WT protein that suggested 0.3 g/L of

the resin is necessary to chelate 90% of trace metals. An excess of 0.2 g/L of the resin is used to

account for variability in the amounts of pure protein obtained. The procedure removed trace

metals from gp74 leaving only one equivalent of Zn2+

as determined by ICP–AEOS. The

Page 23 of 74

dialyzed protein was then concentrated to a final concentration of about 800 µM for gp74–WT.

The mutant gp74 proteins were prone to precipitation and thus, lower concentrations were

obtained for mutant gp74 proteins.

Protein concentrations were determined from A280 readings in 6 M guanidinium

hydrochloride (Gdm HCl) [76]. Because the protein would be denatured in 6 M GdmHCl and all

the residues would be completely exposed to the solvent, the extinction coefficient of HK97

gp74 can be estimated as the sum of individual extinction coefficient values of tryptophan (W),

tyrosine (Y), and cysteine (C) residues in 6 M Gdm HCl as described by Pace, et al., 1995 [76].

The extinction coefficient of gp74 in 6M GdmHCl, ε280nm denatured, is calculated as follows:

ε280nm, 6 M GdmHCl (M–1cm–1) = [(nW) (5685 M–1cm–1) + (nY) (1285 M–1cm–1) + (nC) (125 M–1cm–1)],

where nW, nY, and nC are the numbers of tryptophan, tyrosine, and cysteine residues. Given the

sequence of gp74, the extinction coefficient for denatured gp74 is as follows:

ε280nm,denatured gp74 = [(4)(5685 M–1cm–1)+(2)(1285 M–1cm–1)+(4)(125 M–1cm–1)]= 25810 M–1cm–1

The protein concentration determined by A280 readings was also confirmed with amino

acid analysis (Advanced Protein Technology Centre for Amino Acid Analysis Facility, The

Hospital for Sick Children, Toronto). The purity of gp74 was assessed via SDS–PAGE gel and

mass spectrometry (AIMS Mass Spectrometry Laboratory, Toronto). Trace metals were

determined via inductively coupled plasma–atomic emission optical spectroscopy, ICP–AEOS

(Analytical Laboratory for Environmental Science Research and Training, Analest, Toronto).

Page 24 of 74

3. 5. Fluorescence Temperature Melting Studies

The change in intrinsic Trp fluorescence of gp74–WT as a function of temperature was

measured in order to determine melting temperature (Tm) of the protein. Gp74 has four

tryptophan residues. Fluorescence emission spectra were recorded on a Horiba–Jovin Fluoromax

4 fluorimeter. Samples contained 2 µM gp74–WT in 20 mM HEPES, pH 7.0, 5 mM β–

mercaptoethanol. Fluorescence spectra were recorded from 15 to 95 ˚C in 2 ˚C increments with

0.5 ˚C tolerance and 1 min equilibration time. The spectra were recorded with an excitation

wavelength of 280 nm and a slit width of 5 nm. A full emission spectrum was recorded at each

temperature from 300 nm to 450 nm using an emission slit width of 5 nm.

3. 6. CD Spectroscopy

3. 6. 1. Secondary Structure Determination

CD spectra of wild type and mutant gp74 proteins were recorded at 25 ˚C on an Aviv

Biomedical Inc., Lakeview, New Jersey spectrophotometer. Spectra were recorded from 200–

270 nm in 0.5 nm steps with a 0.15 sec averaging time and a bandwidth of 1.0 nm using a 1 cm

path length quartz cell. Each CD spectrum was an average of five scans and was blank corrected

for buffer. Samples were prepared by diluting concentrated samples in 20 mM HEPES, pH 7.0 to

yield samples of 2 µM protein in 20 mM Na+ phosphate, pH 7.0. HEPES buffer results in

scattering in the CD spectrum at wavelengths from 190–210 nm. Typically, the HEPES

concentration was reduced from 20 mM to 0.1 mM and thus scattering was avoided.

3. 6. 2. Thermal Stability Measurements by CD

Thermal denaturation was monitored by the following the change in ellipticity at 221 nm.

Gp74 protein samples were heated from 25 to 91 °C in 2 °C increments, with a 0.5 or 1 min

equilibration time and 0.15 sec averaging time. The melts were carried out in a 1 cm cuvette with

a protein concentration of 2 μM in 20 mM Na+ phosphate, pH 7.0. The reversibility of the

Page 25 of 74

unfolding of gp74 proteins were monitored by heating each 2 µM protein sample from 25 to

91 °C and then cooling the sample from 91 to 25 ˚C in 2 °C increments, with a 0.5 min

equilibration time.

3. 7. HNH Endonuclease DNA Cleavage Assay

DNA digestion assays were performed with 24 µg/mL wild type or mutant gp74,

25 µg/mL lambda phage (λ) DNA, and 500 µM NiCl2 in 20 mM HEPES buffer, pH 7.0 from a

previously established protocol [54]. All samples were prepared on ice. An appropriate amount

of buffer is first added to 1.5 mL Eppendorf tubes to ensure a 200 µL final reaction volume,

followed by the addition of protein and metal, as required. A separate set of tubes are prepared

for individual time points containing a stop solution that consists of final concentrations of 5 mM

EDTA and 1x DNA loading dye. The substrate, λ DNA, was then added to the reaction tubes.

The reaction tubes were gently inverted a few times and were quickly centrifuged for 30 sec at

room temperature. It should be noted that the reaction contents should be mixed by inversion and

a quick centrifugation for equal loading on the gel. Immediately after mixing, 20 µL samples of

each reaction were added to the tubes containing the stop solution (for the 0 hr time point) and

the remaining reaction samples were incubated at 37 ˚C. Additional time points were taken every

hour for 6 hours and placed in a premade stop solution and immediately frozen at –20 ˚C.

Digestion experiments were visualized with 1% agarose gel stained with Sybr Safe. The purity

and oxidation of the gp74 proteins were monitored via SDS–PAGE in reducing and non–

reducing conditions before each assay.

HNH endonuclease experiments using HindIII digested λ DNA for comparison with λ

DNA (undigested) as a substrate were also conducted. HindIII digested λ DNA at a concentration

of 24 μg/mL was used in the assay with 13 μg/mL of gp74–WT. All other conditions were kept

constant as described previously. The time points were recorded from 0–2, 4 and 6 hours.

Page 26 of 74

Two HNH experiments with varying concentrations of Ni2+

ions were performed. The

first experiment was performed to determine the minimum amount of Ni2+

ions needed by gp74

to completely digest λ DNA. The concentration of Ni2+

was varied from 500 μM, 100 μM, and

50 μM. A 13 μg/mL (= 1 μM) of gp74–WT was used with 25 μg/mL λ DNA as substrate, where

all the other reaction components were kept the same as described earlier. The time points were

taken from 0–2, 4 and 6 hours. The second experiment conducted had an increasing

concentration of Ni2+

ions ranging from 1 mM, 2 mM, 5 mM, and 10 mM. All other parameters

were the same as described earlier [54].

3. 8. Rate of DNA Digestion

Two experiments were performed to the determine rate of DNA digestion. In the first

experiment, the rate of λ DNA (25.6 pM) digestion by gp74–WT (1.8 µM) was monitored via

UV spectroscopy in the presence of Ni2+

(500 µM) in 20 mM HEPES, pH 7.0. The change in

absorbance was monitored at 260 nm at various intervals between 0 and 4.5 hours. The 0 hr time

point, obtained as soon as the reaction was mixed, was used as the reference.

The second experiment was set up with 1.8 µM gp74–WT, 25.6 pM λ DNA and 500 µM

Ni2+

ions in 20 mM HEPES, pH 7.0. Time points were taken every 10 min for 40 min, followed

by time points at 60 min and 90 min. A stop solution consisting of a final concentration of

15 mM EDTA and 1x DNA loading dye was used to stop the reaction at each time point. Initially

this experiment was visualized with a low percent agarose (0.4 %) gel that was run for 190 min

at 40 V. We used a 0.4 % agarose gel so that the relative quantity of the undigested λ DNA at

each time point could be estimated using a high molecular weight DNA ladder ranging from

48,502 to 10,171 bp. This caused several problems resulting from melting of the gel. Therefore

for ease–of–handling the gels the visualization was performed using a 1 % agarose gel that was

Page 27 of 74

run for 30 min at 100 V. The relative quantity of the undigested DNA at each time point was

estimated using lanes that were loaded with 375, 250, 125, and 50 ng λ DNA.

