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BiophysicalcharacterizationandunfoldingofLEF4factorofRNApolymerasefromAcNPV

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Biophysical Characterization and Unfolding of LEF4 Factor of RNAPolymerase from AcNPV

Sheeba Rasheedi,1 Madhuri Suragani,2 Soghra K. Haq,2 Sudip Ghosh,2 Nasreen Z. Ehtesham,2

Seyed E. Hasnain3,4,5

1 Laboratory of Molecular and Cellular Biology, Centre for DNA Fingerprinting and Diagnostics,

Hyderabad, Andhra Pradesh 500 076, India

2 Molecular Biology Unit, National Institute of Nutrition, Hyderabad, Andhra Pradesh 500 007, India

3 Vice Chancellor, University of Hyderabad, Hyderabad, Andhra Pradesh 500 046, India

4 Institute of Life Sciences, University of Hyderabad Campus, Hyderabad, Andhra Pradesh 500 046, India

5 Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, Karnataka 560 064, India

Received 6 August 2008; revised 6 December 2008; accepted 10 February 2009

Published online 9 March 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bip.21180

This article was originally published online as an accepted

preprint. The ‘‘Published Online’’ date corresponds to the

preprint version. You can request a copy of the preprint by

emailing the Biopolymers editorial office at biopolymers@wiley.

com

INTRODUCTION

The Autographa californica nuclear polyhedrosis virus

(AcNPV) encodes around 150 proteins, some of

which are involved in the regulation of expression

from viral gene promoters along with the participa-

tion of some host factors. The hyperactive polyhedrin

promoter (Ppolh) is activated by binding of polyhedrin pro-

moter binding protein (PPBP) within the basal Ppolh nucle-

Biophysical Characterization and Unfolding of LEF4 Factor of RNAPolymerase from AcNPV

Correspondence to: Seyed E. Hasnain; e-mail: vc@uohyd.ernet.in or Nasreen Z.

Ehtesham; e-mail: nasreen_e1@hotmail.com

ABSTRACT:

Late expression factor 4 (LEF4) is one of the four subunits

of Autographa californica nuclear polyhedrosis virus

(AcNPV) RNA polymerase. LEF4 was overexpressed in

Escherichia coli and recombinant protein was subjected

to structural characterization. Chemical induced

unfolding of LEF4 was investigated using intrinsic

fluorescence, hydrophobic dye binding, fluorescence

quenching, and circular dichroism (CD) techniques. The

unfolding of LEF4 was found to be a non-two state,

biphasic transition. Intermediate states of LEF4 at 2M

GnHCl and 4M urea shared some common structural

features and hence may lie on the same pathway of

protein folding. Steady-state fluorescence and far-UV CD

showed that while there was considerable shift in the

wavelength of emission maximum (kmax), the secondary

structure of LEF4 intermediates at 2M GnHCl and 4M

urea remained intact. Further, temperature induced

denaturation of LEF4 was monitored using far-UV CD.

This study points to the structural stability of LEF4 under

the influence of denaturants like urea and temperature.

Although LEF4 is an interesting model protein to study

protein folding intermediates, in terms of functional

significance the robust nature of this protein might reflect

one of the several strategies adapted by the virus to

survive under very adverse environmental and

physiological conditions. # 2009 Wiley Periodicals, Inc.

Biopolymers 91: 574–582, 2009.

Keywords: LEF4; AcNPV; fluorescence quenching;

GnHCl; urea; Stern-Volmer equation

VVC 2009 Wiley Periodicals, Inc.

574 Biopolymers Volume 91 / Number 7

otide determinant.1,2 This factor participates in transcription

from very late gene promoters.3,4 The participation of Sp-like

cellular factors and involvement of TFIID-Sp protein interac-

tion in transcription regulation of polyhedrin promoter has

been reported.5,6 Guarino et al.7 identified virus-encoded

RNA polymerase complex consisting of four subunits-LEF4,

LEF8, LEF9, and P47 constituting the holoenzyme. Although

LEF4 does not show any distinct RNA polymerase sequence

motif, it is reported to harbor 50mRNA capping activity.8 It

has been found that LEF4 protein has both RNA 50-triphos-

phatase and guanylyltransferase activities in vitro.

