Half-of-the-sites reactivity of F235C {lambda}-repressor: implications for the structure of the whole repressor

Sumita Bandyopadhyay1, Sunanda Deb1, Sudeep Bose1 and Siddhartha Roy,1

Department of Biophysics, Bose Institute, P-1/12 C.I.T. Scheme VII M, Calcutta 700 054, India


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Note added in proof
 References
 
A site-directed mutation, F235C, was created at the penultimate residue of the {lambda}-repressor. Measurement of dimer–monomer dissociation constant suggested that dimer–monomer dissociation of the mutant repressor is similar to that of the wild-type. Affinity towards a single operator OR1 is also similar to that of the wild-type repressor. The mutant repressor gene in a multi-copy plasmid confers immunity towards infection by a cI- lambda phage, suggesting preservation of functional integrity. Far-UV circular dichroism spectra show no major change in the secondary structure. Fluorescence quenching experiments, however, suggest increased exposure of some tryptophan residues. The urea denaturation profile indicates decreased stability of a part of the C-terminal domain. Under non-denaturing conditions, cysteine-235 shows half-of-the-sites reactivity, i.e. on average only one out of two cysteine-235 residues in the dimer shows reactivity towards sulfhydryl reagents. Fluorescence energy transfer between randomly labeled donor and acceptor fluorescent probes indicates that only one sulfhydryl per dimer is reactive, suggesting true half-of-the-sites reactivity. The structural role of the C-terminal tail in the whole repressor dimer is discussed.

Keywords: asymmetry/fluorescence/lambda-repressor/sulfhydryl


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Note added in proof
 References
 
The {lambda}-repressor plays crucial and versatile roles in the regulation of life cycle of the bacteriophage {lambda}. It is a repressor of the promoter PR and activator of the promoter PRM (Ptashne, 1992Go). It cooperatively binds to several pairs of operator sites in multi-partite operators and recognizes them with different affinities (Senear et al., 1986Go). It is thus a model system for many aspects of protein–DNA and protein–protein interactions that forms the underlying basis of more complex regulatory systems of eukaryotes.

DNA-induced conformational changes in DNA binding proteins are well known. The {lambda}-repressor is one of the better characterized systems in that respect (Saha et al., 1992Go). We have previously shown that the repressor conformation is different when bound to different operator sites (Deb et al., 2000Go). Although biological functions of such conformational changes are not well understood, it may be important for specificity of multi-partite complex formation. Given the widespread occurrence of DNA-induced conformational change and multi-partite binding sites, it is of general importance to understand the nature of the DNA-induced conformational change.

The C-terminal tail of {lambda}-repressor is an interesting region although its role in the functions of the repressor alluded above is not well understood, but may be involved in transmission of DNA-induced conformational changes, among others. Several mutations in the tail region have been isolated that are known to cause impairment of monomer–monomer interaction (S228N) (Burz et al., 1994Go) and cooperativity (T234K) (Whipple et al., 1994Go), suggesting that the tail may be important in protein–protein interactions. The change of fluorescence properties of another residue in this locale, W230, on going from dimer to tetramer strongly suggests a role of this region in the dimer–dimer interaction in the unliganded state (Bandyopadhyay et al., 1995Go, 1996Go).

The crystal structure of the isolated C-terminal domain (although not the whole repressor) of the {lambda}-repressor is now known (Bell et al., 2000Go). Although core of the structure is consistent with all the solution data, some solution data on tail residues do not readily reconcile with the published structure. Owing to the flexible conformation of the tail region, it is possible that the crystal conformation is one among several possible conformations. The crystal and NMR structure of the homologous protein UmuD' has also led to a similar dispute (Peat et al., 1996Go; Ferentz et al., 1997Go). Considering the importance of the putative roles of the tail region on many aspects of the {lambda}-repressor function, we decided to introduce a fluorescence probe in the C-terminal region.

Previously, we have shown that none of the cysteine residues of {lambda}-repressor is reactive towards sulfhydryl reagents (Banik et al., 1992Go). Thus, the introduction of a cysteine in the C-terminal tail region by site-directed mutagenesis may create a unique fluorescence probe attachment point. We chose residue 235 for mutagenesis on the basis of several arguments. No mutant having any defective phenotype has ever been isolated in this residue. In addition, as fluorescence probes are aromatic in nature, an aromatic residue was chosen so that fluorescent-labeled mutant protein would be similar to the wild-type residue. This paper reports the creation of a site-directed mutant F235C and its use in the elucidation of the role of the C-terminal region of {lambda}-repressor.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Note added in proof
 References
 
Materials

6-[Fluorescein-(5 and 6)-carboxamido]hexanoic acid succinimidyl ester and fluorescein maleimide were purchased from Molecular Probes (Eugene, OR), IPTG, DTNB, proteinase K, chymotrypsin, trypsin, subtilisin and PMSF from Sigma Chemical (St. Louis, MO) and QAE-Sephadex from Pharmacia (Uppsala, Sweden). All other chemicals were of analytical grade.

