CH339K

Proteins: Primary Structure, Purification, and Sequencing

-Amino Acid-Amino Acid

•All amino acids as incorporated are in the L-formAll amino acids as incorporated are in the L-form• Some amino acids can be changed to D- after Some amino acids can be changed to D- after incorporationincorporation• D-amino acids occur in some non-protein moleculesD-amino acids occur in some non-protein molecules

C

HOOC

NH2

R H C

HOOC

NH2

RH

L-amino acid D-amino acid

I prefer this layout, personally…

2 Amides

The Acidic and the Amide Amino Acids Exist as Conjugate Pairs

Ionizable Side Chains

Hydrogen Bond Donors / Acceptors

Disulfide formation

4-Hydroxyproline Collagen

5-Hydroxylysine Collagen

6-N-Methyllysine Histones

-Carboxygultamate Clotting factors

Desmosine Elastin

Selenocysteine Several enzymes (e.g. glutathione peroxidase)

Modified Amino AcidsModified Amino Acids

A Modified Amino Acid That Can Kill You

Diphthamide (2-Amino-3-[2-(3-carbamoyl-3-trimethylammonio-propyl)-3H-imidazol-4-yl]propanoate)

Histidine

• Diphthamide is a modified Histidine residue in Eukaryotic Elongation Factor 2

• EF-2 is required for the translocation step in protein synthesis

Diphthamide Continued – Elongation Factor 2

Corynebacterium diphtheriaeCorynebacterium diphtheriae CorynebacteriophageCorynebacteriophage

Diphtheria Toxin Action

• Virus infects bacterium• Infected bacxterium

produces toxin• Toxin binds receptor on

cell• Receptor-toxin complex

is endocytosed• Endocytic vessel

becomes acidic• Receptor releases toxin• Toxin escapes

endocytic vessel into cytoplasm

• Bad things happen

• Diphtheria toxin adds a bulky group to diphthamide

• eEF2 is inactivated• Cell quits making

protein• Cell(s) die• Victim dies

Diphtheria Toxin Action

Other Amino Acids

Every Every -amino acid has at -amino acid has at least 2 pKa’sleast 2 pKa’s

PolymerizationPolymerization

GG00’ = +10-15 kJ/mol’ = +10-15 kJ/mol

In vivoIn vivo, amino acids are , amino acids are activatedactivated by coupling to by coupling to tRNAtRNA

Polymerization of activated Polymerization of activated a.a.:a.a.:GGoo’ = -15-20 kJ/mol’ = -15-20 kJ/mol

• In vitro, a starting amino acid In vitro, a starting amino acid can be coupled to a solid matrixcan be coupled to a solid matrix• Another amino acid withAnother amino acid with

• A protected amino groupA protected amino group• An activating group at the An activating group at the carboxy groupcarboxy group

• Can be coupledCan be coupled• This method runs backwards This method runs backwards from in vivo synthesis (Cfrom in vivo synthesis (C N) N)

Peptide Bond

Resonance stabilization of peptide bond

Cis-trans isomerization in prolines

•Other amino acids have a trans-cis ratio of ~ 1000:1Other amino acids have a trans-cis ratio of ~ 1000:1•Prolines have cis:trans ratio of ~ 3:1Prolines have cis:trans ratio of ~ 3:1•Ring structure of proline minimizes Ring structure of proline minimizes GG00 difference difference

Physical Methodsor

How to Purify and Sequence a

Weapons-Grade Protein

First Question

How do I measure the amount of protein I have?

UV Absorption Spectrophotometry

Beer-Lambert Law

clTA

I

IT cl

o

log :Absorbance

elyalternativor

10 :nceTransmitta

c = concentrationc = concentrationl = path lengthl = path length = extinction coefficient= extinction coefficientAn Absorbance = 2 means that only 1% of the incident beam is An Absorbance = 2 means that only 1% of the incident beam is getting through. getting through.

Transmittance and Absorbance

Absorbance vs. Concentration Transmittance vs. Concentration

Second Question

How can I spot my protein in the great mass of different proteins?