3. 9. Expression and Purification of TEV Protease

TEV protease is needed to remove the 6x His tag in the purification of gp74 proteins. A

sample of a plasmid expressing 6x His–TEV was obtained from the laboratory of Karen L.

Maxwell (CCBR, Toronto). The 6x His–TEV protease was expressed in E. coli BL21 (DE3)

cells in LB media with 100 µg/ml ampicillin. Cells were grown at 37 °C with shaking at 250 rpm

until OD600 was approximately 0.5, at which point the temperature was reduced successively to a

final temperature of 16 ˚C when OD600 reached 0.7–0.8. Following 30 min of incubation at

16 ˚C, protein expression was induced overnight with 1 mM IPTG. The next day, cells were

harvested and stored at –20 ˚C until purification.

The TEV protease was purified via Ni2+

affinity chromatography by following the same

protocol as described for HK97 gp74. TEV protease expression and purification was verified via

SDS–PAGE. The elution fractions containing the TEV protease were pooled and dialyzed with

5 mM EDTA against 50 mM Tris HCl, pH 7.0, 50 mM NaCl, and 5 mM β–mercaptoethanol

overnight at 4 ˚C. The dialyzed TEV protease was then concentrated to 1 mg/mL. For storage at

–80 ˚C, glycerol at a final concentration of 50% (v/v) was added to the concentrated TEV

protease. The protein was then aliquoted in 500 μL fractions and stored at –80 C until required.

4. Results and Discussion

4. 1. Structure–Based Sequence Alignment

Phyre2 is a Protein Homology/analogY Recognition Engine that predicts a 3D structure

of a protein using the primary sequence as a query [71]. This program searches approximately 10

million known sequences to detect evolutionary related homologues of the query sequence using

the PSI–BLAST program [71]. Phyre2 then collects all the evolutionary related homologues and

Page 28 of 74

converts them into a Hidden Markov model (HMM). The HMM predicts all mutations that may

have occurred over time in the query sequence to determine an evolutionary fingerprint [71].

Phyre2 also performs a PSI–BLAST search and an HMM calculation for the sequences of all

structures in the protein data bank (PDB). Phyre2 then aligns the HMM of the query sequence to

the HMM data bank of sequences for all known structures to find the sequence in the PDB with

the greatest similarity to the query sequence [71]. This step generates a sequence alignment

between the query protein sequence and the target protein from the PDB, and subsequently

generates a 3D homology model. Using this technique, the Phyre2 program can generate

accurate models even with very low sequence homology of <15% [71]. Phyre2 generated a

homology model of HK97 gp74 based on Geobacter metallireducins GS–15 protein, which

Phyre2 determined to be evolutionary related to gp74. GS–15 is a suitable template for the

homology model because it contains a putative Zn2+

finger and HNH residues and has a

sequence identity of 22.4 % with gp74. The structure of GS–15 was solved as part of a Protein

Structure Initiative Phase 2 (PSI–2) by Northeast Structural Genomics Consortium (NESG).

There is no further information of this protein in literature. In examining the model created by

Phyre2, we see evidence of the HNH motif hallmarks. However, Phyre2 predicts that gp74 is

structured is regions which are predicted to be disordered by PONDR. For example, although

GS-15 contains an N-terminal a-helix, many of these residues are missing in gp74 and thus this

region in gp74 is predicted to be disorderd. Prediction of protein structure by programs such as

Phyre2 is highly dependent on the sequence alignment between the target (i.e. gp74) and

template (GS-15) proteins. In order to verify, and possibly improve the model, we sought to

generate our own alignment and homology model using ClustalW and MODELLER,

respectively.

Page 29 of 74

ClustalW is a tool that compares relatedness and conservation between protein and DNA

sequences [72]. The structural information of a protein is used to construct a structure–based

alignment in ClustalW. By taking into account the structure of the target sequence (i.e. GS–15),

gaps are inserted into the query sequence (i.e. gp74) at positions that correspond to disordered

regions, such as loops and termini, in the target sequence. The ClustalW alignment between gp74

and GS–15 indicated that the conserved residues C26, C29, H43, N73, C78, H81, and H82 in

gp74 correlate in GS–15 with residues C37, C40, H52, N69, C74, C77, and N78, respectively

(Figure 10). Additionally, we used PONDR to confirm the identity of disordered regions [73].

Further, the sequence alignment obtained from ClustalW differs from that obtained from Phyre2

in the following manner: Firstly, the sequence alignments between gp74 and GS–15 obtained

from Phyre2 and ClustalW show differences at the N–terminus. The sequence obtained from

Phyre2 shows an N–terminal α–helix. However, the PONDR prediction of gp74 displays

disorder in the same region. A similar situation occurs when comparing the C–terminal

sequences in the alignment. Because the sequence identity between GS–15 and gp74 is very low

at the termini, we wanted to create a homology model of gp74 that would not take into account

the secondary structure of GS–15 and would take into account the information from PONDR.

MODELLER is a program that generates homology models using protein sequences.

MODELLER calculates dihedral angles of non–hydrogen atoms and the distance between atoms

based on the template protein and applies it to the query protein [74, 77, 78]. The location of the

density of each protein atom allows a homology model to be generated. The homology model of

gp74 (Figure 11) was generated in MODELLER using the sequence alignment from ClustalW

and the structure of GS–15 as inputs. The structure of gp74 is illustrated with a ββα–fold flanked

on one side by two helices and the other side with disordered C–terminal amino acid residues.

Page 30 of 74

Figure 10. Structure–based sequence alignment of HK97 gp74 based on the structure of with G.

metallireducins GS–15 protein using ClustalW. (Hyder, B. Z. and Kanelis, V.; 2012, unpublished). The secondary structure of GS–15 is shown above, where blue cylinders represent α–helices, blue

arrows represent β–strands, and blue lines represent unstructured regions. These structural constraints are

applied to gp74 in MODELLER. The disordered regions (Disorder) indicated by ‘D’ were predicted using

PONDR [73] and secondary structure predictions (SS Pred) indicated by ‘A for α–helices and B for β–

sheets’ were predicted by ClustalW for gp74 [71]. Negatively charged residues are in red, positively

charged residues are in dark blue, hydrophobic residues are in green, polar uncharged residues are in light

blue, helix breaking residues are in orange, aromatic residues are in cyan, cysteine residues are in dark

red, and histidine residues are in pink. An “*” indicates that the residues in that column are identical in all

sequences in the alignment. A “:” indicates that conserved substitutes are observed and a “.” indicates that

semi–conserved substitutions are observed. The conserved HNH residues H43, N73, and H82 and the

cysteine residues that form a putative Zn–finger C26

XXC29

and C78

XXH81

are highlighted by red boxes.

Figure 11. A homology model of HK97 gp74

based on Geobacter metallireducins GS–15

protein (2QGP). The HNH motif is shown in cyan whereas

the rest of the protein is shown in grey. The

HNH motif is comprised of 2 β–strands,

containing the first His (H43 as a general base)

and Asn residue (N73 for secondary structure

stabilization), followed by an α–helix that

contains the second His residue (H82 for metal

binding site). The side chain of residues H23,

H43, H47, H81, H82, and H110 are shown in

blue, N57, N73, and N101 are shown in green,

C26, C29, and C78 are shown in yellow, and

D42 is shown in colour red. This figure was

generated using MolMol [75] (Hyder, B. Z. and Kanelis, V.; 2012, unpublished).

Page 31 of 74

4. 2. Expression and Purification of HK97 gp74

The 6x His–gp74 protein is highly expressed in both BL21 (DE3) and BL21 (DE3)

STAR cells. Upon IPTG induction, a protein of size 16 kDa is expressed (Figure 12a, lane 3).

