From the Department of Biology, Massachusetts Institute of
Technology, Cambridge, Massachusetts 02139
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INTRODUCTION |
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.
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MATERIALS AND METHODS |
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-
-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-
-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-
-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
-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
-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
Gu was modeled as a linear
function of urea concentration
Gu =
GuH20
m × [urea], where
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
-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.
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RESULTS |
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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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.
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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
-helices and
-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
-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 -mercaptoethanol, and 100 mM NaCl. The
denatured sample also contained 9.5 M urea. Excitation was at 280 nm.
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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
(
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
D reaction without significantly populated intermediate
states. A global fit of all the experimental denaturation data gave a
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
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 -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
GuH20 = 6.0 (±0.1)
kcal/mol, m = 1.16 (±0.02)
kcal/mol·M.
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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 -mercaptoethanol, and 100 mM NaCl.
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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
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
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.
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DISCUSSION |
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
-helix/
-sheet secondary structure.
In equilibrium experiments, TraY denatures cooperatively in an apparent
two-state, N
D fashion with a
Cm of 5.3 M urea and a
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
-ribbon or strand followed by two
-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
-helix and
-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
-strands to form an antiparallel
-sheet that packs against the
-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
-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 N
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
-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.
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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.