Electrophoresis

d

- - -

-

+

-

-

+

ChargedMolecule

(Charge q)

dV

F = qV/d

Gel matrix

f

q

E

v

qEfv

v

f

fvF

E

q

qEd

VqF

b

f

or

:mequilibriuAt

velocity

tcoefficien frictional

strength field

charge

f

q

E

v

M

qv

The frictional coefficient The frictional coefficient f f depends on the size of the depends on the size of the molecule, which in turn depends upon the molecular mass, molecule, which in turn depends upon the molecular mass, so:so:

i.e. the velocity depends on the charge/mass ratio, which i.e. the velocity depends on the charge/mass ratio, which varies from protein to proteinvaries from protein to protein

Polyacrylamide Gels

Polyacrylamide gel electrophoresis of whole cell proteins of three strains of lactic acid bacteria.

Agarose

Gelidium sp.Gelidium sp.

SDS binds to proteins at a constant ratio of 1.4 g SDS/g proteinSDS binds to proteins at a constant ratio of 1.4 g SDS/g protein

Na+

OS

OCH2

CH

2

CH2

O

O

CH

2

CH2

CH

2

CH2

CH

2

CH2

CH

2

CH2

CH3

SDS PAGESodium Dodecyl (Lauryl) Sulfate

Constant q/M ratioConstant q/M ratio

rf logM

1 R

Disulfide cleavage

Disulfide cleavage and chain separation

+ ME

Isoelectric Point

Abrin A - Predicted Charge

-40

-30

-20

-10

0

10

20

30

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0

pH

Ch

arg

e o

n P

rote

in

Predicted pI5.088

Isoelectric FocusingIsoelectric Focusing

pH

Carrier Ampholytes

• Amphoteric Electrolytes

• Mixture of molecules containing multiple amino- and carboxyl- groups with closely spaced pIs

• Partition into a smooth, buffered pH gradient

Separation by pISeparation by pI

Isoelectric Focusing

BelowBelow the pI, a protein has a positive charge and migrates the pI, a protein has a positive charge and migrates toward the cathodetoward the cathodeAboveAbove the pI, a protein has a negative charge and migrates the pI, a protein has a negative charge and migrates toward the anodetoward the anode

Isoelectric Focusing Foot Flesh Extracts from Pomacea flagellata and Pomacea patula

catemacensis

STOPHERE

Protein Purification Steps1 unit = amount of enzyme that catalyzes 1 unit = amount of enzyme that catalyzes conversion of 1 conversion of 1 mol of substrate to product in 1 mol of substrate to product in 1 minuteminute

1 unit = amount of enzyme that catalyzes 1 unit = amount of enzyme that catalyzes conversion of 1 conversion of 1 mol of substrate to product in 1 mol of substrate to product in 1 minuteminute

Purification visualized

Example:Purification of Ricin

Georgi Markov1929-1978

Ricinus communisRicinus communis – castor oil – castor oil plantplant

Ricin

Ricin B chainRicin B chain(the attachment bit)(the attachment bit)

Ricin uptake and release1.1. endocytosis by coated pits and endocytosis by coated pits and

vesicles or, vesicles or, 2.2. endocytosis by smooth pits and endocytosis by smooth pits and

vesicles. The vesicles fuse with an vesicles. The vesicles fuse with an endosome. endosome.

3.3. Many ricin molecules are returned to Many ricin molecules are returned to the cell surface by exocytosis, or the cell surface by exocytosis, or

4.4. the vesicles may fuse to lysosomes the vesicles may fuse to lysosomes where the ricin would be destroyed. where the ricin would be destroyed.

5.5. If the ricin-containing vesicles fuse If the ricin-containing vesicles fuse to the Trans Golgi Network, (TGN), to the Trans Golgi Network, (TGN), there ís still a chance they may there ís still a chance they may

6.6. return to the cell surface. return to the cell surface. 7.7. Toxic action will occur when RTA, Toxic action will occur when RTA,

aided by RTB, penetrates the TGN aided by RTB, penetrates the TGN membrane and is liberated into the membrane and is liberated into the cytosol.cytosol.