This is in agreement with the molecular weight of 6x His–gp74 that is 15.3 kDa. Unfortunately,

most of the wild type and mutant gp74 proteins are expressed in the insoluble fraction (Figure

12b, lanes 2 and 4). However, the small amount of the soluble protein could be purified by Ni2+

affinity chromatography (Figure 12b, lanes 3 and 5). Since HK97 gp74 was expressed as a fusion

protein with a 6x–His tag, the expressed 6x His–gp74 protein can bind to the Ni2+

resin with

higher affinity than most E. coli proteins and the untagged gp74. However, as a metal–binding

protein, even the untagged gp74 protein can bind a Ni2+

column (Moodley and Kanelis;

unpublished). Many of the non–specific proteins bound to the Ni2+

resin were removed using a

wash buffer containing 20 mM imidazole (Figure 12b, lane 8), whereas 6x His–gp74 was eluted

with a buffer containing 400 mM imidazole (Figure 12b, Lane 10 and 11). The wash buffer also

contained 150 mM NaCl, which helped to prevent non–specific proteins binding to the resin and

2 mM of β–mercaptoethanol to ensure that the four Cys residues in gp74 are reduced. Oxidation

of these Cys residues could result in both intra– and intermolecular disulfide bond formation, and

precipitation of the protein. The inclusion of 5 mM benzamidine and 150 μM of PMSF in the

lysis and purification buffers are used to inhibit serine proteases, while 5 mM 6–aminocaproic

acid is used to inhibit carboxypeptidases. Further, a mild anionic detergent deoxycholic acid is

used to solubilize bacterial cell membrane [79–81].

Although the 6x His–gp74 wild type and mutant fusion proteins could be purified by Ni2+

chromatography, the Ni2+

column elution fractions contained some impurities. Thus, further

purification was necessary, as determined before in our laboratory [54]. As shown previously,

after removing the 6x His tag from 6x His–gp74 using TEV protease (Figure 12c), the protein

Page 32 of 74

can be purified to homogeneity via size exclusion chromatography (Figure 13a and 13b). A

Superdex 75 column was chosen because it possess the optimal separation range, from 3000 kDa

to 70,000 kDa, to purify a protein with a molecular weight of 13 kDa, such as gp74.

Additional purification steps were attempted in order to expedite the process by which

highly pure protein is achieved. Thus, we chose to include cation exchange purification step

Figure 12.Purification of HK97 gp74. 17% SDS–PAGE was used to monitor the expression and

purification of gp74–WT. A. 6x His–gp74, shown as a schematic

diagram above the gel, over–expression is induced with 1mM

IPTG. The post–IPTG lane displays an induced protein of

~16 kDa. B. Samples taken during cell lysis and purification by

immobilized Ni2+

column chromatography. Pellet 1 and

Supernatant 1 are the insoluble and soluble fractions respectively

after the first sonication. Pellet 2 and supernatant 2 are taken after

the second sonication. Elutions 1-5 are samples from 5 mL

elution fractions from the Ni2+

column. 8 µL of each sample was

loaded on the gel. C. Cleaved gp74, shown as a schematic

diagram above the gel, with pre– (lane 3) and post–TEV (lane 4)

digestion. A 10 µL of 5 mL of each sample was loaded on the

gel. The samples loaded onto the above gels were denatured with

5x SDS reducing buffer and boiled at 95˚C for 5 min. The

electrophoresis was done at 135V for 90 min, and the gels stained

for 20 min with Coomassie Brilliant Blue and de–stained with

30% ethanol.

B A

C

Page 33 of 74

before size exclusion column. The pI of gp74 is 9.3. Therefore, the protein is positively charged

at pH 7, the pH at which the cation exchange purification was conducted. In addition to

potentially removing impurities, a cation exchange column would also concentrate the protein,

which is necessary before application to a size exclusion column, and thus would eliminate a

time consuming concentration step. Elution of the protein is monitored by UV absorbance at

280 nm, the wavelength corresponding to the absorbance of aromatic residues. Gp74 elutes at

50 mL with 400 mM NaCl (Figure 13c). The elution fractions containing the protein, confirmed

via SDS–PAGE gel (not shown), were pooled, concentrated, and further purified to homogeneity

via size exclusion chromatography. However, we did not continue with this procedure because

there is no advantage in purification and concentration of protein was still required.

In order to assess whether gp74 purified as shown previously, the catalytic activity of each gp74–

WT elution from the Superdex 75 gel filtration column was assayed, as shown previously

(Figure 14) [54]. The digestion assay was performed with 25 µg/mL λ DNA and 500 µM NiCl2

in 20 mM HEPES buffer, pH 7.0. In each reaction 10 µL of the gp74 fraction was used in a total

reaction volume of 200 µL. A volume of 10 µL was chosen, so that gp74 concentration would be

58 µg/mL in the reaction tube corresponding to the elution fraction with highest gp74

concentration. For the control reaction consisting of λ DNA and gp74 only, 10 µL of gp74 was

used from the elution fraction displaying the highest peak at 280 nm. Time points were taken

every hour for 5 hours. As expected for an HNH endonuclease, and as shown previously for

gp74 [54], no DNA digestion is observed unless reactions contained both gp74 and a divalent

metal ion such as Ni2+

(Figure 14). The lack of DNA digestion for fractions that do not contain

gp74 indicates that the endonuclease activity is due to gp74 and not another protein which co–

purified with gp74.

Page 34 of 74

Figure 13. Further purification of gp74–WT via size exclusion and cation exchange chromatography.

A. An absorbance trace of gp74–WT eluted from Superdex 75 column was measured at A280nm

(mAU) and plotted against elution volume (mL). B. A 17% SDS gel shows 0.5 mL fractions obtained

from the elution volume of the size exclusion column. The loaded samples were denatured with 5x SDS

reducing buffer, boiled at 95˚C for 5 min. The gel was electrophoresed at 135 V for 90 min, stained for 20

min with Coomassie Brilliant Blue and de–stained with 30% ethanol for overnight. C. An absorbance

trace of cation exchange chromatography of gp74–WT monitored at A280 nm (blue line). The salt

concentration was as conductivity by the AKTA–purifier during elution (red line).

Gp74 activity has also been demonstrated to be dependent on the amount of divalent

metal ions used [54], as with other metalloenzymes. I also tested my purified gp74 against

different concentrations of metals. The concentration of Ni2+

ions tested consisted of two ranges:

1, 1–10 mM and 2, 50–500 µM. Ni2+

was used as a reference since it displays the most efficient

gp74 mediated DNA digestion [54]. As with previous preparations of gp74, it was observed that

at higher concentrations of Ni2+

(5 mM and 10 mM), the endonuclease activity of gp74 was

inhibited [54] (Figure 15a). This indicates that purified gp74 behaves as previously documented

-50

250

550

850

1150

1450

1750

2050

2350

0 5 10 15 20 25

UV

Ab

sorb

ance

(m

AU

)

Elution Volume (mL)

250

300

350

400

450

500

550

-100

0

100

200

300

400

37 42 47 52 57 62

Mo

lari

ty o

f N

aCl (

mM

)

UV

Ab

sorb

ance

(m

AU

)

Elution Volume (mL) mAU [NaCl]

A B

C

Page 35 of 74

[54], and is also similar to other HNH proteins that also display a decrease in activity in the

presence of high divalent metal ion concentrations [54, 57, 62]. Further, complete digestion was

observed with 500 μM Ni2+

within 2 hours, it took about 3–4 hours for 100 μM Ni2+

to

completely digest DNA, whereas with 50 μM Ni2+

some of the DNA was still left undigested at

the 4 hour time point (Figure 15b). This data suggests that gp74 is active at all of the tested

concentrations of Ni2+

ions. However, the time gp74 takes to digest λ DNA increases as the

concentration of the Ni2+

ions decreases. Further, due to time restraints 500 μM Ni2+

was used

Figure 14. Gp74 mediated digestion of λ DNA in the presence of Ni

2+ ions.

The HNH endonuclease activity of the eluent fractions from the Superdex 75 gel filtration column was

tested. Digestion reactions were allowed to proceed for 5 hours. A 25 µg/mL of λ DNA and 500 µM of

Ni2+

were used in this assay with 20 mM HEPES, pH 7.0. A 10 µL of each gp74 elution fraction was

used. Thus, the concentration of gp74 varied in each reaction as with the elution. Gp74 in the control

reactions was taken from the fraction containing the highest gp74 concentration. Digestion reactions were

visualized with a 1 % agarose gel stained with Sybr safe (1: 10000).