Oligonucleotides

Oligonucleotides were synthesized with a 5'-amino link (X = hexylamino group) on an Applied Biosystems (ABI) Model 381A DNA synthesizer using chemicals purchased from ABI and purified by reversed-phase chromatography. For preparation of fluorescein-labeled oligonucleotides, the purified oligonucleotides (5'-X CTA TTT TAC CTC TGG CGG TGA TAA TGG TT-3' and its complement) were then labeled in 500 µl of solution containing 1 M sodium carbonate–bicarbonate buffer (pH 9)–DMF–water (5:2:3) containing 6-[fluorescein-(5 and 6)-carboxamido]hexanoic acid succinimidyl ester. Reaction was carried out for 20 h at room temperature. After incubation, the reaction mixture was loaded on to a Sephadex G25 column (pre-equilibrated in 0.1 M phosphate buffer, pH 8.0) and eluted with the same buffer. The oligonucleotides were annealed before use.

Construction of F235C mutant {lambda}-repressor gene

F235C mutation was created by a PCR-directed mutagenesis procedure. Two primers were used for the mutagenesis procedure: (1) internal primer overlapping with the internal HindIII site and (2) the mutagenic primer partly overlapping with the C-terminal tail region containing F235C mutation and partly overlapping the non-coding region. This primer contained additional mutations on the 5' side creating a HindIII site in the non-coding region. PCR with these primers, using the plasmid pEA305 (carrying wild-type {lambda}cI under tac promoter control) as template showed a unique band of the right size in the agarose gel. This band was purified and digested with HindIII. Plasmid pEA305 was digested with HindIII and the digested PCR band was ligated and transformed into DH5{alpha} strain and selected for ampicillin resistance. Twenty-three ampicillin-resistant colonies were selected and grown and analyzed for protein production after IPTG induction on SDS–PAGE. Nine out of the 23 colonies showed an intense band having mobility identical with that of the wild-type {lambda}-repressor. Plasmids were purified from five of these colonies and all of them appeared to have the right size as judged from mobility on agarose gel. When sequenced by the dideoxy method, all five plasmids showed the presence of F235C mutation and no other additional mutations. This plasmid will be referred to as pSR235.

Isolation and purification of F235C {lambda}-repressor

DH5{alpha}IqF' bearing F235C {lambda}-repressor gene under tac promoter control (pSR235) was grown and induced like that of the wild-type {lambda}-repressor. The protein purification was similar to that for the wild-type and was described in detail previously (Deb et al., 1998Go). The cell lysate supernatant was mixed with 50 ml of SB [10 mM Tris–HCl, pH 8, containing 1 mM calcium chloride, 0.1 mM EDTA, 0.1 mM 2-mercaptoethanol, 5% glycerol (v/v)] containing 50 mM KCl and 50 ml of pre-swollen QAE Sephadex A-50 in SB containing 50 mM KCl. The mixture was stirred for 1 h at 4°C, then poured into a column and allowed to settle. The column was washed with 50 ml of SB containing 50 mM KCl. The protein was eluted with a linear gradient of 100 ml of SB buffer containing 50 mM KCl and 100 ml of SB buffer containing 600 mM KCl. The hydroxyapatite column was run as described previously (Saha et al., 1992Go). SDS–PAGE at this stage indicated a single band of 26 kDa. The stored protein was dialyzed against 0.1 M potassium phosphate buffer, pH 8.0, before use. Repressor concentration is always expressed in terms of monomer unless stated otherwise.

Chemical modification and anisotropy measurement

Acrylodan labeling was carried out as described previously (Bandyopadhyay et al., 1996Go). The protein concentration of the modified protein was determined by A280 measurement with appropriate subtraction for incorporated acrylodan. Fluorescein maleimide labeling was carried out at a protein concentration of 10 µM in 0.1 M potassium phosphate buffer, pH 8.0, for 30 min at room temperature. The protein was then dialyzed exhaustively and the incorporation ratio measured. The incorporation ratio was calculated using an extinction coefficient of 83 000 M-1 cm-1 at 490 nm. FM-labeled F235C repressor was then serially diluted with the same buffer, the same buffer containing 0.5 µM BSA and the same buffer containing 0.5 µM wild-type {lambda}-repressor and the anisotropy was measured as described previously (Bandyopadhyay et al., 1996Go). For measurement of OR1 binding by repressor, 2 nM fluorescein-labeled OR1 oligonucleotide was titrated with increasing concentrations of wild-type or F235C {lambda}-repressor. The solution conditions were 0.1 M potassium phosphate buffer, pH 8.0, containing 0.1 M NaCl. The temperature was 25°C.

Fluorescence methods

All fluorescence spectra were measured with a Hitachi F 3010 spectrofluorimeter having a spectra addition and subtraction facility. The excitation and emission bandpasses were 5 nm, unless mentioned otherwise. Fluorescence quenching studies were performed as described previously (Bandyopadhyay et al., 1995Go). The excitation wavelength was 295 nm and the emission was measured at 340 nm. Urea denaturation was carried out in 0.1 M phosphate buffer, pH 8.0, containing an appropriate amount of urea. F235C repressor was incubated in urea-containing buffer overnight at 25°C and fluorescence spectra were recorded at 25°C. The excitation wavelength was 295 nm and the fluorescence intensity at 340 and 350 nm and the position of the emission maximum were noted.

