Department of Biophysics, Bose Institute, P-1/12 C.I.T. Scheme VII M, Calcutta 700 054, India
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Abstract |
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Keywords: asymmetry/fluorescence/lambda-repressor/sulfhydryl
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Introduction |
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DNA-induced conformational changes in DNA binding proteins are well known. The -repressor is one of the better characterized systems in that respect (Saha et al., 1992
). We have previously shown that the repressor conformation is different when bound to different operator sites (Deb et al., 2000
). 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 -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 monomermonomer interaction (S228N) (Burz et al., 1994
) and cooperativity (T234K) (Whipple et al., 1994
), suggesting that the tail may be important in proteinprotein 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 dimerdimer interaction in the unliganded state (Bandyopadhyay et al., 1995
, 1996
).
The crystal structure of the isolated C-terminal domain (although not the whole repressor) of the -repressor is now known (Bell et al., 2000
). 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., 1996
; Ferentz et al., 1997
). Considering the importance of the putative roles of the tail region on many aspects of the
-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 -repressor is reactive towards sulfhydryl reagents (Banik et al., 1992
). 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
-repressor.
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Materials and methods |
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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 carbonatebicarbonate buffer (pH 9)DMFwater (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 -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 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
strain and selected for ampicillin resistance. Twenty-three ampicillin-resistant colonies were selected and grown and analyzed for protein production after IPTG induction on SDSPAGE. Nine out of the 23 colonies showed an intense band having mobility identical with that of the wild-type
-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 -repressor
DH5IqF' bearing F235C
-repressor gene under tac promoter control (pSR235) was grown and induced like that of the wild-type
-repressor. The protein purification was similar to that for the wild-type and was described in detail previously (Deb et al., 1998
). The cell lysate supernatant was mixed with 50 ml of SB [10 mM TrisHCl, 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., 1992
). SDSPAGE 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., 1996). 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
-repressor and the anisotropy was measured as described previously (Bandyopadhyay et al., 1996
). For measurement of OR1 binding by repressor, 2 nM fluorescein-labeled OR1 oligonucleotide was titrated with increasing concentrations of wild-type or F235C
-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., 1995). 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 CMrepressor 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., 1992, 1993
).
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 cI- (
c47) and 1 mM IPTG (final concentration). Escherichia coli cells (RR1) alone, bearing pSR235, pEA305 (Amann et al., 1983
), plasmid containing S228N
-repressor (pS228N; monomerdimer defective) gene under tac control and ts2
-repressor [(Jana et al., 1999
); 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 -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, 199799, version 2.0 beta 4). The following equations were used to fit the data:
For DNA binding with coupled monomerdimer equilibrium of the protein:
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For monomerdimer equilibrium of the protein:
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Results |
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Biological activity of -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 1
shows the titration of fluorescein end-labeled OR1 oligonucleotide with wild-type and F235C
-repressor. The experiments were conducted at higher ionic strengths (1/2
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 I
.
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Another aspect of -repressor function is the monomerdimer equilibrium. It is known that the
-repressor defective in monomerdimer 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, 1991
). It is known (Burz and Ackers, 1996
) that S228N mutation weakens monomerdimer association very significantly, thus underlining the role of the C-terminal tail region in the monomermonomer 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 2
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 5100 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 dimermonomer 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 monomerdimer 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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The structural integrity of the mutant repressor was assessed by spectroscopic measurements. Figure 3 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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Sulfhydryl reactivity of F235C -repressor
Table II shows the reactivity of cysteine residues of F235C
-repressor towards DTNB. DTNB when mixed with F235C repressor, in the concentration range 115 µM, reacts with
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
-repressor has been shown to be totally unreactive (Banik et al., 1992
).
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Establishment of the phenomenon of 0.5 reaction stoichiometry in the F235C -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
-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
3 and
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., 1992
). 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 II
, both the reagents also react with the
-repressor with a stoichiometry of
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 II). 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 donoracceptor 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 coumarinfluorescein pair on the same dimer. A similar strategy has been used to detect dimermonomer 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 4550 Å (Bell et al., 2000
). This is also the approximate distance between the two furthest points on the crystal structure. Figure 8
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 coumarinfluorescein pair is 42.5 Å, an energy transfer efficiency of
50% would have been anticipated. The upper limit of energy transfer was estimated to be
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 donoracceptor 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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Discussion |
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In the recent crystal structure of the C-terminal domain of -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 dimermonomer dissociation constant to that of the wild-type protein. Unchanged single operator binding by the mutant repressor is also consistent with unchanged dimermonomer equilibrium [due to coupling of dimermonomer equilibrium with DNA binding (Weiss et al., 1987
)]. 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 dimermonomer 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., 1995). 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., 1996
). Recently, a homology-modeled structure of
-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 domaindomain 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., 1992). 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 dimermonomer 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, 1998). It is possible that such an asymmetric bending is important for promoter activation. In bacteriophage
, all six operator sites have imperfect symmetry. The role of such imperfect symmetry in the functioning of
-repressor is not known. In contrast to the lac and gal situation, it has been noted (Sarai and Takeda, 1989
) that
-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.
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Note added in proof |
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Notes |
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Acknowledgments |
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References |
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Received May 25, 2001; revised December 10, 2001; accepted January 29, 2002.