Page 36 of 74

for all of the activity assays. This suggests that a lot of metal is needed for gp74 digestion of its

substrate probably because low affinity of nickel binding to the metal–binding residues [54] and

also that λ DNA is not the substrate in vivo.

Figure 15. The HNH endonuclease activity of gp74 is affected upon altering Ni2+

ions concentrations. Gp74–mediated λ DNA digestion in the presence of Ni

2+ ions with varying concentrations at A, 1, 2, 5, and

10 mM and B, 500, 100, and 50 µM. Reaction time points were taken every hour from 0–6 hours for panel A

and at 1 hr, 2hr, and 4 hr interval for panel B. 20 µL of each reaction was placed in a 10 µL of stop solution

consisting of final concentration of 1x DNA loading dye and 5 mM EDTA. 12.5 µL of these samples were

loaded onto 1 % agarose gel. The gel was run for 30 minutes at 100 V and viewed with Sybr Safe stain.

The mutant proteins gp74–H82A (Figure 16) and gp74–D42A (data not shown) were

purified similar to gp74–WT protein. As with the wild type, the mutant proteins are primarily

found in the insoluble fraction, although some soluble protein was expressed and could be

isolated with the Ni2+

affinity column. Further, the mutant proteins eluted at the same volume

from the Superdex 75 column, indicating that the mutations did not cause aggregation of the

proteins. Additional gp74 mutant proteins (gp74–N73A, gp74–H43A, and gp74–H43A/H82A)

were also expressed and purified. However, unlike gp74–WT, gp74–H82A, and gp74–D42A,

4 hr 2 hr 1 hr

A

B

Page 37 of 74

these additional mutants were prone to precipitation after removal of the 6x His tag. Because

gp74 requires divalent metal ions for activity, removal of the 6x His tag was essential for our

studies. Thus, studies of the gp74–N73A, gp74–H43A, and gp74–H43A/H82A mutants requires

new sample conditions to be determined that promote sample solubility.

For each endonuclease activity assay, the purity and oxidation state of gp74 was tested in

reducing and non–reducing conditions via SDS PAGE. The expected position of the gp74 on the

gel is between protein standard marker bands of 16 and 6 kDa (around 13 kDa), as the molecular

mass of gp74 is 13025 Da (Figure 17). The gel further shows that the gp74 protein has not

oxidized to form any intermolecular disulfide bridges, where the intramolecular oxidation can be

tested using a DTNB assay. The reducing and non–reducing samples were spaced out to avoid

any diffusion of the reductant between the lanes. This gel was also run prior to preparing samples

for CD, ICP, and mass spectrometry.

Figure 16.Purification of HK97 gp74–H82A.

Samples taken during cell lysis and

purification by immobilized Ni2+

column

chromatography. Pellet 1 and Supernatant 1 are

the insoluble and soluble fractions respectively

after the first sonication. Pellet 2 and

supernatant 2 are taken after the second

sonication. Elutions 1-5 are samples from 5 mL

elution fractions from the Ni2+

column. 8 µL of

each sample was loaded on the gel. The samples

loaded onto the above gels were denatured with

5x SDS reducing buffer and boiled at 95˚C for

5 min. A 17% SDS–PAGE was run at 135V for

90 min, and the gels were stained for 20 min

with Coomassie Brilliant Blue and de–stained

with 30% ethanol.

Page 38 of 74

4. 3. Total Protein Content and Purity Assessment via Mass Spectrometry

Throughout HK97 gp74 research experience, several issues were encountered that were

dealt with by persistent troubleshooting. Firstly, most of the gp74 proteins are insoluble but there

is a need to acquire a lot of protein to conduct the series of experiments mentioned above such

as, ICP–AEOS, mass spectrometry, fluorescence studies, CD thermal stability experiments,

several HNH endonuclease activity assays. We wished to conduct all the experiments from a

single preparation protein so that results regarding stability and activity can be analyzed knowing

metal–loaded state of the protein. Initially, 2 L of bacterial culture was grown in 100% M9

minimal media and the protein was purified. The initial amounts obtained were determined to be

about 2.6 mg from 2 L of gp74–WT and even lower amounts were obtained for the mutants. Due

to different levels of soluble expression and solubility of the wild type and mutant gp74 proteins,

different amounts of purified proteins are obtained (Table 3). We have optimized gp74–WT

expression and purification to 10.8 mg/L of culture grown in 6 L of minimal media (and 7 mg/L

culture grown in 2 L of 100% LB). Slightly lower amounts of protein can be obtained for gp74–

H43A and gp74–H82A proteins. However, a much lower amount of protein was obtained for

gp74–N73A and H43A/H82A proteins, due to aggregation during the purification. In order to get

Figure 17. A 17% SDS gel for

gp74–WT with reducing (SDS) and

non–reducing conditions (NR SDS).

The gel samples contained 1x SDS

sample buffer with and without β–

mercaptoethanol, and boiled at 95˚C

for 5 min. The gel was

electrophoresed at 135 V for 90

min, stained for 20 min with

Coomassie Brilliant Blue and de–

stained with 30% ethanol.

Page 39 of 74

enough protein so that all experiments could be conducted on single sample, 6 L of culture were

used for the expression of each protein, except for gp74–D42A which was purified from 3 L of

culture.

Table 3: Gp74 protein concentrations were determined using absorbance at 280 nm.

HK97 gp74

Soluble Protein Obtained

Grown in 100% M9,

minimal media

Soluble Protein Obtained

Grown in 100% LB,

enriched media

mg of protein/L of culture mg of protein/L of culture

WT 2.69 3.46

H43A 1.15 2.81

N73A 0.41 1.13

H82A 1.66 N.D.

H43A/H82A 0.43 1.13

D42A1 0.34 2.85

N.D.: Not determined

ESI–mass spectrometry was used to confirm the identity and assess the purity of each

gp74 protein (Table 4). The expected molecular weight of each gp74 protein compared favorably

with the molecular weight determined via mass spectrometry.

Table 4: Purity assessment of gp74 proteins via mass spectrometry

HK97 gp74

protein

Mass Spectrometry

Expected (E)

Mass

(Da)

Actual (A)

Mass

(Da)

Difference

(E – A)

WT 13024.9 13025 – 0.1

H43A 12958.9 12955 + 3.9

N73A 12981.9 12979 + 2.9

H82A 12958.9 12959 – 0.1

D42A 12980.9 12981 – 0.1

H43A/H82A 12892.8 12890 +2.8

4. 4. Metal dependence of gp74 expression

Minimal media recipes generally have trace quantities of divalent metals for optimal

growth and expression of proteins. In order to determine which metals are required for

expression of soluble gp74 and to determine if we could express the protein in the absence of any

Page 40 of 74

metals, we conducted an experiment where gp74–WT was grown in M9 minimal media with and

without metals Mg2+

, Ca2+

, and/or Zn2+

. Cellular growth was arrested at OD600 ~0.4, there was no

cellular growth, and thus there was no protein expression in the absence of Mg2+

ions (Figure

18a: lane 7; 18b: lane 2 and 7) regardless of the presence of Ca2+

or Zn2+

ions (Figure 18a: lane 5

and 9; 18b: lane 4). This is probably because DNA replication enzymes are Mg2+

dependent [82],

Figure 18. SDS–PAGE is showing pre– and

post–IPTG inductions of gp74–WT in M9

minimal media with various divalent metals.

A concentration of 0.1 mM Ca2+

, 1 mM Mg2+

,

and/or 10 μM Zn2+

was used for all of the above

minimal growths. A. Gp74 was expressed using

minimal media containing all metals (lanes 2, 3),

lacking Ca2+

(lanes 4, 5), lacking Mg2+

(lanes 6,

7), or lacking Zn2+

(lanes 8, 9). B. Gp74 was

expressed using minimal media containing only

Zn2+

(lanes 1, 2), only Mg2+

(lanes 3, 4), only Ca2+

(lanes 6, 7), or lacking all metals (lanes 8, 9).

C. Purification of 6x His–gp74–WT expressed in

minimal media lacking Zn2+

. Samples are as

described in Figure 12. A 10 µL of each sample

was loaded on the gel.

