Biophysical Characterization of the TraY Protein of Escherichia coli F Factor*

Joel F. SchildbachDagger , Clifford R. Robinson§, and Robert T. Sauer

From the Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The TraY protein is required for efficient bacterial conjugation by Escherichia coli F factor. TraY has two functional roles: participating in the "relaxosome," a protein-DNA complex that nicks one strand of the F factor plasmid, and up-regulating transcription from the traYI promoter. The traY gene was cloned, and the TraY protein was expressed, purified, and characterized. TraY has a mixed alpha -helix and beta -sheet secondary structure as judged by its circular dichroism spectrum, is monomeric, and undergoes reversible urea denaturation with Delta Gu = 6 kcal/mol at 25 °C. The kinetics of protein unfolding and refolding, as measured by changes in fluorescence, are complex, suggesting the presence of intermediates or of heterogeneity in the folding reaction. TraY has been classified as a member of the ribbon-helix-helix family of transcription factors but is unusual in appearing to have tandem repeats of the beta alpha alpha motif in the same polypeptide chain. The data presented here show that folding and assembly of the functional (beta alpha alpha )2 unit occurs as an intramolecular reaction and not by cross-folding between different polypeptide chains.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Bacterial conjugation is a process by which conjugative plasmid DNA is transferred in single-stranded form from a host to a recipient bacterium (for review, see Refs. 1 and 2). For the Escherichia coli K-12 Sex Factor F (F factor or F plasmid), most of the gene products required for conjugation are encoded within the tra or transfer region, a 33-kilobase pair segment of the plasmid (1). An integral step in F factor conjugation is relaxosome formation. The relaxosome is a complex composed of three different DNA-binding proteins that assemble at a specific F plasmid site. The complex nicks one plasmid DNA strand in preparation for plasmid unwinding and transfer of the nicked strand to the recipient. The name relaxosome is derived from the ability of the complex to relax the supercoiled F factor DNA via nicking (3, 4). Two tra gene products (TraY and TraI) and integration host factor form the relaxosome (5, 6). All three of these proteins are required for efficient nicking of the plasmid DNA, yet only TraI possesses an endonucleolytic activity (5-9). The roles of TraY and integration host factor in relaxosome formation are unclear, although both bind to DNA in a sequence-specific manner and are known to bend or distort the DNA (10-12). In addition to its role in relaxosome formation, TraY binds a sequence near its own promoter, up-regulating transcription (13-15). In this paper, we describe the biophysical characterization of TraY as a first step in determining the structural basis for the dual activities of TraY.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cloning of traY Gene-- The traY gene was amplified by PCR1 using genomic DNA from E. coli Hfr strain CAG5051 (16). The oligonucleotides (5'-GCCGAGGTGCATATGAAAAGATTTGGTACACGTTCT-3', complementary to the antisense strand, and 5'-CGCGAATTCCTAGAGTGTATTAAATGTTATATC-3', complementary to the sense strand) contained NdeI and EcoRI sites, respectively, to facilitate cloning. Oligonucleotides were synthesized on an Applied Biosystems 380C DNA synthesizer. The PCR reaction volume (100 µl) contained 500 nM of each primer, 200 µM dNTPs, and 5 units of Vent DNA polymerase (New England Biolabs). The PCR reaction included an initial denaturation for 4 min at 94 °C; three cycles of denaturation for 1 min at 94 °C, annealing for 45 s at 45 °C, and extension for 45 s at 72 °C; and then 27 cycles of denaturation at 94 °C for 60 s, annealing at 60 °C for 30 s, and extension at 72 °C for 45 s. The resulting product was digested with NdeI and EcoRI and ligated to the NdeI-EcoRI backbone fragment of pAED4 (17), an expression vector utilizing a T7 promoter. E. coli strain X90 (18) was transformed with the ligation product, ampicillin-resistant transformants were selected, and plasmid DNA was purified from individual candidates. The identity of the cloned gene and the fidelity of PCR were confirmed by DNA sequencing of the plasmid insert by the dideoxy termination method using T7-promoter- and T7-terminator-specific primers. Sequencing was performed using Sequenase Version 2.0 (U. S. Biochemical Corp.) or by automated sequencing by the Biopolymers Laboratory at the Massachusetts Institute of Technology.