Ricin Action

• Ricin and related enzymes remove an adenine base from the large ribosomal RNA

• Shut down protein synthesis

The possibility that ricin might be used as an asymmetric warfare weapon has not escaped the attention of the armed services.

The last time I was qualified to know for sure, there were no effective antidotes.

Significant Terrorist Incidents Involving Chemical and Biological Agents

Year Organization Agents

1946 DIN("Revenge" in Hebrew; also Dahm Y'Israel Nokeam, "Avenging Israel's Blood")(Germany)

Arsenic Compounds

1970 Weather Underground(United States)

Tried to obtain agents from Ft. Detrick by blackmailing a homosexual serviceman.

1972 R.I.S.E (United States)

Typhoid, diphtheria, dysentery, meningitis and several others to be delivered by aerosol.

1974 Aliens of America(Alphabet Bomber)(United States)

Nerve Agents

1980 R.A.F. (Rote Armee Faktion) (Germany)

Botulinum toxin 

1984 Rajneshee Cult (United States) Salmonella enterica serovar typhimurium

1991 Minnesota Patriots Council(United States)

Ricin

1990-1995 Aum Shinrikyo(Japan)

Bacteria and viral agents, toxins, organophosphorus nerve agents.

1995 Aryan Nation (United States)

Yersinia pestis

1995 The Covenant and the Sword (United States)

Ricin

1998 Republic of Texas(United States)

Bacterial and viral agents

2001 Unknown (United States) Bacillus anthracis

2003-2004 Fallen Angel (United States) Ricin

RawExtract

(NH4)2SO4

Cut

Affinity GelFiltration

Salting In – Salting out

iz

ic

zcI

i

i

n

iii

ion on charge

ion ofion concentrat

2

1 :Strength Ionic

1

2

• salting in: Increasing ionic strength increases protein solubility

• salting out: Increasing further leads to a loss of solubility

Salting in – salting out

The solubility of haemoglobin in different electrolytes as a function of ionic strength.Derived from original data by Green, A.A. J. Biol. Chem. 1932, 95, 47

Solubility reaches minimum at pI

Salting in: Counterions help prevent formation of interchain salt links

Salting out: there’s simply less water available to solubilize the protein.

Different proteins have different solubilities in (NH4)2SO4

Lyotropic ChaotropicSeries

Cations: N(CH3)3H+> NH4+> K+> Na+> Li+> Mg2+>Ca2+> Al3+>

guanidinium / urea

Anions: SO42−> HPO4

2−> CH3COO−> citrate > tartrate > F−> Cl−> Br−> I−> NO3

−> ClO4−> SCN−

1) Bring to 37% Saturation – ricin still soluble, many other proteins ppt

2) Collect supernatant3) Bring to 67% Saturation – ricin ppt, many remaining

proteins still soluble4) Collect pellet5) Redissolve in buffer

Dialysis and Ultrafiltration(How do you get the %@$&#! salt out?)

RawExtract

(NH4)2SO4

Cut

Affinity GelFiltration

Separation by chromatographySeparation by chromatographyBasic Idea:Basic Idea:You have a You have a stationary phasestationary phaseYou have a You have a mobile phasemobile phaseYour material partitions out between Your material partitions out between the phases.the phases.

Affinity Chromatography

Structure of AgaroseAgarose is a polymer of agarobiose, which in turn consists of one unit each of galactose and 3,6-anhydro-a-L-galactose.

Ricin sticks to galactose, so store-bought agarose acts as an affinity column right out of the bottle, with ricin binding the beads while other proteins wash through.