C

A B

Page 41 of 74

so the absence of Mg2+

would render these enzymes non–functional, thus limiting cell growth.

The expression of gp74 has little dependence on the inclusion of Ca2+

or Zn2+

in the media,

although the protein obtained was in very small quantities. It should be noted that in the absence

of Zn2+

, the insolubility of gp74 increased and therefore there was no protein obtained in the

elution fractions (Figure 18c: lane 1, 3, 10). This is likely due to misfolding of gp74. Binding of

Zn2+

to the CXXC and CXXH motifs is essential for folding of the Zn2+

–finger, which may be

essential for folding of gp74

4. 5. Gp74 and Proteolysis

It was noticed for a few months that the gp74 proteins were undergoing proteolysis

during expression and/or purification as there were proteins of two different sizes induced

(Figure 19, lane 2). Multiple attempts were made to resolve this issue. Firstly, purification

buffers were remade and including fresh protease inhibitors, because initially it seemed as if the

proteolysis was occurring in the sonication step. However, there was no change in proteolysis.

Secondly, fresh competent cells were prepared, as we have seen aberrant protein expression in

the lab when the competent cells are older. However, the proteolysis of gp74 continued. The Ni2+

column used for Ni2+

affinity chromatography was extensively cleaned on the basis that

proteases may be stuck to the resin. Further, a freshly packed column with new Ni2+

–resin was

even used, but again gp74 eluted from this column as a proteolyzed product. When the cell type

was changed to BL21 (DE3) STAR cells from BL21 (DE3) cells, no proteolysis was observed.

However, this was not reproducible. Therefore, in order to test whether or not this is consistent

when cells are grown in rich media (LB), the BL21 (DE3) STAR cells were grown in 100% LB.

No proteolysis or any degradation was observed. No degradation was also observed for gp74–

D42A, H43A, N73A, H82A, and H43A/H82A proteins. This can be explained using different

stages of bacterial growth. When bacterial culture is grown in minimal media, the bacterial

Page 42 of 74

growth can reach stationary phase fairly quickly (at an OD600 ~ 1.0) due to lack of nutrition and

the shorter duration of log phase. However, when the same bacterial culture is grown in rich

media the duration of log phase is increased due to abundance of nutrients, and thus it takes

much longer to reach stationary phase (at an OD600 ~2.0). So if the cells were induced in

stationary phase (OD600 ~ 1.1), they would have already begun to die in the minimal media

growth culture and hence, we see proteolysis with gp74. This was also consistent with the small

overnight culture (OD600 ~ 1.3), which was used to make 1 L day cultures. This implies that the

cells have already decreased their efficiency to express more protein. However, when the cells

were induced in the log phase (OD600 ~ 0.7–0.8), there was no proteolysis.

4. 6. Gp74 and Storage

Once the protein is purified, it is concentrated to > 2 mg/mL and is stored in a buffer

containing 20 mM HEPES, pH 7.0, 5 mM β–mercaptoethanol at 4 °C. Storage of the protein in a

buffer lacking NaCl is necessary for the HNH endonuclease activity assays, as salt has been

found to inhibit gp74 HNH activity [54]. Further, high salt concentrations result in noisy CD

spectra at wavelengths of 190 nm to 210 nm. However, over time the protein continuously

aggregates when concentrated to 2 mg/mL until there is around 1 mg/mL left in the solution.

Figure 19. SDS–PAGE showing

degraded gp74–WT grown in BL21

DE3 cells (lane 2) versus the full–

length gp74–WT grown in BL21

DE3 STAR cells (lane 4).

Page 43 of 74

This is probably due to lack of sample stability and the absence of salt in the buffer. The gp74

fractions eluted from Supredex 75 in 50 mM HEPES, pH 7.0, 150 mM NaCl, and 5 mM β–

mercaptoethanol have shown no aggregation for months when stored at 4 ˚C. Further, gp74–WT

is active when diluting these fractions so that the salt final concentration in HNH assays is

7.5 mM (Figure 14). Therefore, I suggest storing gp74 in a buffer containing 50 mM NaCl.

4. 7. ICP Analysis

In order to determine the metal–loaded state of the wild type and mutant gp74 proteins,

each protein sample was analyzed by ICP–AEOS (Table 5). ICP–AEOS analysis was only

performed on gp74–WT and its mutants gp74–D42A and gp74–H82A. ICP–AEOS data indicates

that 1 equivalent of Zn2+

is bound to all gp74–WT preparations (n = 3) studied. The gp74–WT

preparations lack any bound Ni2+

. Similar results were also obtained for one preparation each of

gp74–D42A and gp74–H82A. The other mutant proteins, gp74–H43A, gp74–N73A, and gp74–

H43A/H82A, were not analyzed because adequate quantities of these proteins could not be

obtained. ICP–AEOS requires 4.5 mL of 30 µM of sample, which is greater than the soluble

amount of protein obtained for these mutant proteins. The lack of Ni2+

bound indicates that the

HNH motif does not have a Ni2+

ion constitutively associated with it. Further, one equivalent of

Zn2+

is likely bound to the putative Zn2+

finger formed by C26

XXC29

and C78

XXH81

motifs in

gp74. Our HNH assays, which show that divalent metal ions (i.e. Ni2+

and Zn2+

[54], Figure 14)

must be added to the reaction for the DNA digestion, confirm that metal ions are not

constitutively associated with the HNH motif. Further, Zn2+

–fingers have a high affinity for Zn2+

ranging from 10–6

to 10–12

M [83–86]. In contrast, HNH motifs have varying affinities for metal

with some binding metal tightly in the absence of DNA and some needing the substrate from

high affinity [86]. Because gp74 binds Ni2+

with weak affinity [54], we assume that all divalent

Page 44 of 74

metal ions will bind the HNH motif with weak affinity. The low amounts of other metals

detected may be due to trace metal ions in glassware, plastics, or water.

When gp74–WT was grown in 100% rich media (LB), it was observed that there was

only 77.5 % Zn2+

bound to the protein. Because we get higher levels of expression of gp74 in

LB, these data suggest that there are not enough Zn2+

ions in the media to load the Zn2+

–finger of

the expressed gp74. In order to generate a sample fully loaded with Zn2+

, excess Zn2+

ions were

added by dialysis and the protein sample was sent for ICP analysis after chelating excess metal

ions. The analysis indicated that there is 2: 1 Zn2+

bound to gp74 (>200%), suggesting that both

metal–binding sites are occupied in the protein including the putative Zn2+

–finger and the metal–

binding site within the HNH motif. An HNH assay is necessary to confirm this hypothesis.

Table 5: Determination of percent (%) metal–bound for gp74–WT and mutants via ICP–AEOS.

Elements

in Sample

WT D42A H82A

Trial 1 Trial 2 Trial 3 Grown

in LB

Reloaded

with Zn2+

Trial 1 Trial 1

Ba 0.19 – – – – – 0.07

Cu 0.84 – – – – – 1.42

K – – 3.41 – – 2.55 8.36

Mg 0.27 – – – – 1.37 3.43

Ni 0 0 0 0 0 0 0

Zn 107 131 114 77.5 232.5 102 99.88

Each value is an average of three different readings from each 30 µM protein sample

4. 8. Fluorescence Studies

Intrinsic tryptophan (Trp) fluorescence was used initially to determine the melting

tempertaure (Tm) of gp74–WT and the mutants of gp74. In a folded protein, a Trp residue may be

buried in the interior of a protein. Thus, the accessibility of the Trp residue to the solvent is

limited, which minimizes quenching and results in maximum Trp fluorescence. Unfolding of a

protein is expected to fully expose the Trp residue to the solvent, which results in collisional

quenching of the Trp fluorescence. Figure 20 shows emission spectra of gp74–WT. As expected,

Page 45 of 74

Figure 20. Gp7–WT denaturation studies monitored by intrinsic Trp fluorescence.

Fluorescence spectra were recorded of 2 µM of gp74–WT in 20 mM HEPES, 5 mM βMe, pH 7.0.