The stability of a native protein is a function of external

variables such as pH, temperature, and solvent composition

as they disrupt different kinds of non-covalent interactions

that are important for the intrinsic stability. Therefore, analy-

sis of the role of such variables in the formation of the native

structure of a protein is a prerequisite in describing the forces

that are responsible for conformational stability. Such studies

involve monitoring of conformational changes due to pertur-

bation of a protein molecule by various agents such as guani-

dine hydrochloride (GnHCl), urea, and temperature. We

report here the biophysical characterization of recombinant

late expression factor 4 (LEF4) of AcNPV, in an attempt to

understand its structure-function relationship. This report

describes the unfolding pattern of the protein factor in the

presence of denaturants like GnHCl, urea, and temperature.

Our results based on comprehensive biophysical analysis sug-

gest that LEF4 is an unusually stable protein.

RESULTS AND DISCUSSIONThe baculovirus AcNPV very late polyhedrin protein is criti-

cal for virus dissemination and survival under adverse envi-

ronmental as well as physiological condition of the insect

host gut. This protein is transcriptionally regulated by an

RNA polymerase complex, which includes LEF4 as a subunit.

We chose to conduct biophysical studies on the purified

recombinant LEF4 to shed some light on its structural stabil-

ity. An elaborate study was performed to evaluate the confor-

mational changes associated with GnHCl and urea induced

unfolding. Thermal denaturation of LEF4 as monitored by

far-UV circular dichroism (CD) was also carried out.

lef4 was cloned and over-expressed as His-tagged protein

in Escherichia coli and the recombinant protein was purified

to homogeneity as described in materials and methods. The

induced fusion protein was mainly present in the soluble

fraction of E. coli cell lysate at 188C, hence purification was

carried out under native conditions. The purified protein

migrates in accordance with its calculated molecular mass of

54 kDa when subjected to SDS-polyacrylamide gel electro-

phoresis. The purity of the preparation was found to be 95%

as evident from the coomassie staining (refer Figure 1).

Biophysical Characterization of LEF4

Three-dimensional structure of a protein obtained through

the folding of a polypeptide chain from an ensemble of fairly

loose and disordered conformation, requires precise physical

interactions between various residues that make up a polypep-

tide chain. The stability of a protein is thus a consequence of

the strength of these various non-covalent forces. Therefore,

protein folding can be studied in terms of an equilibrium of a

two-state transition of the folded to unfolded form of the

polypeptide. These transitions can be studied by measuring

the populations of the folded and unfolded forms by gradually

changing the concentration of chemical denaturants (GnHCl,

urea, etc.), temperature, and pH. The unfolding process can

be analyzed by probing the changes in the intrinsic fluores-

cence and far-UVCD spectra as a function of denaturant.

Far-UV CD: The CD spectrum of LEF4 in far-UV region

is shown in Figure 2A. In the far-UV region, CD spectra of

proteins are particularly sensitive to protein secondary struc-

ture. The native LEF4 protein showed prominent minima

around 208 and 217 nm suggesting that the protein contains

a-helical and b-sheet rich regions. The helical content of the

protein accounts for 14% of the total secondary structure,

calculated as described in materials and methods.

Fluorescence spectra: Intrinsic fluorescence spectrum pro-

vides a sensitive means of characterization of proteins and

their conformations. The spectrum is determined chiefly by

the polarity of the environment of the tryptophan and tyro-

sine residues and by their specific interactions. LEF4 polypep-

tide contains four tryptophan and 27 tyrosine residues (Figure

FIGURE 1 SDS-PAGE profile of purified recombinant His-tagged

LEF4 protein from E. coli. Lane 1 is protein molecular size marker

whereas lanes 2–6 represent 10 ll each of different 1 ml elutions of

the protein collected during affinity purification. The LEF4 protein

band of size 54 kDa is marked by an arrow.