For energy transfer measurements, fluorescein maleimide-labeled F235C repressor was prepared as described above. A similar protocol was used to prepare coumarin maleimide and mixed (CM/FM)-labeled F235C repressor. The excitation spectra for all three were determined at identical probe concentrations and the CM–repressor spectrum was subtracted from the mixed labeled spectra to nullify the intensity due to direct excitation of donor. Calculation of R0 and energy transfer efficiency can be found elsewhere (Banik et al., 1992Go, 1993Go).

Immunity test

The composition of the EMBO agar plates was as follows: 0.5% NaCl, 1% Bacto-tryptone, 0.2% potassium dihydrogenphosphate, 0.2% MgCl2, 1.5% Bacto-agar, ampicillin, 0.04% eosin and 0.0065% methylene blue. Two EMBO agar plates were seeded with phage {lambda}cI- ({lambda}c47) and 1 mM IPTG (final concentration). Escherichia coli cells (RR1) alone, bearing pSR235, pEA305 (Amann et al., 1983Go), plasmid containing S228N {lambda}-repressor (pS228N; monomer–dimer defective) gene under tac control and ts2 {lambda}-repressor [(Jana et al., 1999Go); temperature sensitive mutant] gene were spotted on an LA/AMP plate and two of the above-mentioned EMBO agar plates. The LA/AMP plate and one EMBO agar plate were grown at 32°C and the other EMBO agar plate at 42°C.

Circular dichroism

Circular dichroism spectra were measured with a JASCO J-600 spectropolarimeter using a 1 mm pathlength cuvette for far-UV and a 1 cm pathlength cuvette for near-UV measurements. F235C (1 µM) and wild-type {lambda}-repressor in 0.1 M potassium phosphate, pH 8.0, were used for spectral measurements. A scan speed of 100 nm/min was used. The temperature was ambient (25 ± 1°C). The measurements were performed in 0.1 M potassium phosphate buffer, pH 8.0, at ambient temperature (25 ± 1°C).

DTNB reaction

F235C repressor was exhaustively dialyzed with two changes against 0.1 M potassium phosphate buffer, pH 8.0. The protein at a given concentration was taken in the sample cuvette and the same amount of buffer against which the protein was dialyzed was taken in the reference cuvette of a double-beam Shimadzu UV-160 spectrophotometer. DTNB was added to both the cuvettes at a final concentration of 0.1 mM and the reaction was monitored for 1 h. For protease digestion experiments, proteinase K, chymotrypsin, trypsin and subtilisin were added to a 5 µM repressor solution (mutant or wild-type) at a protein:protease ratio of 50:1 (each protease) and incubated at 25°C for 96 h. The sulfhydryl reactivity was then measured as described above. The same amount of buffer against which the protein was dialyzed was incubated with same mixture of proteases and was used in the reference cell.

Stopped-flow fast kinetic measurements

The kinetics of DTNB reaction were measured using an SX18.MV kinetic spectrometer from Applied Photophysics (UK) by monitoring the absorbance at 412 nm. The dead-time of the instrument was 2 ms. The two solutions were mixed at a ratio of 10:1 (protein:DTNB). The final concentrations were 1.6 and 100 µM for protein and DTNB, respectively. The temperature was 25 ± 1°C. The absorbance increase as a function of time was fitted to a first-order rate equation using Sigma Plot to obtain the pseudo-first-order rate constant.

Curve fitting

The data fitting was done using Sigma Plot (Jandel Scientific) or Kyplot (Koichi Yoshioka, 1997–99, version 2.0 beta 4). The following equations were used to fit the data:

For DNA binding with coupled monomer–dimer equilibrium of the protein:

where Aobs is the observed anisotropy, A{infty} is the anisotropy at infinite protein concentration, A0 is the anisotropy at zero protein concentration, [Ptot] is the total concentration of repressor in terms of monomer, K is the association constant of the repressor and the operator and K' is the monomer–dimer association constant.

For monomer–dimer equilibrium of the protein:

where Aobs is the observed anisotropy, Am is the anisotropy of the monomer, Ad is the anisotropy of the dimer, Kd is the dissociation constant and [Ptot] is the total monomer concentration.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Note added in proof
 References
 
Functional characterization of F235C {lambda}-repressor

Biological activity of {lambda}-repressor is measured by its DNA-binding properties: binding to single operator sites and multiple operator sites. We measured the binding of F235C repressor to a single operator OR1 by fluorescence anisotropy. Figure 1Go shows the titration of fluorescein end-labeled OR1 oligonucleotide with wild-type and F235C {lambda}-repressor. The experiments were conducted at higher ionic strengths (1/2{Sigma}ciZi2) to reduce the binding affinity, since the DNA concentration has to be kept around or below Kd [in 0.1 M potassium phosphate buffer (dianionic at this pH), pH 8.0, containing 0.1 M NaCl]. Both the profiles show saturation and can be fitted to a single site binding equation. The extracted binding constants are (3.4 ± 0.3)x108 and (3.9 ± 0.18)x108 M-1 for the wild-type and F235C repressor, respectively. As a control, we measured the binding of a DNA-binding defective repressor (K4C) under the same conditions. This repressor shows an anisotropy increase at much higher concentrations (about two orders of magnitude), indicating the effectiveness of the assay (data not shown). We also measured the binding of wild-type, F235C and a non-cooperative mutant Y210C to a double operator fragment containing OR2 and OR3, using the method of fluorescence anisotropy as stated above. Although detailed analysis of cooperativity is not possible owing to the unavailability of individual site-loading information, an approximate and qualitative idea of cooperative interaction may be obtained from the average binding affinity of wild-type (full cooperativity) and the Y210C (no cooperativity) repressors for such a template. Based on such an analysis, we conclude that F235C may be moderately cooperativity impaired, falling somewhere between the two extremes (data not shown). This is consistent with the experiments reported in Table IGo.