A. Fluorescence emission spectra of gp74 were recorded using an excitation wavelength of 280 nm an

excitation slit width of 5 nm. Emission spectra were collected from 300 nm to 450 nm using an emission

slit width of 5 nm at both 15 ˚C and 95 ˚C. B. The unfolding of gp74 was monitored by fluorescence

emission at 345 nm from 15 ˚C to 95 ˚C in 2 ˚C increments with 0.5 ˚C tolerance and 1 min equilibration

time. The excitation and emission slit widths were as described for panel A.

when gp74 is heated to 95 ˚C, the intrinsic Trp fluorescence of the protein significantly

decreases, indicating that the protein is unfolded at high temperature. Thus, the intrinsic Trp

fluorescence as a function of temperature can be used to monitor unfolding and determine the

Tm. However, because the graph of fluorescence change as a function of temperature do not

display a sigmoidal curve (Figure 20b), we could not use Trp fluorescence melting data to

calculate the Tm for gp74. A sigmoidal curve is expected for cooperative protein unfolding.

Comparison with a sigmoidal curve indicates that the gp74 unfolding curve lacks the initial

baseline for the folded protein and resembles only part of the sigmoidal curve for cooperative

protein unfolding. Thus, the data indicate that either gp74 does not unfold cooperatively or that

gp74 is partially unfolded even at 15 ˚C. However, NMR data obtained on gp74 at 25 ˚C are

indicative of a folded protein [53]. Alternatively, the deviation of the melting curve of gp74 from

the expected sigmoidal curve may be due to the fact that there are four Trp residues and the

instrinsic Trp fluorescence would monitor a change in the environment of all of the four Trp

A B

Page 46 of 74

residues, where two Trp residues may already be surface exposed indicating a partially unfolded

gp74 protein. Our homology model indicates that W69 and W111, which are located within the

loop of the ββα–fold and the disordered C–terminus of the protein, respectively, are 100%

surface exposed. Residues W12 and W74 are more than 80% surface exposed. Residue W12 is

located at the N–terminal α–helix and W74 is located within the ββα–fold. With respect to the

predicted tertiary structure from our homology model, W12 and W74 are in close proximity with

K71, whereas W69 is near residues D51 and F68 in space. The charged residues and other

aromatic residues are within 5Å of the Trp resdiues likely affects the overall Trp fluorescence

spectrum. Further, as the protein unfolds, these interactions will be broken, and thus the Trp

fluorescence spectrum upon unfolding will also be modulated by these tertiary contacts and not

just by solvent exposure.

Further, when the denatured gp74 was cooled back to 15 ˚C, a considerable drop to the

intial intensity was observed, indicating that the unfolding of gp74 is irreversible. Similar results

are obtained with 2 μM gp74–WT in 20 mM Na+ phosphate, pH 7.0 instead of 20 mM HEPES,

pH 7.0 (not shown) and when protein was heated from 5 ˚C to 95 ˚C instead of 15 ˚C to 95 ˚C.

4. 9. Circular Dichroism Spectroscopy Analysis

CD spectroscopy was used to investigate protein secondary structure of the gp74–WT

and gp74 mutant proteins (Figure 21a). As shown previously [54], the CD spectrum of gp74–WT

displays double minima around 208 nm and 221 nm that are characteristic of a folded protein

that is primarily comprised of α–helices. The CD spectra of gp74–D42A and gp74–H82A mutant

proteins are similar to that for the wild type protein, indicating that mutation of these two

residues predicted to be in the metal binding site of the HNH motif do not disrupt the α–helical

structure of gp74. However, the CD spectra of gp74–H43A and gp74–H43A/H82A show

markedly decreased mean residue ellipticity values, suggesting that the H43A mutation causes a

Page 47 of 74

loss in structural integrity of the protein. Alternatively, the low ellipticity values may be due the

lack of sample stability (i.e. aggregation and precipitation). The CD spectra of the gp74 proteins

are acquired using low concentrations of protein (i.e. 2 M). Thus, any precipitation of the

protein would likely not be visible, but would still affect the CD signal. Any precipitate would

also affect our ability to calculate the mean residue ellipticity because the amount of protein in

solution would not be known.

-16000

-14000

-12000

-10000

-8000

-6000

-4000

-2000

0

2000

200 210 220 230 240 250 260 270

Mo

lar

Ellip

tici

ty (

de

g.cm

2 /d

mo

l)

Wavelength (nm)

WT H82A D42A H43A H43A/H82A

-16000

-14000

-12000

-10000

-8000

-6000

-4000

-2000

0

21 29 37 45 53 61 69 77 85 93

Mo

lar

Ellip

tici

ty (d

eg.

cm2/d

mo

l)

Temperature (˚C)

25–90°C

90–25°C

-16000

-14000

-12000

-10000

-8000

-6000

-4000

-2000

0

23 33 43 53 63 73 83 93

Mo

lar

Ellip

tici

ty (

de

g.cm

2 /d

mo

l)

Temperature (˚C)

WT H82A D42A H43A H43A/H82A

Figure 21. CD spectra of gp74 proteins.

CD spectra were recorded of 2 µM of either

wild type or mutant gp74 proteins in 20 mM Na+

phosphate, pH 7.0. A. CD spectra of gp74–WT

and mutant proteins. Data was collected at 25 °C

from 200 nm to 270 nm in 0.5 nm increments

with a 0.15 sec averaging time. CD spectra are

an average of 5 scans. All CD spectra were

blank corrected. B. Molar ellipticity values at

221 nm for the unfolding (25 ˚C to 90 °C) and

refolding (90 ˚C to 25 °C) of gp74–WT with

30 sec equilibration time. C. Comparison of the

unfolding of gp74 proteins from 25 ˚C to 90 °C

with 1 min equilibration time. Data was

collected from 25 ˚C to 90 °C at 221 nm in 2 °C

increments with a temperature dead band of

0.5 °C and a 0.15 sec averaging time for figure

panels B and C.

A B

C

Tm ~55 ˚C

Page 48 of 74

4. 9. 1. Melting Temperature of HK97 gp74

Because Tm values for wild type and mutant gp74 proteins could not be determined using

intrinsic Trp fluorescence, the unfolding of the gp74 proteins was assessed by monitoring the

change in CD signal at 221 nm (Figure 21). Unlike data observed using Trp fluorescence, the CD

melting curves display a sigmoidal dependence, indicating that gp74 unfolds cooperatively and

further that the fluorescence data is complicated by the existence of four Trp residues in different

environments. Similar to that observed with Trp fluorescence, it was found that the unfolding

was irreversible (Figure 21b). Thus, ΔG and the other thermodynamic variables could not be

obtained. However, the Tm of gp74–WT was estimated to be about 55 °C based on the mid–point

of transition [87] (Figure 21c). Similar values were obtained for gp74–D42A and gp74–H82A

mutant proteins. The Tm values could not be obtained on gp74–H43A and gp74–H43A/H82A

due to low mean ellipticity values for these proteins at all temperatures. This is because samples

precipitated during denaturation experiment, and thus further analysis could not be performed.

4. 10. HNH Endonuclease Assays by HK97 gp74

4. 10. 1. Gp74 and Mutations

In order to test whether the mutations of H82A and D42A compromise the activity of

gp74, we compared the HNH endonuclease activity of the gp74 mutants to gp74–WT. As done

previously, the HNH assays were conducted using λ phage DNA as a substrate in the presence of

Ni2+

ions and were analyzed via agarose gel electrophoresis [54]. As with other HNH assays a

series of control experiments were carried out. As seen previously, DNA digestion is not

observed in controls with λ DNA alone, λ DNA with Ni2+

, and λ DNA with either gp74–WT [54]

or mutant gp74 proteins (Figure 14, 22, 23). Incubation of λ DNA, divalent metal ion, and wild

type or mutant gp74 at 37 °C results in λ DNA digestion (Figure 14, 22, 23). Examination of the

amount of λ DNA digestion at specific time points suggests that the gp74–H82A and gp74–

Page 49 of 74

D42A mutants have reduced HNH activity compared to the wild type protein (Figure 22 and 23).