Biophysical Characterization of LEF4 575

Biopolymers

2B). Therefore, intrinsic fluorescence properties of LEF4 were

studied by exciting the protein at 280 and 295 nm (Figure

2C). The emission spectrum in the range 300–400 nm after

exciting the protein at 280 nm is due to Trp and Tyr residues

and energy transfer due to Tyr residues, while the spectrum af-

ter exciting the protein at 295 nm is only due to Trp residues

since Tyr residue neither excites nor emits at this wavelength.

LEF4 showed strong maximum fluorescence at 342 nm when

the excitation wavelength was 280 nm. When the excitation

wavelength was increased to 295 nm, where only Trp fluores-

ces, the LEF4 fluorescence maximum was red shifted by 5 nm

along with significant quenching of the intensity. This indi-

cates that the four Trp residues of LEF4 are in different envi-

ronments and behave as different fluorescing species.9

GnHCl Induced Denaturation

Changes in the tertiary and secondary structure of LEF4 pro-

tein as a consequence of denaturation in GnHCl were moni-

tored by fluorescence and far-UV CD, respectively.

Intrinsic fluorescence measurement: Tryptophan fluores-

cence measurement (Figures 3A and 3B) shows the GnHCl

induced changes in the tryptophanyl fluorescence emission

parameters [fluorescence intensity and wavelength of emis-

sion maximum (kmax), respectively] of LEF4 in the range

0–6M of the denaturant after exciting the protein at 295 nm.

As seen in these figures, unfolding of LEF4 by GnHCl does

FIGURE 2 (A) Far-UV CD spectrum of LEF4. (B) Amino acid

sequence of LEF4. The Trp residues are marked in bold whereas Tyr

residues are underlined. (C) Fluorescence spectra of LEF4 by excit-

ing the protein at 280 nm (blue) and 295 nm (red).

FIGURE 3 GnHCl induced unfolding of LEF4. (A) Change in

intrinsic fluorescence intensity with increasing concentration of the

denaturant. Excitation wavelength was 295 nm. (B) Plot of wave-

length of maximum emission as a function of denaturant concen-

tration (0–6M) when excited at 295 nm. Error bars indicate SE.

576 Rasheedi et al.

Biopolymers

not follow two-state transition. The native protein has a kmax

at 347 nm. At low GnHCl concentrations (upto �1M),

the fluorescence emission intensity is decreased along with a

�3 nm red shift. In the concentration range 1.2–2M GnHCl,

the intensity increases while no change in kmax was observed.

On the other hand, a red shift of around 4 nm from 350.4

to 354.4 nm is seen in the GnHCl concentration range of

2–3.6M. It is consistent with the exposure of tryptophan

residues to the polar environment by protein unfolding.

These results clearly suggest that the gross conformational

changes in the protein leading to unfolding followed biphasic

transition.