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Fig. 1. Fluorescein end-labeled OR1 titration of {lambda}-repressor, wild-type (open circles) and F235C mutant (closed circles). The solid lines represent the best fit to the coupled monomer–dimer and operator binding as described in the Materials and methods section. The solution conditions were 0.1 M potassium phosphate buffer, pH 8.0, containing 0.1 M NaCl. The temperature was 25°C. The DNA concentration was 2 nM.

 

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Table I. Growth of E.coli carrying various plasmids in the absence and presence of {lambda}cI- phage under various conditions
 
The functional integrity of the F235C repressor is of particular concern since it has been reported that T234K mutant repressor has a dimerization defect and non-cooperative phenotype (Whipple et al., 1994Go). The functional integrity of the F235C repressor was tested by whether pSR235 can confer immunity to {lambda}cI- phage infection to E.coli cells bearing the plasmid under inducing conditions. The results in Table IGo show that cells bearing plasmids carrying F235C in addition to wild-type repressor genes (induced with IPTG) are resistant to the {lambda}cI- phage, suggesting that the F235C repressor is functionally active. In the control experiments, it was shown that cells carrying plasmids that encode a monomer–dimer defective {lambda}-repressor (S228N) were unable to confer resistance. We also observed that a temperature-sensitive mutant gene, ts2, was able to confer resistance only at the permissive temperature. The ts2 mutant shows weakly defective operator binding properties ({sigma}3-fold) at the permissive temperatures, but strongly defective binding properties (>10-fold) at non-permissive temperatures (Jana et al., 1999Go). It has been shown by Ackers and co-workers (Burz and Ackers, 1994Go, 1996Go) that S228N saturates the wild-type OR1–OR2 at low nanomolar concentrations, in vitro. Even in such a case, multi-copy S228N bearing plasmid is unable to resist cI- phage infection under the present conditions. This may suggest that resistance of F235C to cI- phage infection is unlikely to be due solely to excess production of a defective repressor from multi-copy plasmids and is consistent with only a weak cooperativity defect.

Another aspect of {lambda}-repressor function is the monomer–dimer equilibrium. It is known that the {lambda}-repressor defective in monomer–dimer association is incapable of proper binding to operators. From various measurements, it has been estimated that the dimer dissociation constant of the wild-type repressor is around 10 nM (Koblan and Ackers, 1991Go). It is known (Burz and Ackers, 1996Go) that S228N mutation weakens monomer–dimer association very significantly, thus underlining the role of the C-terminal tail region in the monomer–monomer interaction. In order to estimate the dimer dissociation constant, the F235C repressor was labeled with fluorescein maleimide. The anisotropy of the fluorescein-labeled F235C repressor is shown in Figure 2Go as a function of repressor concentration. The anisotropy decreases upon dilution with buffer alone (data not shown) and buffer containing 0.5 µM BSA. It is clear that the repressor undergoes dissociation in the concentration range 5–100 nM. Under the same conditions, if the fluoresceinated F235C repressor is diluted with unlabeled wild-type repressor without any change in the total repressor concentration, the fluorescence anisotropy remains virtually unchanged. When the data are fitted to a dimer–monomer dissociation equation, a value of 17 nM was obtained for the dissociation constant. This value is very close to that obtained by others, therefore suggesting that the F235C mutation has no significant effect on monomer–dimer equilibrium (Koblans and Ackers, 1991). This is in agreement with the fact that F235C shows no defect in single operator binding.



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Fig. 2. Fluorescence anisotropy of FM-labeled F235C {lambda}-repressor as a function of labeled repressor concentration. The solid circles represents when the FM labeled repressor was diluted with buffer containing 0.5 µM BSA and the open circles represent when the FM-labeled repressor was diluted with buffer containing 0.5 µM wild-type repressor. The solid line is the best-fit line to a monomer–dimer equation. The experiments were carried out in 0.1 M potassium phosphate buffer, pH 8.0, at 25°C. The excitation wavelength was 490 nm and emission wavelength 515 nm.

 
Structural characterization of F235C {lambda} -repressor

The structural integrity of the mutant repressor was assessed by spectroscopic measurements. Figure 3Go shows the far-UV circular dichroism spectra of the mutant and the wild-type repressor. The far-UV circular dichroism spectra of the two repressors are almost identical, suggesting no major change in the secondary structures of the mutant protein.



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Fig. 3. Far-UV circular dichroism spectra of wild-type (solid line) and F235C {lambda}-repressor (dash-dotted line). The spectra were obtained in a 1 mm pathlength cuvette in 0.1 M potassium phosphate buffer, pH 8.0, at ambient temperature. The protein concentrations were 1 µM. The scan speed was 100 nm/min.