Begin adding 0.2 M Begin adding 0.2 M LactoseLactose

RawExtract

(NH4)2SO4

Cut

Affinity GelFiltration

B

AB

BAASS SS

SS

Ricinus communis Agglutinin (RCA)MW = 120,000

RicinMW = 60,000

Castor Beans contain two proteins that bind galactose

Gel Filtration

Gel Filtration

Gel Filtration (aka Size Exclusion)

VmVm = = matrixmatrix volume volume

VoVo = = voidvoid volume volume

VpVp = = porepore volume volume

VtVt = = totaltotal volume volume

VeVe = = elutionelution volume volume

(1a) Vt = Vo + Vp or(1a) Vt = Vo + Vp or

(1b) Vp = Vt - Vo(1b) Vp = Vt - Vo

(2) Ve = Vo + Kav*Vp(2) Ve = Vo + Kav*Vp

Combining 1b with 2Combining 1b with 2

You knew I couldn’t leave it at that…

0t

oeav VV

VVK

• a and b represent the effective separation range

• c corresponds to the exclusion limit

Kav

Fig. 3. Fig. 3. Measurement of molecular weight of native NAGase enzyme of green crab by gel Measurement of molecular weight of native NAGase enzyme of green crab by gel filtration on Sephadex G-200: standard proteins (empty circles); green crab NAGase filtration on Sephadex G-200: standard proteins (empty circles); green crab NAGase (filled circle). (filled circle).

From Zhang, J.P., Chen, Q.X., Wang, Q., and Xie, J.J. (2006) From Zhang, J.P., Chen, Q.X., Wang, Q., and Xie, J.J. (2006) Biochemistry (Moscow)Biochemistry (Moscow) 7171(Supp. 1) (Supp. 1) 855-859.855-859.

Note:Note: smaller = slowersmaller = slower, , whereas in SDS-PAGE, whereas in SDS-PAGE, smaller = fastersmaller = faster..

NoteNote

RCARCA

RicinRicin

Gel Filtration Separation of RicinGel Filtration Separation of Ricin

RawExtract

(NH4)2SO4

Cut

Affinity GelFiltration

Okay, Now Let’s Sequence the A-Chain

Bovine InsulinBovine Insulin21 residue A chain21 residue A chain31 residue B chain31 residue B chainConnected by disulfidesConnected by disulfides

In order to sequence the protein, the In order to sequence the protein, the chains have to be separatedchains have to be separated

Chain Separation

• Interchain disulfide broken by high concentrations of ME

• Chains are about the same size – but can take advantage of different pIs– B-Chain pI ~ 5.3– A-Chain pI ~ 7.2

Ion Exchangers

•Apply ME – treated ricin to DEAE-cellulose at pH 7•At pH 7:

•A chain (pKa 7.2) is essentially uncharged, •B chain (pKa 4.8) is highly negative

•A chain washes through the column•B chain sticks, eluted with gradient of NaCl

2-D Electrophoresis (an aside)

• Can use two different properties of a protein to separate electrophoretically

• For analysis of cellular protein content, often use 2-dimensional electrophoresis:

• 1st dimension is isoelectric focusing

• 2nd dimension is SDS PAGE

2-D Electrophoresis (cont.)

• Can use other protein properties to separate– Simple PAGE at 2 different pHs– PAGE and SDS PAGE

Sequencing with Phenylisothiocyanate

• Applied Biosystems 492 Procise Protein Sequencer

Chain Cleavage: Cyanogen Bromide

C-Terminal Sequencing

• Carboxypeptidases are enzymes that chew proteins from the carboxy terminus

• Can incubate a protein (preferably denatured – more later) with a carboxypeptidase

• Remove aliquot at intervals (time course)

• Run amino acid analysis of aliquots

C-Terminal Sequencing of Rat Plasma Selenoprotein

From Himeno et al (1996) J. Biol. Chem. From Himeno et al (1996) J. Biol. Chem. 271271: 15769-15775.: 15769-15775.