Since LEF4 harbors 27 tyrosyl residues distributed

throughout the polypeptide chain, we investigated the tyrosyl

fluorescence to get additional insight into the structural fea-

tures of the protein. Figure 4 shows the GnHCl induced

unfolding of LEF4 as monitored by fluorescence intensity

306/350 ratio after exciting the protein at 280 nm. Like Trp

fluorescence, GnHCl induced unfolding monitored by Tyr

fluorescence was also a biphasic transition. An increase

in fluorescence intensity 306/350 ratio in the concentration

regions 0–1M and 2.8–6M GnHCl was noted which indicates

that Tyr residues have moved away from some of the Trp res-

idues and energy transfer from Tyr to Trp has decreased

pointing to unfolding of LEF4. Similar observations were

made in other proteins during chemical denaturation.10,11

Far-UV CD: The far-UV CD studies on LEF4 protein were

performed to assess the effect of the denaturant (GnHCl) on

the secondary structure of the protein. The spectra obtained

for the protein in the absence and presence of 0.2M, 0.5M,

1M, 2M, and 6M GnHCl concentrations are depicted in Fig-

ure 5. In the presence of 0.2M GnHCl, the mean residual el-

lipticity (MRE) was considerably decreased. At 0.5M concen-

tration of the denaturant, the MRE values at 208 and 217 nm

were decreased further. Interestingly, at 2M GnHCl concen-

tration, the far-UV CD spectrum showed increased ellipticity

values almost equivalent to the native protein (Table I). The

protein appears to acquire a native-like secondary structural

conformation. With further increments in the denaturant

concentration, the ellipticity at 217 nm was lost sharply (data

not shown). The curve obtained for the completely dena-

tured protein in 6M GnHCl is overlaid for comparison.

FIGURE 4 GnHCl denaturation profile of LEF4 as monitored by

fluorescence intensity (F.I.) 306/350 ratio by exciting the protein at

280 nm. Error bars indicate SE.

FIGURE 5 Far-UV CD spectra of LEF4 in presence of 0M (black),

0.2M (purple), 0.5M (pink), 1.0M (green), 2.0M (red), and 6.0M

(blue) GnHCl.

Table I Comparison of Different Structural Parameters of LEF4*

Parameters Native GnHCl (2M) GnHCl (6M) Urea (4M) Urea (8M)

Trp F.I. at 350 nm 200.556 0.25 163.916 3.00 170.906 5.30 206.25 6 5.27 210.756 1.01

Trp kmax (nm) 347.156 0.14 350.406 0.49 354.876 0.12 351.28 6 0.17 353.256 0.14

FI (306/350) 0.33 6 0.01 0.356 0.00 0.42 6 0.00 0.33 6 0.00 0.33 6 0.00

ANS F.I. at 480 nm 211.166 2.05 177.736 2.28 110.396 4.74 165.57 6 3.91 144.706 1.59

MRE (deg � cm2/dmol) at 225 nm 25616.1 25148.9 2232.4 25795.5 23611.4

KSV (/M) 3.84 5.95 ND** 5.57 ND**

* The data are expressed as mean6 standard deviation (n5 3).

** ND: Not determined.

Biophysical Characterization of LEF4 577

Biopolymers

ANS binding: Changes in ANS fluorescence are frequently

used to detect non-native intermediate conformations of

proteins.12 The fluorescence emission of ANS is known to

markedly increase when the dye binds to exposed hydropho-

bic clusters of proteins.13,14 The extent of ANS binding to

LEF4 at different GnHCl concentrations is shown in Figure 6.

As can be seen from this figure, ANS binding to the protein

is maximal at 0M GnHCl and significantly less at higher con-

centrations of GnHCl. These results indicate that the native

LEF4 contains significant amount of exposed hydrophobic

clusters, which are disrupted upon unfolding. A considerable

decrease in ANS binding was observed with increase in

GnHCl concentration from 0–0.4M GnHCl pointing to the

disruption of hydrophobic surface in some part of the pro-

tein due to unfolding. Steep increase in ANS fluorescence in-

tensity was observed above 1.2M GnHCl reaching a peak at

2M GnHCl. Enhanced ANS fluorescence at 2M GnHCl as

compared to the unfolded protein in the presence of 6M

GnHCl and native-like secondary structure content (Table I)

points to the existence of a molten globule-like intermediate

state at around 2M GnHCl concentration.15–18

Urea Induced Denaturation

Changes in the tertiary and secondary structure of LEF4 pro-

tein, as a consequence of denaturation by urea, were moni-

tored by fluorescence and far-UV CD, respectively.