 
Tryptophan fluorescence is a sensitive monitor of the structural integrity of a protein. In previous studies we extensively used the fluorescence characteristics of the three tryptophan residues of {lambda}-repressor, which are situated in the C-terminal domain and the hinge region, to study structural perturbations (Bandyopadhyay et al., 1995Go). The composite emission maximum of the tryptophans of the wild-type {lambda}-repressor is 340 nm (Bandyopadhyay et al., 1995Go). In contrast, the emission maximum of F235C {lambda}-repressor is around 346 nm, suggesting a significant change in the tryptophan environment resulting in greater solvent exposure. Figure 4Go shows the Lehrer plot (Lehrer, 1971Go) of the quenching of tryptophan fluorescence of {lambda}-repressor by acrylamide. In contrast to the plots of the wild-type {lambda}-repressor, both the plots at protein concentrations of 0.5 µM (largely dimeric for the wild-type repressor) and 10 µM (largely tetrameric for the wild-type repressor) pass through 1, indicating that unlike the wild-type repressor there is no significant non-quenchable component present in F235C repressor. The Stern–Volmer constants, KSV, calculated from the plot, are 14 and 4.5 M-1 for 0.5 and 10 µM, respectively. At 0.5 µM the value of KSV is significantly higher than the value for the wild-type repressor, suggesting increased exposure of some tryptophan residues. At 10 µM the value of KSV is significantly larger than that of the major portion of the tryptophan fluorescence for the wild-type, suggesting increased exposure of some tryptophan residues. It is noteworthy, however, that compared with the dimer the KSV value is significantly reduced in the tetramer, suggesting that like that of the wild-type repressor, some tryptophan residues shift to a more inaccessible environment upon tetramer formation.



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Fig. 4. Lehrer plot of acrylamide quenching of tryptophan fluorescence of F235C {lambda}-repressor at 0.5 (solid triangles) and 10 µM (solid circles) concentration. The excitation wavelength was 295 nm and the emission was monitored at 340 nm. The quenching was carried out in 0.1 M potassium phosphate buffer, pH 8.0, at 25°C. The excitation and emission bandpasses were 5 nm each.

 
This is strongly supported by the emission maximum shift in the dimeric and tetrameric concentrations as shown in Figure 5Go. The emission maximum of the tryptophans of F235C repressor shifts weakly to the red upon addition of acrylamide at 0.5 µM, whereas the emission maximum shifts to almost 340 nm upon addition of acrylamide, at 10 µM repressor concentration. This suggests that like that of the wild-type repressor, there are more blue-shifted tryptophans in the tetrameric F235C repressor than in the dimeric form (Bandyopadhyay et al., 1995Go). We therefore conclude that like that of the wild-type repressor, F235C repressor undergoes tetramer formation in this concentration range (perhaps weaker than the wild-type) with a shift of one or more tryptophans to the apolar environment. It is likely that this tryptophan is W230 (Bandyopadhyay et al., 1995Go). This is consistent with the weaker than normal cooperative interactions deduced above.



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Fig. 5. Shift of wavelength maximum of tryptophan fluorescence of F235C {lambda}-repressor at 0.5 µM (solid triangles) and 10 µM (solid circles) upon addition of increasing concentrations of acrylamide. The excitation wavelength was 295 nm and the emission wavelength was 340 nm. The excitation bandpass was 10 nm and the emission bandpass was 1.5 nm. The quenching was carried out in 0.1 M potassium phosphate buffer, pH 8.0, at 25°C.

 
Since the introduction of the mutation has some effect on the tryptophan fluorescence, we investigated its effect on the stability of the C-terminal domain. Figure 6Go shows the urea denaturation profile of the F235C {lambda}-repressor [for the denaturation profile of wild-type {lambda}-repressor, see Figure 7Go in a previous paper (Deb et al., 1998Go)]. We have shown that the F340/F350 ratio can be used as a sensitive parameter for the denaturation (Banik et al., 1992Go). In the wild-type {lambda}-repressor, this ratio starts at {sigma}1.07 and levels off at {sigma}0.87 in two distinct phases of approximately equal amplitudes, centered around 3 and 6.5 M urea. The intermediate has an F340/F350 ratio of {sigma}0.98. The corresponding emission maxima shift from 340 to {sigma}355 nm with an in-between plateau around 346 nm (Banik et al., 1992Go). We will call this intermediate intermediate I. In the native F235C repressor, the F340/F350 ratio starts at {sigma}0.98 and the emission maximum starts at {sigma}345 nm (data not shown), suggesting a conformation of the intermediate I type. The denaturation profile shows no significant transition around 3 M that can be detected by tryptophan fluorescence, supporting the view that the mutant repressor may already be in an intermediate state. The second transition in high urea concentration, in which the F340/F350 ratio shifts from {sigma}0.98 to {sigma}0.88, is more likely to be a partial unfolding of the C-terminal domain because the lack of total sulfhydryl reactivity under these conditions suggests that a core of the C-terminal domain survives. This transition in the F235C repressor is significantly shifted to the lower urea concentration and is centered around 4.5 M urea. This suggests that the stability of a part of the C-terminal domain is significantly affected by the mutation F235C.