TraY Protein Expression and Purification-- E. coli strain BL21(DE3) (19) was transformed with the pAED4-traY plasmid, and transformants were tested for TraY expression upon induction with isopropyl-1-thio-beta -D-galactopyranoside. Single colonies were picked and inoculated into 5 ml of LB broth containing 100 µg/ml ampicillin (LB-amp). Cultures were grown at 37 °C to an A600 of approximately 0.5, 1 ml was transferred to another tube, and protein expression was induced by the addition of 100 µg/ml isopropyl-1-thio-beta -D-galactopyranoside. After 2 h of additional growth, cell cultures were mixed 1:1 with loading buffer and loaded onto 10% polyacrylamide Tris-Tricine gels (20). Induction was deemed successful by the presence of a band at the appropriate molecular weight in the induced sample. Frequently, transformants showed no TraY production upon induction, or protein expression was lost over time. Once a transformant that stably expressed TraY was identified, it was used to inoculate a 500-ml culture of LB-amp, and the culture was grown to an A600 of 0.5. The cells were then chilled for 10 min on ice, pelleted by centrifugation (3500 × g, 10 min, 4 °C), resuspended in 10 ml of LB-amp with 15% glycerol, and frozen in 0.5-ml aliquots. For protein purification, 500 ml of LB-amp was inoculated with 50 µl of the frozen cells, and the culture was grown to an A600 of 0.4. Protein expression was induced by the addition of isopropyl-1-thio-beta -D-galactopyranoside to a concentration of 100 µg/ml. Cells were grown for 3 h post-induction and harvested by centrifugation (3500 × g, 30 min, 4 °C), and the pellets were frozen at -80 °C prior to purification. Cell pellets were thawed at 4 °C, resuspended in buffer A (20 mM sodium phosphate (pH 7.4), 1 mM EDTA, 5 mM dithiothreitol) plus 100 mM NaCl, and phenylmethylsulfonyl fluoride was added to a final concentration of 50 µM. Cells were lysed by sonication, and the resulting solution was centrifuged at 4 °C for 10 min at 10,000 × g. MgCl2 and DNaseI (Boehringer Mannheim) were added to the supernatant to final concentrations of 8 mM and 10 µg/ml, respectively, the solution was incubated on ice for 2 h, and insoluble material was removed by centrifugation for 10 min at 10,000 × g. The supernatant was applied to a 5-ml HiTrap heparin column (Pharmacia Biotech Inc.) equilibrated in buffer A plus 100 mM NaCl. A gradient from 100 to 1,000 mM NaCl in buffer A was applied to the column using a fast protein liquid chromatography system (Pharmacia), and the elution was monitored by A280. A peak that included TraY eluted at approximately 500 mM NaCl. This peak was applied directly to a 5-ml HiTrap Blue column (Pharmacia) equilibrated in buffer A plus 500 mM NaCl. A gradient from 500 mM to 3 M NaCl in buffer A was run over the column, with TraY eluting at approximately 2 M NaCl. The TraY peak was dialyzed extensively against buffer A plus 100 mM NaCl and concentrated to approximately 2 mg/ml using Centriprep-3 concentrators (Amicon). At this point, TraY protein was greater than 95% pure as assessed by Coomassie staining of SDS-polyacrylamide gels. The protein concentration was estimated by absorbance at 280 nm using an extinction coefficient, calculated from the sequence, of 11,460 M-1 cm-1 (21). The yield of purified TraY protein was approximately 2 mg/liter of cell culture. Amino acid analysis and 10 cycles of N-terminal sequencing of this protein prep were performed by the Biopolymers Laboratory at Massachusetts Institute of Technology. The mass was determined by electrospray ionization mass spectrometry on a Finnigan TSQ7000 triple quadrupole mass spectrometer at the Harvard Microchemical facility at Harvard University.

Spectroscopy-- Fluorescence data were collected using a Hitachi F-4500 fluorescence spectrophotometer. CD spectra were obtained using an AVIV 60DS spectrapolarimeter. UV absorbance spectra were collected using a Hewlett Packard 8452A diode array spectrophotometer. The buffer used for spectroscopy was 20 mM sodium phosphate (pH 7.4), 100 mM NaCl, 1 mM EDTA, and 5 mM beta -mercaptoethanol.