Intrinsic fluorescence measurement: Figure 7A shows the

intrinsic fluorescence emission intensity at 350 nm plotted as

a function of urea concentration after exciting the protein at

295 nm. Slight changes in the intensity are observed through-

out the concentration range of urea (0–8M) scanned. How-

ever, when urea induced unfolding of LEF4 was examined by

the changes in the kmax of emission (Figure 7B), the unfold-

ing transition was found to occur in two steps. First transi-

tion, which is characterized by a red shift of �4 nm occurred

in the urea concentration range 1.5–3M while the second

transition, which is characterized by a red shift of �6 nm

compared to native occurred in the 5–8M urea.

Urea induced unfolding of LEF4 was also monitored by

fluorescence intensity 306/350 ratio after exciting the protein

at 280 nm (refer Figure 8). The unfolding curve was found to

be biphasic but was significantly different from the curve

monitored by wavelength of emission maximum. The differ-

ence in the curve may be due to very heterogeneous nature of

the Tyr environment.

Far-UV CD: The far-UV CD studies suggest that the sec-

ondary structural elements of LEF4 protein are quite resist-

ant to urea denaturation. As seen in Figure 9, the negative

ellipticities in the 217–225 nm range were decreased with

increasing urea concentration. This is due to unfolding of

FIGURE 6 GnHCl induced unfolding profile of LEF4 as moni-

tored by ANS fluorescence at 480 nm by exciting the complex at 380

nm. Error bars indicate SE.

FIGURE 7 Urea induced denaturation of LEF4 protein. (A)

Changes in intrinsic fluorescence intensity at 350 nm with increas-

ing concentration of the denaturant (0–8.0M) when excited at 295

nm. (B) Shift in kmax with increasing urea concentration (0–8.0M)

when excited at 295 nm. Error bars indicate SE.

578 Rasheedi et al.

Biopolymers

secondary structure of the protein. However, even in 8M

urea, most of the secondary structure was still retained. Sur-

prisingly, at 4M urea concentration, the far-UV CD spectrum

showed ellipticity values almost equivalent to the native pro-

tein (Table I). This pattern resembles the intermediate state

obtained at 2M GnHCl.

ANS binding: The urea induced unfolding transition was

monitored by ANS-protein complex fluorescence at 480 nm

after exciting the protein at 380 nm (refer Figure 10). The

urea induced unfolding transition of LEF4 monitored by ANS

fluorescence was also found to be cooperative and biphasic

with an apparent intermediate state around 3.5–4M urea.

Similar intermediate has also been observed in the unfolding

curve monitored by intrinsic fluorescence (Figure 7B).

Taken together, these results indicate that GnHCl and urea

induced unfolding of LEF4 is a two-step process with an accu-

mulation of intermediates at 2M GnHCl and 4M urea, respec-

tively. GnHCl and urea have been used as chemical denatur-

ants to study the stability profile of monomeric and multi-

meric proteins. GnHCl shows ionic and chaotropic effects

whereas urea has only chaotropic effect.19–22 The biophysical

characteristics of the native protein and in the presence of 2M

GnHCl and 4M urea have been summarized in Table I. Similar

values of Trp kmax and fluorescence intensity 306/350 ratio as

well as comparable ANS binding and secondary structure con-

tent suggest the existence of a thermodynamically stable inter-

mediate induced by 2M GnHCl and 4M urea.

To get information about the tryptophanyl micro-

environment and function of the intermediate states at 2M

GnHCl and 4M urea, acrylamide quenching and activity

measurements were carried out.

Acrylamide Quenching

Acrylamide quenching behavior was studied to gain an insight

into the relative surface exposure of tryptophan residues in the

protein as a function of denaturant concentration. Because of

its polar nature, acrylamide does not affect the fluorescence of

completely buried tryptophan residues. It is quite clear from

the Stern-Volmer plot (refer Figure 11) that LEF4 protein in

2M GnHCl is in a relatively expanded conformation (Ksv 5

5.95/M) as compared to the native protein (Ksv 5 3.84/M).