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Fig. 6. Urea denaturation profile of F235C {lambda}-repressor. 1 µM F235C {lambda}-repressor was incubated in a given concentration of urea overnight at room temperature. The fluorescence intensity was then measured at 340 and 350 nm, with excitation at 295 nm. The bandpasses were 5 nm each. The solution conditions were 0.1 M potassium phosphate buffer, pH 8.0, at 25°C.

 


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Fig. 7. Kinetics of reaction of DTNB with F235C {lambda}-repressor. The kinetics of DTNB reaction were measured in an SX18.MV kinetic spectrometer (Applied Photophysics) by monitoring the absorbance at 412 nm. The vertical axis represents the absorbance change. The final concentrations were 1.6 and 100 µM for protein and DTNB, respectively. The temperature was 25 ± 1°C. The solid line represents the best-fit line to a first-order rate equation.

 
The identical nature of the circular dichroism spectra of the wild-type and the F235C repressor under native conditions suggests that the transition to the intermediate I does not involve a change of secondary structure. Our previous urea denaturation study also suggests that no significant CD change accompanies transition of native wild-type {lambda}-repressor to intermediate I (Banik et al., 1992Go). The nature of the interactions that are disrupted by the F235C mutation remains unclear but may involve the interaction of two domains along with the hinge region. Such interactions have been inferred from DSC studies (Merabet et al., 1998Go). Hence it is possible that the C-terminal tail region plays a role in the formation of these putative domain–domain interactions.

Sulfhydryl reactivity of F235C {lambda}-repressor

Table IIGo shows the reactivity of cysteine residues of F235C {lambda}-repressor towards DTNB. DTNB when mixed with F235C repressor, in the concentration range 1–15 µM, reacts with {sigma}0.5 sulfhydryl groups per repressor subunit or one cysteine residue per repressor dimer. This reaction is fast and is mostly over within the mixing time. Under identical conditions, wild-type {lambda}-repressor has been shown to be totally unreactive (Banik et al., 1992Go).


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Table II. Sulfhydryl reactivities of wild-type and mutant {lambda}-repressor under different conditions
 
The rate of reaction of this reactive cysteine with DTNB was examined by the stopped-flow method. Figure 7Go shows the absorbance increase at 412 nm as a function of time. The calculated pseudo-first-order rate constant is 0.2268 ± 0.006 s-1 and the second-order rate constant is (2.17 ± 0.06)x103 M-1 s-1. As a control, reaction of free cysteine with DTNB was measured under identical conditions. The calculated second-order rate constant is 8.44x103 M-1 s-1, only 4-fold higher than C235, thus suggesting a highly exposed nature of the reactive sulfhydryl group (data not shown).

Establishment of the phenomenon of 0.5 reaction stoichiometry in the F235C {lambda}-repressor requires careful standardization of the reaction. To demonstrate that the reaction is indeed measuring the number of cysteine residues correctly, we exhaustively digested the wild-type and F235C {lambda}-repressor with a combination of proteases and measured the total number of cysteine residues. The total number of cysteine residues determined in this way was {sigma}3 and {sigma}4 for the wild-type and the F235C repressor, respectively (the exact difference between the two is 0.92). This agrees with the expected number of cysteine residues, suggesting that the DTNB titration done in this way measures the number of cysteine residues quantitatively (Banik et al., 1992Go). To determine whether other sulfhydryl reactive reagents show similar stoichiometry, we incubated the sulfhydryl-specific reagents acrylodan and fluorescein maleimide with the F235C repressor. As shown in Table IIGo, both the reagents also react with the {lambda}-repressor with a stoichiometry of {sigma}0.5. The fluorescein maleimide-labeled F235C repressor was exhaustively digested with trypsin and the tryptic peptides were analyzed as described previously (Bandyopadyay et al., 1995). One predominantly fluorescein-labeled peptide peak (>80% fluorescence) is seen in the HPLC elution profile, suggesting that one sulfhydryl group becomes labeled under this condition.

A reaction stoichiometry of 0.5 per subunit may not necessarily imply half-of-the-sites reactivity. One possibility is that there are other unreactive conformers/variants of the protein that may simply constitute about half the amount of protein. One major source of unreactive sulfhydryls is oxidation. However, reduction of the protein with DTT and dialysis under anaerobic conditions with heavy metal chelators to prevent re-oxidation yielded similar results (Table IIGo). To detect other possible variants, we labeled the F235C repressor with a mixture of coumarin maleimide and fluorescein maleimide. Coumarin and fluorescein are an excellent donor–acceptor pair for fluorescence resonance energy transfer. An excess of coumarin maleimide over fluorescein maleimide was used for labeling, which produced excess coumarin label over fluorescein. If the reaction stoichiometry indeed originates from a single reactive sulfhydryl residue on the dimer, reacted fluorescein would not have a donor coumarin partner on the same molecule. In the other case (conformational variants), random labeling would lead to the presence of a coumarin–fluorescein pair on the same dimer. A similar strategy has been used to detect dimer–monomer dissociation in other proteins. In the crystal structure, the two 235 residues are separated by a distance of 15 Å. Even if the tail is allowed a free rotation, the distance is unlikely to exceed 45–50 Å (Bell et al., 2000Go). This is also the approximate distance between the two furthest points on the crystal structure. Figure 8Go shows the excitation spectra of the mixed labeled protein and that of fluorescein maleimide-labeled F235C repressor. It is clear that there is only a small difference between the two. Since the R0 for the coumarin–fluorescein pair is 42.5 Å, an energy transfer efficiency of {sigma}50% would have been anticipated. The upper limit of energy transfer was estimated to be {sigma}3.5%. This small degree of energy transfer may occur from weak association of the labeled dimers. Hence it is likely that the probes are on different dimer molecules, indicating that the reaction stoichiometry may have originated from half-of-the-sites reactivity of C235. Although the above discussion implied the labeled repressors to be dimeric, a quaternary structure of a tetramer may be envisaged. However, a tetramer would have at least two reactive sulfhydryl residues in the same molecule and may give rise to donor–acceptor pairs having distances where significant energy transfer can take place. This would be contrary to the observed very low, near-zero, energy transfer efficiency.