Equilibrium Unfolding and Refolding-- For urea denaturation experiments, equimolar solutions of TraY were made using 20 mM sodium phosphate (pH 7.4), 100 mM NaCl, 1 mM EDTA, 5 mM beta -mercaptoethanol, and either 0 or 9.5 M urea. For experiments using intrinsic fluorescence, the starting solutions (2 µM TraY) were mixed in ratios to produce solutions ranging from 0 to 9.5 M urea at intervals of 0.475 M. Solutions were allowed to equilibrate at ambient temperature for 30 min, transferred to a cuvette thermostated at 25 °C, and allowed to equilibrate until no change in fluorescence was observed. The fluorescence emission at 345 nm (excitation 280 nm) was averaged for 30 s, and the emission spectrum from 300 to 420 nm was also recorded. For experiments using circular dichroism, the starting solutions (5 µM TraY) with either 0 or 9.5 M urea were placed in a cuvette thermostated at 25 °C, and the urea concentration was changed incrementally by removing an aliquot from the cell and replacing it with an equal volume of the other solution. Solutions were allowed to equilibrate until no signal change was observed, and then the ellipticity at 234 nm was averaged for 30 s and recorded. Ellipticity at 234 nm rather than 222 nm was used because of the greater signal-to-noise ratio in high urea concentrations. Urea denaturation was reversible, and the data fit well to a two-state transition between native (N) and denatured (D) monomers (Ku = [D]/[N]). The urea dependence of Delta Gu was modeled as a linear function of urea concentration Delta Gu = Delta GuH20 - m × [urea], where Delta GuH20 is the Gibbs free energy of unfolding in the absence of urea. Data were fit using the nonlinear curve-fitting function of Kaleidagraph 3.0 (Synergy Software). Thermal denaturation of the TraY protein was found to be irreversible.

Kinetics of Unfolding and Refolding-- Kinetic measurements of TraY unfolding and refolding reactions were performed using an Applied Photosystems SpectraKinetic stopped-flow fluorescence instrument equipped with a 300-nm cutoff filter. Measurements were made at 25 °C in buffer containing 20 mM sodium phosphate (pH 7.4), 100 mM NaCl, 1 mM EDTA, 5 mM beta -mercaptoethanol, and different concentrations of urea. For unfolding experiments, a solution of TraY denatured in buffer plus 8 M urea was mixed with solutions containing 0-2.4 M urea in a 1:5 ratio to give final urea concentrations of 1.3-3.3 M. In refolding experiments, a solution of TraY in 4 M urea was mixed in a 1:5 ratio with solutions containing 7.6-10 M urea to give final urea concentrations of 7-9 M. Final TraY concentrations ranged from 1 to 12 µM. At the starting and ending concentrations, the TraY population was greater than 95% native or denatured. For jumps into the refolding/unfolding transition zone, solutions of 6 µM TraY in buffer plus 4 M urea (native) or buffer plus 8 M urea (denatured) were mixed with solutions of higher or lower urea to give final concentrations of 5-6 M. Kinetic data for refolding and unfolding required the use of double exponential functions to achieve good fits. Data points within the mixing dead time of the instrument (~1 ms) were not used in the fitting procedures.

Analytical Ultracentrifugation-- Experiments were performed using a Beckman Optima XL-A centrifuge. TraY samples of 1 or 12 µM in 20 mM sodium phosphate (pH 7.4), 100 mM NaCl, 1 mM EDTA, 5 mM dithiothreitol were centrifuged overnight at 18,000 and 27,500 rpm, respectively. Absorbance readings were performed at 1 h intervals to determine that the samples had reached equilibrium. Absorbance profiles were obtained at 222 nm (1 µM TraY) or 280 nm (12 µM TraY), with absorbances ranging from 0.1 to 0.7. Ten scans were averaged and analyzed according to Laue et al. (22) to determine apparent molecular weights. A partial specific volume of 0.7407 was calculated according to Cohn and Edsall (23) for use in the analysis.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

TraY Purification-- The gene encoding F factor TraY was amplified by PCR, cloned into an expression vector, expressed in E. coli strain BL21(DE3), and purified (see "Materials and Methods"). As shown in Table I, the N-terminal sequence, amino acid composition, and subunit molecular weight determined by electrospray ionization mass spectrometry of the purified protein are in excellent agreement with values predicted from the gene sequence. The purified protein was active, as judged by its ability to bind specifically and strongly (Kd ~ 1 nM) to an oligonucleotide corresponding to the recognition sequence at oriT (13) in a gel electrophoretic mobility shift assay (not shown). As shown in Fig. 1, analytical ultracentrifugation data for TraY fit well to a monomer model but fit extremely poorly to a dimer model. These results indicate that the solution form of TraY is predominantly monomeric.