LEF4 in 4M urea (Ksv 5 5.57/M) also exhibits a similar degree

of compactness as that observed in 2M GnHCl, suggesting that

the two intermediate states share common structural features.

It is to be noted that for all the three plots in Figure 11 the

value of r2 (regression coefficient) is � 0.9, which is acceptable.

ATPase Assay

Having shown that LEF4 protein at 2M GnHCl and 4M urea

has native-like secondary structure and also displays loss of

FIGURE 8 Urea denaturation profile of LEF4 as monitored by

fluorescence intensity (F.I.) 306/350 ratio by exciting the protein at

280 nm. Error bars indicate SE.

FIGURE 9 Changes in far-UV CD spectra of LEF4 protein in

presence of 0M (black), 2M (red), 4M (pink), 5M (green), 6M

(blue), and 8M (brown) urea.

FIGURE 10 Urea induced unfolding profile of LEF4 as monitored

by ANS fluorescence at 480 nm by exciting the complex at 380 nm.

Error bars indicate SE.

Biophysical Characterization of LEF4 579

Biopolymers

tertiary structure with exposed hydrophobic clusters, the two

intermediate states were checked for their functional activity.

As mentioned earlier, the activity of LEF4 can be assayed by

monitoring its ability to hydrolyze nucleoside triphosphate

into nucleoside diphosphate and inorganic phosphate (Pi). It

was found that the protein at 2M GnHCl and 4M urea was

functionally inactive (refer Figure 12). This possibly is due to

the overall conformational changes in the tertiary structure

of the protein in 2M GnHCl and 4M urea, as evident from

the shift in kmax (Figures 3B and 7B). Similar observation

was made when the intermediates of homodimeric class lglutathione transferases in urea and GnHCl were found to be

catalytically inactive despite having native-like secondary

structure.23 This indicates that the enzyme is susceptible to

inactivation before complete unfolding of the protein in both

the cases. The denaturation pattern of soybean seed acid

phosphatase in presence of chaotropic agents also showed

similar trend.24 Hence, the intact tertiary structure is

required for the protein to be functionally active.

Temperature Induced Denaturation

The temperature-mediated denaturation of LEF4 was studied

by far-UV CD. We tested the unfolding properties of LEF4 as

a function of temperature by monitoring the changes in ellip-

ticity at 208 and 222 nm. Figure 13A shows the MRE changes

followed at 208 and 222 nm. These comparisons showed that

FIGURE 11 Stern Volmer plot of LEF4 native (l), in presence of

2M GnHCl (*) and 4M Urea (q).

FIGURE 12 Effect of GnHCl and urea on ATPase activity of

LEF4. Recombinant protein treated with 0M denaturant (lane 2),

2M GnHCl (lane 3), 6M GnHCl (lane 4), 4M urea (lane 5), and 8M

urea (lane 6) was used to measure the activity. Lane 1 is the control

with no protein.

FIGURE 13 Thermal unfolding of LEF4. (A) Plot of temperature-

induced changes in secondary structure of the protein in the range

from 30–908C as monitored by far-UV MRE values at 208 (p) and222 nm (O). Error bars indicate SE. (B) Far-UV CD spectra of LEF4

with increasing temperature: 308C (pink), 508C (blue), 708C (red),

858C (green), and 908C (black).

580 Rasheedi et al.

Biopolymers

the protein resists unfolding along a wide range of tempera-

ture. No significant change was observed even upto 858C sug-

gesting that LEF4 is a considerably thermostable protein. Visi-

ble aggregates of the protein were observed at temperature

above 858C with a sharp decrease in the MRE. This decrease

in MRE values may be a reflection of reduced protein concen-

tration due to visible precipitation at this temperature. The

corresponding far-UV CD spectra are shown in Figure 13B.

The spectrum at 858C is almost similar to the native protein

spectrum at 258C with the 208 nm ellipticity slightly decreased

indicating the thermostable nature of this protein.