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Fig. 8. Measurement of energy transfer efficiency between coumarin and fluorescein in a mixed-labeled F235C {lambda}-repressor. Excitation spectra of coumarin and fluorescein-labeled F235C (solid line) and fluorescein-labeled F235C repressor (dashed line). The excitation spectrum of an equal concentration of CM labeled repressor was subtracted from the mixed labeled spectrum to nullify the fluorescence intensity due to direct excitation of coumarin. The emission wavelength was fixed at 550 nm. The solution conditions were 0.1 M potassium phosphate buffer, pH 8.0, and 25°C. Bandpasses were 5 nm each.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Note added in proof
 References
 
The half-of-the-sites reactivity of C235 is an intriguing feature. There are three possible explanations for this strong half-of-the-sites reactivity in F235C {lambda}-repressor, pre-existing asymmetry in the dimer (Figure 9A and BGo), negative cooperativity in the dimer (Figure 9CGo) and pre-existing asymmetry in the tetramer. From a structural point of view, the last explanation is most easily reconciled with what is known about {lambda}-repressor structure. In that model, both of the 235 sulfhydryls would be exposed in the {lambda}-repressor dimer and one C-terminal tail region from each dimer would interact to form the tetramer, thus burying one pair of C235 in the dimer–dimer interface. It has indeed been shown previously that the C-terminal tail region of the repressor is involved in the formation of tetramer in the absence of DNA (Bandyopadhyay et al., 1996Go). However, for a number of other important reasons, we do not favor this explanation. We have shown that wild-type {lambda}-repressor in phosphate buffer shows a dimer–tetramer transition in this concentration range (Banik et al., 1993Go; Bandyopadhyay et al., 1996Go). The tetramer dissociation constant of wild-type repressor is {sigma}2 µM (Bandyopadhyay et al., 1996Go). Although we have not directly measured the tetramer–dimer dissociation, data from a number of experiments suggest that this association is only modestly weaker in F235C than in the wild-type. The anisotropy versus repressor concentration curve at submicromolar concentrations can be fitted well to a monomer–dimer dissociation equation suggesting that at low micromolar concentrations the repressor is still a dimer (Figure 2Go). However, when the acrylodan-labeled repressor is titrated with unlabeled repressor, there is a significant shift of the emission maximum at repressor concentrations of {sigma}10 µM or higher. This indicates tetramerization of the repressor at around the concentration range where the wild-type repressor forms a tetramer (Bandyopadhyay et al., 1996Go). At two different protein concentrations, the emission maximum shift due to acrylamide quenching is different and the difference is qualitatively similar to that of the wild-type repressor, except the initial red shift (Figure 5Go). Clearly, different emission maximum shift patterns at two different concentrations of F235C repressor is suggestive of change in its quaternary structure and taken with the data mentioned above is strongly indicative of a similar dimer-tetramer equilibrium property to that of the wild-type repressor.



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Fig. 9. Cartoon diagram explaining probable origins of half-of-the-site reactivity in {lambda}-repressor. The medium-sized gray circle is the sulfhydryl reagent, the black dot is the sulfhydryl group, the light line depicts the flexible tail and the ball and stick figure is the rest of the repressor. (A) Side-by-side association in which one of the sulfhydryl groups is buried in the interface. (B) Equilibrium between two conformers of the subunit; in one conformation the sulfhydryl group is buried. The association between two conformers (hetero-association) is significantly tighter than that between each of the conformers (homo-association), resulting in predominance of asymmetric structure. (C) Symmetric structure but reaction of sulfhydryl group of one of the subunits results in burial of the other.