                              
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Table I
Composition and molecular weight of purified TraY


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Fig. 1.   Analytical centrifugation profile of TraY at 12 µM, 18,000 rpm, 25 °C. Circles represent experimental data points collected at 280 nm. The solid line represents the expected profile for monomeric TraY with a molecular mass of 15,183 Da. The dashed line represents the expected profile for a dimeric form of TraY with a molecular mass of 30,366 Da. Assuming a partial specific volume of 0.7407, the experimental data shown are best fit to an apparent molecular mass of 14,208 Da. Abs, absorbance; r2, radius squared.

Spectroscopy-- The CD spectrum of native TraY is shown in Fig. 2. The strong negative ellipticity at 208, 215, and 222 nm suggests that the protein is composed of a mixture of alpha -helices and beta -sheets. The fluorescence emission spectra of native TraY has a maximum near 345 nm as expected from a Trp residue that is largely solvent-exposed (Fig. 3). TraY contains one Trp and four Tyr residues, but the latter side chains do not contribute to fluorescence in the 320-360 range. TraY denatured in 9.5 M urea shows a significant decrease in fluorescence intensity (Fig. 3). The minor peak at 310 nm in the spectrum of denatured TraY is due to Raman scattering of water, based on the position of the peak and shifts in this position in spectra arising from different excitation wavelengths.


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Fig. 2.   Circular dichroism spectrum of native TraY. The spectrum was taken at 25 °C using a 0.1-cm cuvette containing 10 µM TraY in 20 mM sodium phosphate (pH 7.4), 1 mM EDTA, 5 mM beta -mercaptoethanol, and 100 mM NaCl. mdeg, millidegrees.


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Fig. 3.   Fluorescence spectra of native and denatured TraY. Spectra were taken at 25 °C using 2 µM TraY in 20 mM sodium phosphate (pH 7.4), 1 mM EDTA, 5 mM beta -mercaptoethanol, and 100 mM NaCl. The denatured sample also contained 9.5 M urea. Excitation was at 280 nm.

Equilibrium Unfolding and Refolding-- Denaturation of TraY by urea was monitored by changes in circular dichroism ellipticity at 234 nm or fluorescence emission at 345 nm (Fig. 4). From 0 to 4 M urea, the protein is largely native, and at urea concentrations above 6.5 M, the protein is fully denatured. Table II lists values for the free energy of denaturation in the absence of urea (Delta GuH20) and the change in free energy with urea (m) obtained from non-linear least squares fits of individual denaturation experiments. These values are similar for experiments monitored by CD and by fluorescence (Fig. 4, Table II), which were performed at different TraY concentrations (5 and 2 µM, respectively). This concurrence is expected if TraY denaturation is an N right-arrow D reaction without significantly populated intermediate states. A global fit of all the experimental denaturation data gave a Delta GuH20 value of 6.0 (± 0.1) kcal/mol and an m value of 1.16 (± 0.02) kcal/mol·M.


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Fig. 4.   Equilibrium urea denaturation of TraY. The unfolding of TraY was monitored by change in circular dichroism ellipticity at 234 nm (CD) or in fluorescence intensity (Fl) at 345 nm arising from excitation at 280 nm. The resulting data were normalized to fraction of unfolded protein and plotted. The solid line indicates the best fit to four combined equilibrium denaturation experiments, with Delta GuH20 = 6.0 (±0.1) kcal/mol, m = 1.16 (±0.02) kcal/mol·M].

                              
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Table II
Stability parameters for equilibrium urea denaturation of TraY
Denaturation experiments were performed at 25 °C in 20 mM sodium phosphate (pH 7.4), 100 mM NaCl, 1 mM EDTA, 5 mM beta -mercaptoethanol. Unfolding was followed by changes in circular dichroism ellipticity at 234 nm (CD) or fluorescence emission at 345 nm following excitation at 280 nm (Fl). A global fit to all data yielded Delta GuH20 = 6.0 (±0.1) kcal/mol, m = 1.16 (±0.02) kcal/mol·M.