It takes about 2 hours for the gp74–WT to completely digest λ DNA, whereas it takes about

4 hours for gp74–H82A and 4 hours for gp74–D42A. In case of the gp74–H82A and gp74–

D42A mutations, a loss of activity is not necessarily expected because only one residue of the

metal–binding site is altered in each one of the mutants. A further reduction in activity is

expected for a D42A/H82A double mutant. Further, there may be additional residues in the

metal–binding site of the HNH motif in gp74, as observed for other HNH endonucleases. For

example, the crystal structure of the E7 colicin bound to Zn2+

displays a metal–binding site

formed by H544, H569, and H573 [86]. The E7 colicin residues H544 and H569 correspond to

D42 and H82, respectively, in gp74, while H573 is located C–terminal to the HNH motif. Gp74

also contains a His residue, H110, which is located C–terminal to the HNH motif (Figure 11).

Thus, it is possible that gp74 also contains additional metal–binding site residues and that

complete inhibition of HNH activity would require mutation of multiple amino acids.

Preliminary data were also acquired for gp74–N101A (not shown), which was initially

thought to be a potential secondary structure stabilization residue until gp74–N73A was purified.

The gp74–N101A activity assays illustrated similar activity as the gp74–WT, where λ DNA was

completely digested in 2 hours.

The effect of mutation of H43 and N73 could not be studied to due instability of the

gp74–H43A and gp74–N73A samples. However, we would predict that mutation of H43 would

result in a protein that is not catalytically active. Further, mutation of N73 should also

compromise activity, as this residue is expected to orient the catalytic His residue during the

endonuclease reaction [58, 86]. A survey of other sample conditions, such as storing samples in a

buffer containing some NaCl as described above, is necessary for further work on these mutants.

Page 50 of 74

Figure 22. The effect of the H82A mutation on gp74 activity. Lambda phage DNA was incubated with either gp4–WT (lanes indicaed by orange arrows)or gp74–H82A

(lanes indicated by blue arrows) in the presence of Ni2+

ions. Reaction time points were taken at 0 hr, 0.5 hr, 1

hr, 1.5 hr, 2 hr, 4 hr, and 24 hr. At each time point, 20 µL of each reaction was placed in 10 µL of stop solution

consisting of a final concentration of 5 mM EDTA and 1x DNA loading dye. 15 µL of these samples were

loaded onto 1 % agarose gel. The gel was run for 30 minutes at 100 V and visualized with Sybr Safe stain (1:

10000). Control reactions, consisting of either λ DNA alone, λ DNA with either gp74–WT or gp74–H82A, or

λ DNA with Ni2+

, were also incubated. At the 4 hour time point, samples in lanes for gp74-WT and gp74-

H82A were switched.

Figure 23. The effect of the D42A mutation on gp74 activity. Lambda phage DNA was incubated with either gp4–WT (lanes indicaed by orange arrows) or gp74–D42A

(lanes indicaed by orange arrows) in the presence of Ni2+

ions. Reaction time points were taken at 0 hr, 1 hr, 2,

hr, and 4 hr. At each time point, 20 µL of each reaction was placed in 10 µL of stop solution consisting of a final concentration of 5 mM EDTA and 1x DNA loading dye. 15 µL of these samples were loaded onto 1 %

agarose gel. The gel was run for 30 minutes at 100 V and visualized with Sybr Safe stain (1: 10000). Control

reactions, consisting of either λ DNA alone, λ DNA with either gp74–WT or gp74–D42A, or λ DNA with

Ni2+

, were also incubated.

1 hr 0.5 hr 0 hr

1 hr 0 hr 2 hr 4 hr

1.5 hr 2 hr 4 hr 24 hr

Page 51 of 74

4. 10. 2. HindIII Digested λ DNA as a Substrate for Cleavage

Some of our digestion experiments using λ phage DNA displayed long–lived species of

approximately 3 kB, implying that although gp74 cleaves λ phage DNA at multiple places, there

may be some preference for specific sites. In order to gain some insight into the gp74–mediated

digestion sites of λ DNA, an activity assay using HindIII digested λ phage DNA as a substrate

was conducted. We were hoping that not all HindIII digested fragments would be cleaved by

gp74 and thus, we may be able to narrow down segments of λ DNA that were not digested by

gp74. However, all fragments of HindIII digested λ phage DNA were digested to the same extent

by gp74 (Figure 24). Thus, different DNA substrates need to be investigated to determine the

gp74 endonuclease recognition sequence requirements.

Figure 24. HindIII digested λ DNA versus λ DNA as a substrate for endonuclease digestion by gp74.

HindIII digested λ DNA was digested with gp74–WT. Reaction time points were taken at 0hr, 1 hr,

2 hr, 4hr and 6hr. 20 µL of each reaction was placed in a 10 µL of stop solution consisting of a final

concentration of 5 mM EDTA and 1x DNA loading dye. 15 µL of these samples were loaded onto 1 %

agarose gel. The gel was run for 30 min at 100V and viewed with Sybr Safe stain. Control reactions,

consisting of HindIII digested λ DNA alone, HindIII digested λ DNA with gp74–WT, HindIII digested λ

DNA with Ni2+

, and λ DNA with Ni2+

were also incubated.

1 hr 2 hr

4 hr 6 hr

0 hr

Page 52 of 74

4. 11. Rate of DNA Digestion by HK97 gp74

Because our engineered mutants display catalytic activity, we sought to use the

differences in the rates of DNA digestion between the wild type and mutant gp74 proteins to

quantify the loss of activity by the mutations. Two different types of experiments were

performed in order to characterize the rate of DNA digestion by gp74–WT. Figure 25a shows

an increase in change in absorbance (ΔA) of the free bases at 260 nm. This is expected as the λ

DNA is digested by gp74 the free nucleic acid bases are released, and thus as time increases so

does the concentration of the free bases. However, the ΔA values of free bases released upon

gp74 digestion of λ DNA is much smaller than noted in literature by E9 colicin (ΔA from 0.05 to

0.3) [88]. Also, the differences in these values are very small, complicating analysis. Therefore,

gel electrophoresis was used to determine the rate of DNA digestion by gp74, based on the

amount of λ DNA left undigested by gp74. Figure 25b shows a 1 % agarose gel with decreasing

amounts of undigested DNA by gp74–WT as a function of time (min). Densitometry was used to

determine the amount (ng) of full length λ DNA left undigested in each line (Figure 25c).

Although these experiments were designed to determine the rate of λ DNA digestion by gp74–

WT, the acquired data could not be fit to an equation to determine the absolute rate of digestion

(Figure 25c). This may be because gp74 digests λ DNA promiscuously at multiple sites, and thus

the λ DNA digestion products are available as substrates until all λ DNA is digested. However, a

relative rate of digestion could be determined using densitometry of bands showing undigested λ

DNA by gp74–WT, where the acquired data could be compared to the mutant gp74 proteins.

There were several issues that were encountered repetitively with our attempts to quantify

the amount of undigested DNA using densitometry. Firstly, unequal loading of the samples on to

the gel was problematic and as a consequence comprised data analysis. This was solved by

mixing reactants by inversion and a quick centrifugation as described earlier in the methods

Page 53 of 74

Figure 25. Rate of DNA digestion by gp74 using

densitometry.

A. The rate of λ DNA (25.6 pM) digestion by

gp74–WT (1.8 µM) monitored at 260 nm in the

presence of 500 µM Ni2+

ions in 20 mM HEPES,

pH 7.0. Time point ‘0’ was used as a reference

point. B. An HNH endonuclease assay where

25.6 pM of λ DNA is digested by 1.8 µM gp74–

WT in the presence of 500 µM Ni2+

ions in 20 mM

HEPES, pH 7.0 for 90 minutes. A 1% agarose gel

was run for 35 min at 100 V and visualized with

Red safe stain. C. The absolute quantity of λ DNA

(ng) left undigested per time (min) by gp74 is

plotted based on the gel in panel B.

section. Secondly, we ran into problems when trying to quantify the amount of full length λ

DNA in solution. Originally, we attempted to use a high molecular weight DNA ladder (48,502

to 10,171 bp) for calibration. Because of the large sizes of DNA in the high molecular weight

ladder, we needed a low percent (0.4 %) agarose gel to resolve all bands. This low percent gel

was run at 40 V for 190 min, during which time the gel started to melt. Further, the DNA bands

became very diffuse, complicating densitometry. In the end, we used 1 kB molecular weight

DNA ladder and four different amounts of λ DNA were used to estimate the amount of

undigested substrate left per time. For these studies, we used a 1 % agarose gel. Finally, it was

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0 45 90 135 180 225 270

A 2

60

(n

m)

Time (min)

0

75

150

225

300

375

0 15 30 45 60 75 90

Am

ou

nt

of

Un

dig

est

ed

DN

A (

ng)

Time (min)

A

C

B

Page 54 of 74

also noted that using a final concentration of 5 mM EDTA in the stop solution was not enough to

completely stop the reaction. This was because the DNA in reaction samples loaded on the gel

was being digested during electrophoresis due to high temperatures. Several experiments were

performed to fully sequester Ni2+

ions by increasing amounts of EDTA, which is added to the

stop solution to abolish the activity of gp74 at each time point. Thus, determining the right

amount of ETDA (15 mM) in the stop solution was necessary to reproducibly perform

densitometry on the full length DNA left undigested by gp74.