Tandem Mass Spectrometry can also be used to determine peptide sequences

MOLECULAR EVOLUTION

 Time of Divergence|-------------|-------------|------------|------------|-------------|------------| ┌───────────────────────────────Shark │ │ ┌─────────────────────Perch └─────────┤ │ ┌─────────────Alligator └───────┤ │ ┌──────Horse └──────┤ │ ┌───Chimp └──┤ │ └───Human|-------------|-------------|------------|------------|------------|------------|------------|------------|Sequence Difference 

Sequence differences among vertebrate hemoglobins

Neutral Theory of Molecular Evolution• Kimura (1968)

• Mutations can be:– Advantageous– Detrimental– Neutral (no good or bad phenotypic effect)

• Advantageous mutations are rapidly fixed, but really rare

• Diadvantageous mutations are rapidly eliminated

• Neutral mutations accumulate

What Happens to a Neutral Mutation?

• Frequency subject to random chance

• Will carrier of gene reproduce?

• Many born but few survive– Partly selection– Mostly dumb luck

• Gene can have two fates– Elimination (frequent– Fixation (rare)

Genetic Drift in Action

Ow!

Our green genes are evolutionarily superior!

Never mind…

Simulation of Genetic Drift• 100 Mutations x 100 generations:

• 1 gets fixed• 2 still exist• 97 eliminated (most almost immediately)

Rates of Change

CLOCK MOLECULAR a becan on accumulati change Therefore

CONSTANT. ison accumulati change Therefore

fixation. ofy probabilit theimesmutation t ofy probabilit on theonly depends R

out. cancels size population Therefore

1

size population torelatedboth are and and

ratefixation

ratemutation

:where

Rate Overall

T

NR

NR

NRR

R

R

RRR

F

M

FM

F

M

FMT

Protein Evolution RatesDifferent proteins have different rates

Protein Evolution RatesDifferent proteins have different rates

Rates (cont.)

• Slow rates in proteins critical to basic functions• E.g. histones ≈ 6 x 10-12 changes/a.a./year

Rates (cont.)

Fibrinopeptides

• Theoretical max mutation rate• Last step in blood clotting pathway• Thrombin converts fibrinogen to fibrin

Fibrinopeptides keep fibrinogens from sticking together.Fibrinopeptides keep fibrinogens from sticking together.

Rates (cont.)

• Only constraint on sequence is that it has to physically be there

• Fibrinopeptide limit ≈ 9 x 10-9 changes/a.a./year

Relationships among plant hemoglobinsArredondo-Peter, Raul, et al (1998) Plant Physiol. 118: 1121-1125

Amino acid sequences of Amino acid sequences of several ribosome-inhibiting several ribosome-inhibiting proteinsproteins

Phylogenetic trees built from the amino acid sequences of type 1 RIP or A chains (A) and B chains (B) of type 2 RIP (ricin-A, ricin-B, and lectin RCA-A and RCA-B from castor bean; abrin-A, abrina/b-B, and agglutinin APA-A and APA-B from A. precatorius; SNAI-A and SNAI-B, SNAV-A and SNAV-B, SNAI'-A and SNAI'-B, LRPSN1-A and LRPSN1-B, LRPSN2-A and LRPSN2-B, and SNA-IV from S. nigra; sieboldinb-A, sieboldinb-B, SSAI-A, and SSAI-B from S. sieboldiana; momordin and momorcharin from Momordica charantia; MIRJA from Mirabilis jalapa; PMRIPm-A and PMRIPm-B, PMRIPt-A and PMRIPt-B from Polygonatum multiflorum; RIPIriHol.A1, RIPIriHol.A2, and RIPIriHol.A3 from iris hybrid; IRAr-A and IRAr-B, IRAb-A and IRAb-B from iris hybrid; SAPOF from S. officinalis; luffin-A and luffin-B from Luffa cylindrica; and karasurin and trichosanthin from Trichosanthes kirilowii)

Hao Q. et.al. Plant Physiol. 2010:125:866-876Hao Q. et.al. Plant Physiol. 2010:125:866-876

Phylogenetic tree of Opisthokonts, based on nuclear protein sequencesIñaki Ruiz-Trillo, Andrew J. Roger, Gertraud Burger, Michael W. Gray & B. Franz Lang (2008) Molecular Biology and Evolution, Jan 9