CONCLUSIONThe results presented in this study have increased our knowl-

edge of the partially folded intermediate states of LEF4 protein

thereby pointing to the use of the viral coded LEF4 protein as

a model to study protein folding intermediates. LEF4 is appa-

rently a very stable protein as far as its conformation is con-

cerned. It resists total structural changes in the presence of

chemical denaturants like urea. It also resists temperature

induced unfolding. During the very late gene expression when

polyhedrin is over transcribed (representing over 50% of the

total cell protein) the polh transcription machinery necessarily

has to be very robust to bring about rapid rounds of transcrip-

tion within a very short time. The extreme stability of LEF4

will naturally be very useful in such situations. Several exam-

ples are known in biological system where a single protein per-

forms more than one unrelated functions in the cell. BV/ODV-

E26 is one such viral protein that is reported to be present in

different functional states in AcNPV infected cell and hence is

described as a multifunctional protein factor.25 The possibility

of LEF4 participating in tasks other than transcription, there-

fore, is highly conceivable. Given the fact that lef4 expression

overlaps with the time the virus prepares itself for occlusion, it

may possibly have a role in protecting the virus from any kind

of stress. The robust nature of this protein might reflect one of

the many tools adapted by the virus to survive under very

adverse environment. Therefore, it is likely that LEF4 is one of

the several protein factors that acquire additional functions to

assist the viruses for their survival in host free environment.

MATERIALS AND METHODS

MaterialsGnHCl was purchased from USB whereas urea was from Sigma

Aldrich. Very high purity acrylamide was obtained from USB. 8-

Anilino-1-naphthalenesulphonic acid (ANS) was purchased from

Sigma-Aldrich. All other reagents were of high quality grade.

Cloning of lef4 ORF in Expression VectorPolymerase chain reaction (PCR) was performed by adding 50 ng of

Bacmid (from Gibco BRL) as template to a 50 ll of total reactioncontaining 100 ng each of forward and reverse primers (FP:

cttGGATCCatggactacggcgattttgtg; RP: ttcCTCGAGttaatttggcac-

gattcggtc), 0.2 mM dNTPs (Amersham Pharmacia) and 0.1 U of Accu

DNA polymerase (Sigma Aldrich). PCR consisted of initial denatura-

tion step at 948C for 5 min followed by repeated steps (10 cycles) of

denaturation at 948C for 60 s, annealing at 508C for 60 s and poly-

merization at 688C for 3 min, followed by 25 cycles of denaturation

at 948C for 60 s, annealing at 608C for 60 s and polymerization at

688C for 3 min. Final extension was carried out at 688C for 7 min.

PCR amplified product was ligated in the pBSK1 vector (Stratagene)

linearized with SmaI. The lef4 gene was excised from pBSK1 using

BamHI and XhoI and subcloned under T7 promoter into pET28a

(Invitrogen) vector that was linearized with same restriction enzymes.

Expression and Purification of Recombinant LEF4

from E. colilef4 ORF was expressed under T7 promoter in pET28a using Bl21

(DE3) cells and 0.1 mM IPTG at 188C for 24 h. The induced culture

was then harvested and the cell pellet resuspended in extraction

buffer [20 mM Tris (pH 8.0) and 300 mM NaCl] and sonicated. The

lysed cells were centrifuged at 13000 rpm for 20 min. Recombinant

protein was found predominantly in the soluble fraction; hence the

supernatant was absorbed to the TALON affinity resin (Clontech)

pre-equilibrated with the same buffer. Washes were given with

10 mM imidazole in extraction buffer. The His-tagged protein was

eluted with 200 mM imidazole in the same buffer. Finally, the puri-

fied protein was dialyzed against 20 mM Tris (pH 8.0) and 100 mM

NaCl and used for fluorescence and CD studies. All the measure-

ments were recorded at 258C.