 
The number of cysteine residues reacted, however, is independent of protein concentration in this concentration range. Most importantly, as shown in Table IIGo, the F235C repressor shows half-of-the-sites reactivity at 37°C at 1 and 10 µM protein concentrations. We have shown previously that at these concentrations, at this temperature, virtually no tetramer or higher order structure is present in the wild-type repressor and this should also be the case with F235C repressor (Banik et al., 1993Go; Burz and Ackers, 1996Go). This suggests that half-of-the-site reactivity originates in the dimer structure. Thus, half-of-the-sites reactivity shown by C235 may be a result of extreme negative cooperativity or pre-existing asymmetry. Spatial proximity of the tail regions of two different subunits may give rise to alteration of conformation of one tail, upon reaction of C235 in the other. The pre-existing asymmetry could be due to side-by-side non-symmetric association of the subunits where one sulfhydryl is buried in interface [e.g. in tubulin, although the subunits are homologous but not identical (Nogales et al., 1995Go)] or association of two subunits in different conformations [e.g. in arrestin (Schubert et al., 1999Go)] in which the reactive sulfhydryl is buried in one of the conformations. Either of the above-mentioned possibilities suggests more complex conformational character of the tail region than visualized in the crystal structure of the isolated C-terminal domain.

In the recent crystal structure of the C-terminal domain of {lambda}-repressor, two tails are seen to be interacting in an anti-parallel fashion. The important question is whether the tail region retains the same conformation in the whole repressor dimer. Some results obtained in solution appear to indicate otherwise. It has been demonstrated here that the F235C mutant has a very similar dimer–monomer dissociation constant to that of the wild-type protein. Unchanged single operator binding by the mutant repressor is also consistent with unchanged dimer–monomer equilibrium [due to coupling of dimer–monomer equilibrium with DNA binding (Weiss et al., 1987Go)]. In the crystal structure of the C-terminal domain, the F235 is buried and interacts with a number of residues in the other subunit (Val225, Phe179, Phe160). Upon substitution with cysteine, residue 235 may become exposed and reactive owing to disruption of the interactions with F235. However, in such a case it is expected that F235C mutation should significantly affect the dimer–monomer dissociation constant if the same arrangement is preserved in the whole repressor, contrary to the results presented here.

Based on acrylamide quenching and chemical modification, we have previously suggested that W230 is highly exposed in the dimer (Bandyopadhyay et al., 1995Go). In the above-mentioned crystal structure, however, W230 is almost totally buried. These results suggest that in the whole repressor dimer, the tails are in different positions (maybe interacting with the other domains). In the absence of the other domains they adopt an alternative conformation seen in the crystal structure. The fact that acrylodan-labeled C235 repressor shows a very significant fluorescence change upon operator binding is also indicative of such a situation (Bandyopadhyay et al., 1996Go). Recently, a homology-modeled structure of {lambda}-repressor dimer has been deposited in the PDB. In this model, tail residues are in different conformations from that in the crystal structure (the rest of the C-terminal domain structure is very similar) and interacts with residues in the hinge region (PDB reference 1gfx). Without implying that this is the actual conformation, we emphasize that alternative conformations of the C-terminal tail region are energetically possible.

The complex nature of the tertiary structure of the whole repressor dimer is also reflected in the complexity of the denaturation profile. It is clear from the denaturation results presented that replacement of F235 with a C results in disruption of some tertiary interactions, possibly a domain–domain interaction. This loss of interaction also affects the stability of part of the C-terminal domain as detected by the shift of emission maxima upon urea denaturation. A similar loss of stability is seen upon removal of the N-terminal domain and the hinge region (Banik et al., 1992Go). This is indicative of interaction of the tail region with a part of the N-terminal domain/hinge and exerting an influence on the stability of part of the C-terminal domain, directly or indirectly. This interaction is unlikely to be inter-subunit in nature as no change in the dimer–monomer dissociation constant or DNA binding is observed.

Does conformational complexity of the tail region point towards asymmetry in the dimer? Certainly there is precedence. Most DNA-binding proteins in prokaryotes are symmetrical dimers that bind to sequences often having imperfect inverted repeats. In lac and gal repressors, it is known that perfectly symmetrical operators have higher binding affinity than the natural ones, which are pseudo-symmetrical. This is indicative of a yet to be understood role of the imperfect symmetry in the natural context. Recently, it has been demonstrated that CRP binds to a site near the galP1 promoter, resulting in asymmetric bending (Pyles and Lee, 1998Go). It is possible that such an asymmetric bending is important for promoter activation. In bacteriophage {lambda}, all six operator sites have imperfect symmetry. The role of such imperfect symmetry in the functioning of {lambda}-repressor is not known. In contrast to the lac and gal situation, it has been noted (Sarai and Takeda, 1989Go) that {lambda}-repressor binds to a more symmetric operator with a weaker affinity. This may be a result of pre-existing asymmetry in the wild-type repressor structure or functional communication between two subunits, where the tail regions play an important role.


    Note added in proof
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Note added in proof
 References
 
A recent determination of the crystal structure of LexA, a homolog of {lambda}-repressor, suggests different conformations of the two subunits within the dimer (Luo et al., 2001Go). This is supportive of a pre-existing asymmetry model described above.


    Notes
 
1 To whom correspondence should be addressed. E-mail: sidroy{at}boseinst.ernet.in Back


    Acknowledgments
 
We acknowledge the CSIR, Government of India, for supporting the research. We also acknowledge a CSIR fellowship to Ms Sunanda Deb. We thank Professor N.C.Mandal for very useful discussions and Dr Sankar Adhya for support.


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 Introduction
 Materials and methods
 Results
 Discussion
 Note added in proof
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Received May 25, 2001; revised December 10, 2001; accepted January 29, 2002.





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