5. Conclusion

Previous work in our laboratory established that HK97 gp74 is indeed a divalent metal

ion–dependent HNH endonuclease. However, experiments needed to be conducted to confirm

the identity of the HNH residues, and thus was the focus of this work. Because analysis of each

protein required multiple experiments, it was necessary to optimize the amount of recombinant

protein obtained for the gp74. Gp74 protein expression and purification were optimized to obtain

2.7 mg of protein per L of culture [54]. Lower amounts were obtained for all the mutants. The

purity of all gp74 proteins was assessed by mass spectrometry, whereas the metal–bound states

of gp74–WT, gp74–D42A, and gp74–H82A were investigated by ICP–AEOS. ICP analysis

suggested there is always Zn2+

bound to the gp74 protein, in both wild type and mutant states.

We hypothesize that the Zn2+

ion is bound to the putative Zn2+

–finger. Fluorescence studies and

CD thermal stability experiments showed that the folding and unfolding of gp74 is irreversible.

CD spectroscopy demonstrated that gp74–D42A and gp74–H82A were folded similar to gp74–

WT. A Tm of about 55 °C was estimated from CD thermal stability experiments for the gp74–

WT, gp74–D42A, and gp74–H82A proteins. HNH endonuclease assays displayed slightly

reduced activities with the gp74–mutants (gp74–D42A and gp74–H82A).

Page 55 of 74

6. Future Work

Additional studies of gp74 mutant proteins are necessary to determine the residues

involved in DNA binding, DNA digestion, and metal–binding. Studies of structural and metal–

binding mutants of gp74, such as gp74–D42A and gp74–H82A may help determine metal–

binding affinities of various metals especially Ni2+

and Zn2+

. It has been shown in this report that

gp74–H43A, gp74–N73A, and gp74–H43A/H82A are highly insoluble and unstable. The effect

of mutations at these residues in gp74 needs to be determined. Additional solution conditions

should be probed in order to study these mutant proteins. Also, the activity assays with natural

substrate is an ongoing work, where the natural substrate yet to be determined.

Ultimately, we wish to obtain a solution structure of gp74–WT by NMR. NMR

experiments require milligrams of highly pure protein. For gp74, we would need to grow E. coli

cultures in 6 L or more of labeled media, which is not cost effective. In order to obtain increased

protein yields without growing large cultures, which is necessary for NMR studies, a protocol is

being established for refolding of insoluble fraction of gp74 in the Kanelis Lab [89]. I supervised

an undergraduate research student for the following studies. The insoluble pellets containing

gp74 protein are first completely unfolded using 6 M guanidium chloride in 20 mM Tris HCl

buffer, pH 7.9, 150 mM NaCl, 5 mM imidazole, and 2 mM β–mercaptoethanol. The solubilized

gp74 inclusion bodies are then loaded onto a Ni2+

affinity column equilibrated with same buffer.

The column is washed with 15 column volumes of 20 mM Tris HCl, pH 7.9, 150 mM NaCl,

20 mM imidazole, 6 M Gdm HCl, 2 mM β–mercaptoethanol. The protein is then eluted from the

column using 20 mM Na+ acetate, pH 4.0, 150 mM NaCl, 6 M Gdm HCl and 2 mM β–

mercaptoethanol. This low pH buffer was used to protonate all histidine residues to disrupt the

bonds between the 6x His tag on the 6x His–gp74 and the Ni2+

on the affinity resin to elute the

protein. Refolding of gp74 was accomplished by dialysis overnight against 20 mM HEPES,

Page 56 of 74

pH 7.0, 150 mM NaCl and 2 mM β–mercaptoethanol. Once the protein is refolded, the 6x His

tag can be removed using TEV protease. Figure 26 shows that CD spectra of gp74 refolded in the

absence or presence of Zn2+

ions has some α–helcial characteristics similar to natively folded

gp74 [89], albeit with small mean ellipticity values, suggesting that the protein is not fully re–

folded.

Structural and conformational changes of gp74 upon metal–binding can be studied using

limited proteolysis. Very low amounts of trypsin and/or chymotrypsin can be used to selectively

digest gp74 in the presence or absence of metals (Ni2+

and Zn2+

). Because these proteolyzing

enzymes are expected to digest gp74 only at surface–exposed sites when used in small quantities

(1: 100), differences in digestion patterns are expected upon metal–binding due to structural

changes. I also supervised our undergraduate research student in these studies. Preliminary

experiments in our laboratory showed that gp74–digested in the absence of any metals and

gp74–digested in the presence of Zn2+

ions by trypsin result in larger fragments than gp74–

digested in the presence of Ni2+

ions (Figure 27 [89]). The SDS–PAGE analysis shows the

presence of four trypsin–digested fragments of gp74–WT from 0 min to 60 min that are reduced

to essentially a single band of high intensity of size 6.8 kDa at 90 min [89]. Similar results were

obtained when gp74–WT was incubated with trypsin in the presence of Ni2+

ions (Figure 27b).

However, when gp74–WT was incubated with trypsin in the presence of Zn2+

ions a different

mode of trypsin digestion is observed (Figure 27c). It was observed that trypsin has initially

digested Zn2+

–bound gp74–WT into fragments of five different sizes, which were then broken

down into two smaller fragments of sizes 9.2 and 8.3 kDa. The different mode of trypsin

digestion of gp74 in the presence of Ni2+

and Zn2+

ions suggest a different mode of metal–

binding by gp74, which is consistent with our work described earlier.

Page 57 of 74

Figure 27. Limited Proteolysis of gp74–WT by

trypsin in the absence of all metals (A), in the

presence of Ni2+

ions (B), or in the presence of Zn2+

ions (C).

The samples contained 1.0 mg/mL of gp74–WT

and 0.1 µg/µL of trypsin in 20 mM HEPES, pH

7.0. 21 µM of Ni2+

(B) or Zn2+

(C) ions were also

added. Time points were taken at intervals 0 min, 5

min, 10 min, 30 min, 60 min, 90 min and 120 min

at 4oC.

This figure is adopted from reference 89.

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-12000

-10000

-8000

-6000

-4000

-2000

0

2000

4000

202 212 222 232 242 252 262 M

ola

r El

lipti

city

Wavelength (nm)

WT - apo

WT - Zn

WT - Native

A B

C

Figure 26. CD spectra of natively folded

gp74 versus gp74 refolded in the absence or

presence of Zn2+

ions.

The CD spectra of gp74 refolded with 10

mM Zn2+

ions (red) and without any metals

added (blue) was obtained. Samples of 2 µM

of gp74 proteins were prepared in 20 mM

Na+ phosphate buffer, pH 7.0. Data was

collected at 25 ˚C from 200–270 nm in 1.0

nm increments with a 0.15 sec averaging

time. CD spectra are an average of three

scans. All CD spectra were blank corrected.

WT-Native (green) is natively folded gp74 as

expressed by E. coli.

This figure is obtained from reference 89.

Page 58 of 74

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3. Mc Grath, S. and van Sinderen D (ed.). (2007). Bacteriophage: Genetics and Molecular Biology

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14. Perna, N. T.; Plunkett, G. 3rd; Burland, V.; Mau, B.; Glasner, J. D.; Rose, D. J.; Mayhew, G. F.;

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