Unfolding and Refolding Rates-- Fig. 5, A and B, show unfolding and refolding trajectories for TraY following jumps from 4 to 7 M urea and 8 to 3.3 M urea, respectively (final TraY concentration = 1 µM, 25 °C (pH 7.4), 100 mM NaCl, photomultiplier tube voltage = 700 V). No change in unfolding or refolding kinetics was observed over a TraY concentration range from 1 to 12 µM. Both reactions occur over a time scale of approximately 10 s under the conditions used and require double exponential functions for an adequate fit (note residuals in Fig. 5, A and B). The kinetics of refolding were similar in pH jump experiments, indicating that the multiphasic kinetics are not unique to urea denaturation. The presence of multiple kinetic phases is consistent with the existence of intermediates in the reactions or conformational heterogenity in the native and/or denatured states. Chemical heterogeneity of TraY was not observed by electrospray mass spectrometry and thus is unlikely to account for the complex kinetic behavior.


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Fig. 5.  

Kinetics of unfolding and refolding of TraY. A, unfolding trajectory for 1 µM TraY following a jump from 4 to 7 M urea. The solid line is the fit to a double exponential with rate 1 of 0.239 s-1, rate 2 of 0.111 s-1, and relative amplitudes of 66 and 34% associated with rate 1 and rate 2, respectively. B, folding trajectory for 1 µM TraY following a jump from 8 to 3.3 M urea. The solid line is the fit to a double exponential with rate 1 of 0.55 s-1, rate 2 of 0.0505 s-1, and relative amplitudes of 66 and 34% associated with rate 1 and rate 2, respectively. The lower panels show the residuals of the fit to single and double exponential functions. Experiments were performed at 25 °C in buffer containing 20 mM sodium phosphate (pH 7.4), 1 mM EDTA, 5 mM beta -mercaptoethanol, and 100 mM NaCl.

The urea dependence of the rate constants obtained from double exponential fits of the unfolding and refolding data are shown in Fig. 6. Although the rate constants obtained from the fits were very reproducible, the ratio of the amplitudes of the fast and slow kinetic phases varied from a ratio of 2:1 to 1:2 for different urea concentrations or experiments. Extrapolating the unfolding data to 0 M urea gives rate constants of 0.017 and 0.0054 s-1 for the fast and slow phases. For the refolding experiments, the extrapolated rate constants are 4.9 s-1 for the fast phase and 0.23 s-1 for the slower phase. Pairing the fastest refolding rate constant with the slowest unfolding rate constant predicts a Delta Gu value of 4 kcal/mol, which is lower than the value obtained in the equilibrium experiments. All other combinations of refolding and unfolding rate constants predict even lower free energies of unfolding and larger discrepancies with the equilibrium Delta Gu value. The discrepancy between the kinetic and equilibrium values suggests that one or more rate constants for a faster refolding step or a slower unfolding step are not detected in the kinetic experiments. No detectable "burst" phase occurred in the refolding experiments (the observed fluorescence intensity at the earliest time was that expected for denatured TraY). It is possible, however, that fluorescence is only sensitive to late events in refolding and thus, that a faster refolding phase is present but undetectable.


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Fig. 6.   Urea dependence of unfolding and refolding rates of TraY. Depicted are the refolding rate 1 (filled circles), refolding rate 2 (filled squares), unfolding rate 1 (open circles), and unfolding rate 2 (open squares). Lines are fits to those points outside the transition (4-6.5 M urea). Extrapolation to 0 M urea gives refolding rate 1 = 4.9 s-1 (r = 0.964), refolding rate 2 = 0.23 s-1 (r = 0.991), unfolding rate 1 = 0.017 s-1 (r = 0.973), and unfolding rate 2 = 0.0054 s-1 (r = 0.958). Error bars indicate standard deviation from the average of two or three experiments.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

TraY has previously been cloned, expressed, and purified (13, 24), and some of its functional characteristics, including DNA binding, have been studied (5, 6, 12-15, 24, 25). The structural and biophysical properties of TraY, however, have been largely unstudied.