Fluorescence MeasurementsAll fluorescence measurements were carried out on a Cary Eclipse

Fluorescence Spectrophotometer. The concentration of LEF4 used

was in the range 0.46–1.84 mM. Cuvette pathlength of 1 cm was

used for fluorescence studies. For intrinsic tryptophan fluorescence,

the excitation wavelength was set at 280 and 295 nm and the emis-

sion spectra recorded in the range of 300–400 nm with 5 and 10 nm

slit widths for excitation and emission, respectively. Binding of ANS

to LEF4 at various denaturant concentrations was studied by excit-

ing the dye at 380 nm and the emission spectra were recorded from

400–600 nm with 10 nm slit width for excitation and emission. The

molar ratio of protein and ANS was 1:100. The error bars in the

plots represent standard error (SE) of mean for three to four inde-

pendent measurements.

CD MeasurementsCD measurements were carried out on a Jasco J-810 spectropo-

larimeter. MRE was calculated as shown by Hackeng et al.26 using

following equation:

MRE ¼ hobs=n 3 l 3 c 3 10

where yobs is observed ellipticity in millidegrees, n is number of

aminoacid residues, l is pathlength in centimeters, and c is molar

Biophysical Characterization of LEF4 581

Biopolymers

concentration. Each spectrum was recorded as an average of five

scans. The far-UV CD spectra were recorded at 200–250 nm wave-

length at a concentration of 3.70 mM in a 1 mm pathlength cuvette.

The values obtained were normalized by subtracting the baseline

recorded for the buffer having same concentration of denaturant

under similar conditions. The percent a-helix was obtained by the

method of Chen et al.27 according to the following equation:

% a-helix ¼ MRE222 � 2340

303003 100

GnHCl/Urea Induced DenaturationUnfolding of LEF4 in the presence of the denaturants was studied

by fluorescence and far-UV CD. Increasing amounts of the fresh

stock solutions (8M GnHCl or 10M Urea) were added to a fixed

concentration of protein in 20 mM Tris (pH 8.0) and 100 mM NaCl

and allowed to equilibrate for 6 h at 258C before recording the

spectra.

Acrylamide QuenchingQuenching of intrinsic tryptophan fluorescence was performed on a

Cary Eclipse Fluorescence Spectrophotometer using a stock solution

of 5M acrylamide. To a fixed amount (1.84 mM) of protein, increas-

ing amounts of acrylamide (0.1–1.0M) were added and the samples

were incubated for 30 min prior to taking the fluorescence measure-

ments. For the intrinsic tryptophan fluorescence spectra, the protein

samples were excited at 295 nm and emission recorded at 344 nm.

The data obtained were analyzed according to the Stern-Volmer

equation.28

Thermal StabilityChanges in the secondary structure of LEF4 with increase in temper-

ature were measured using CD. These measurements were carried

out using a thermostatically controlled cell holder, in a temperature

range of 25–908C with an increment of 5 6 0.108C. The sample was

first equilibrated for at least 15 min at a particular temperature

before an emission spectrum was collected.

ATPase AssayReaction was carried out at 308C for 15 min in a final volume of

5 ll containing 50 mM Tris-HCl (pH 7.8), 5 mM DTT, 1 mM

MnCl2, 50 mM KCl, [c232P]ATP (0.02 lCi), and purified recombi-

nant LEF4 (25 nM); 1 ll of the sample was spotted on poly-

ethyleneimine cellulose TLC plate and run in 0.75M potassium

phosphate buffer (pH 3.6). The intensity of the fastest moving spot

corresponding to inorganic phosphate (Pi) was analyzed using

‘‘Image Quant’’ software.

S.R. was recipient of Senior Research Fellowship from the Council

of Scientific and Industrial Research (CSIR), Government of India.

We are thankful to Basir Ahmad for his help in the data analysis. We

also acknowledge the help provided by Jisha Chalissery in perform-

ing the ATPase assay.

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Reviewing Editor: Alfred Wittinghofer

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