Circular dichroism and intrinsic fluorescence were used to probe native TraY and its denaturation by urea. The circular dichroism spectrum of native TraY suggests a mixed alpha -helix/beta -sheet secondary structure. In equilibrium experiments, TraY denatures cooperatively in an apparent two-state, N right-arrow D fashion with a Cm of 5.3 M urea and a Delta GuH20 value of 6 kcal/mol. The similarity in denaturation curves monitored by CD or fluorescence indicates that changes in the environment of the single tryptophan of TraY (Trp91) and changes in TraY secondary structure occur concurrently. However, the kinetics of TraY unfolding and refolding are not consistent with simple two-state folding behavior, and it seems likely that early folding events may not be detected by changes in fluorescence.

Based on a shared pattern of conserved residues, TraY has been assigned to the ribbon-helix-helix family of transcription factors (26), so named because family members share a common structural motif consisting of a beta -ribbon or strand followed by two alpha -helices (27). This family includes the Arc and Mnt repressors from bacteriophage P22 of Salmonella typhimurium and the MetJ repressor of E. coli. Although structural data are not yet available for TraY, its CD spectrum is consistent with a mixture of alpha -helix and beta -sheet, as expected for a ribbon-helix-helix family member. In the structures of MetJ, Arc, and Mnt (28-33), ribbon-helix-helix motifs from identical subunits interact, allowing the beta -strands to form an antiparallel beta -sheet that packs against the alpha -helices to form a dimeric, roughly globular, DNA-binding domain. By contrast, F factor TraY contains tandem but nonidentical ribbon-helix-helix motifs in a single polypeptide chain. This architecture could, in principle, allow monomeric folding, if the tandem TraY ribbon-helix-helix motifs interact in the same polypeptide chain, or multimeric folding, if motifs from different chains interact. Based on gel-filtration studies, Nelson et al. (13) concluded that F factor TraY is monomeric in solution. Our results confirm this finding. TraY behaves as a monomer in sedimentation equilibrium experiments, and equilibrium and kinetic denaturation and refolding are concentration-independent, as expected if folding is an intramolecular process.

MetJ, Arc, Mnt, and both ribbon-helix-helix motifs of TraY have an invariant Glu residue in the second alpha -helix (Fig. 7). In Arc, this Glu is part of an Arg-Glu-Arg salt-bridge triad. In MetJ and Mnt, the invariant Glu forms a single salt bridge. The N-terminal ribbon-helix-helix motif of TraY could form an Arg35-Glu40-Arg44 salt-bridge triad identical to that found in Arc repressor. In the TraY C-terminal ribbon-helix-helix motif, this triad of residues is Trp91-Glu96-Arg100. If the overall relationship of these three residues is similar to the homologous residues in Arc, then the Nepsilon 1 group of Trp91 could donate a hydrogen bond to the Glu96 carboxylate. In this case, Trp91 would expected to be in a hydrophilic environment in native TraY, both by virtue of its proximity to Glu96 and because it would be partially solvent-exposed. This, in turn, would account for the observed fluorescence emission spectrum of TraY, which suggests that Trp91 is in a substantially hydrophilic environment.


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Fig. 7.   Amino acid alignment of Mnt, MetJ, and Arc with the F factor TraY N-terminal (TraY-N) and C-terminal (TraY-C) halves. The sequences shown form the two alpha -helices of the ribbon-helix-helix fold. Alignments are taken from Breg et al. (29) and Inamoto et al. (34). Hydrogen bonding interactions between the invariant Glu and other residues are indicated schematically.

    ACKNOWLEDGEMENTS

We are indebted to Ernest Frankel, Dennis Rentzeparis, and Wali Karzai for helpful discussions and to Professors Peter Kim and Jonathan King for use of equipment.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant AI-15706 (to R. T. S.), an American Cancer Society post-doctoral fellowship (to J. F. S.), and a National Research Service Award, National Institutes of Health, post-doctoral fellowship (to C. R. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Biology, 144 Mudd Hall, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218. Tel.: 410-516-0176; Fax: 410-516-5213; E-mail: joel{at}jhu.edu.

§ Present address: 3-Dimensional Pharmaceuticals, Inc., Exton, PA 19341.

1 The abbreviation used is: PCR, polymerase chain reaction.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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