NH2-terminal Sequence Truncation Decreases the
Stability of Bovine Rhodanese, Minimally Perturbs Its Crystal
Structure, and Enhances Interaction with GroEL under Native
Conditions*
Richard J.
Trevino
,
Francesca
Gliubich§¶,
Rodolfo
Berni
,
Michele
Cianci§,
John M.
Chirgwin
**,
Giuseppe
Zanotti§, and
Paul M.
Horowitz

From the Departments of
Biochemistry and
** Medicine-Endocrinology, the University of Texas Health Science
Center, San Antonio, Texas 78284, the § Department of
Organic Chemistry, University of Padova and Biopolymer Research Center,
Consiglio Nazionale delle Ricerche, 35131 Padova, Italy, the
¶ Institute of Pharmaceutical Chemistry, University of Milano,
20135 Milano, Italy, and the
Institute of Biochemical Sciences,
University of Parma, 43100 Parma, Italy
 |
ABSTRACT |
The NH2-terminal
sequence of rhodanese influences many of its properties, ranging from
mitochondrial import to folding. Rhodanese truncated by >9 residues is
degraded in Escherichia coli. Mutant enzymes with lesser
truncations are recoverable and active, but they show altered active
site reactivities (Trevino, R. J., Tsalkova, T., Dramer, G.,
Hardesty, B., Chirgwin, J. M., and Horowitz, P. M. (1998)
J. Biol. Chem. 273, 27841-27847), suggesting that the NH2-terminal sequence stabilizes the overall structure. We
tested aspects of the conformations of these shortened species.
Intrinsic and probe fluorescence showed that truncation decreased
stability and increased hydrophobic exposure, while near UV CD
suggested altered tertiary structure. Under native conditions,
truncated rhodanese bound to GroEL and was released and reactivated by
adding ATP and GroES, suggesting equilibrium between native and
non-native conformers. Furthermore, GroEL assisted folding of denatured
mutants to the same extent as wild type, although at a reduced rate.
X-ray crystallography showed that
1-7 crystallized isomorphously
with wild type in polyethyleneglycol, and the structure was highly conserved. Thus, the missing NH2-terminal residues that
contribute to global stability of the native structure in solution do
not significantly alter contacts at the atomic level of the
crystallized protein. The two-domain structure of rhodanese was not
significantly altered by drastically different crystallization
conditions or crystal packing suggesting rigidity of the native
rhodanese domains and the stabilization of the interdomain interactions
by the crystal environment. The results support a model in which loss
of interactions near the rhodanese NH2 terminus does not
distort the folded native structure but does facilitate the transition
in solution to a molten globule state, which among other things, can
interact with molecular chaperones.
 |
INTRODUCTION |
The enzyme rhodanese (thiosulfate:cyanide sulfurtransferase, EC
2.8.1.1) catalyzes in vitro the transfer of sulfur from donors such as thiosulfate to a number of acceptors including cyanide
(1), and it has become an important model for studying protein folding
(2, 3). Rhodanese is monomeric (~32 kDa) and folded into two domains
of very similar size and conformation, connected by a surface loop (4).
The active site is located in the COOH-terminal domain, near the
interdomain interface.
Rhodanese is encoded in the nucleus, translated in the cytosol, and
imported into the mitochondrial matrix without NH2-terminal proteolytic processing, except for the removal of the initiating methionine (5). The NH2-terminal 23 amino acids have been
implicated in rhodanese properties including its unassisted folding,
interactions with molecular chaperones, and mitochondrial import. It
has been suggested, for example, that the disposition of the
NH2-terminal sequence is important for allowing rhodanese
to maintain the non-native conformation that is required for
mitochondrial import (6). Although this sequence has been thought to
contribute to the stability of the active structure of the enzyme (7,
8), its influence on the overall conformational potentials of rhodanese
has never been studied directly. We have used here
NH2-terminal truncations to study the influence of this
region on the structure and function of rhodanese.
The NH2-terminal sequence of rhodanese is located primarily
on the surface of the NH2-terminal domain of the protein
(9-11). The first 9 amino acids contribute to the hydrophobic
interdomain interface. Residues Ser-11 to Gly-22 form an
-helix that
has been suggested to be critical for transport across mitochondrial membranes (12). The influence of the amino-terminal residues on the
rhodanese activity is surprising, since all of the residues required
for catalysis are on the COOH-terminal domain, and the active site
Cys-247 is >18 Å from any of the first 23 NH2-terminal amino acids.
Previous reports suggested that changes within the
NH2-terminal sequence altered the properties of the enzyme.
A point mutation, E17P, within the NH2-proximal
-helix,
facilitated urea denaturation and led to increased proteolytic
susceptibility in vitro (8). When mutants of the enzyme with
sequential NH2-terminal deletions were expressed in
Escherichia coli, the first 10 residues were found to be
necessary for stable, heterologous expression, and longer truncations
were rapidly degraded within the cells (7). Rhodanese mutants with up
to 9 NH2-terminal residues deleted were purified, and their
enzyme kinetic parameters were found to be similar to those of the
full-length enzyme. However, using activity as a criterion, mutants
missing 7 or 9 residues were less resistant to urea perturbation and
exhibited significantly altered active site reactivities.
The present work assessed the biophysical effects of the truncations on
the overall stability of rhodanese. Urea denaturation transitions for
enzymatically active rhodanese with truncations of 3, 7, or 9 NH2-terminal amino acids were monitored using the fluorescence of tryptophan or the hydrophobic probe,
bis-ANS.1 Circular dichroism
was used to compare secondary and tertiary structures under native
conditions. Interactions with GroEL under native conditions were
investigated to evaluate the influence of the truncations on the
ability of rhodanese to adopt conformations that can be recognized by
the molecular chaperone, GroEL. The data are consistent with the
hypothesis that NH2-terminal truncations between residues 4 and 9 produce a fully active enzyme with decreased global stability.
The results are of particular interest, because the x-ray results show
that the native states of all the species are virtually identical.
The rhodanese structure has been studied in some detail, so that
influences of NH2-terminal truncations can be interpreted. Recently, the crystal structure has been refined to 1.36-Å resolution (13). The sulfur-free and sulfur-substituted enzymes crystallized isomorphously. Chemical modifications of the catalytic cysteine led to
limited changes of the protein structure, confined to the enzyme active
site (11). In this work, we have compared the structure of rhodanese
crystallized in a new orthorhombic crystal form with that of the
monoclinic form, and we have determined the structure of an
NH2-terminal-truncated rhodanese (
1-7) in order to
establish the role of NH2-terminal amino acid residues 1-7
on the protein structure.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Wild type and mutant rhodanese species were
purified as described previously (7) and stored as 2.5 M
(NH4)2SO4 pellets containing 5 mM Na2S2O3 and 50 mM NaCl at
70 °C. Some preparations for
crystallography used molecular sieve chromatography as the last step of
the purification (11). Prior to use, they were resuspended in 200 mM Na2HPO4, 50 mM
Na2S2O3, 200 mM
-mercaptoethanol, pH 7.4. Procedures for the purification of
GroEL/ES were modified for optimal yield and purity (14, 15), and these
proteins were stored at
70 °C in 50 mM Tris-HCl, pH
7.8, 10% (v/v) glycerol, and 1 mM dithiothreitol. Urea was
obtained from Bio-Rad Laboratories. Molecular Probes, Inc. supplied the
bis-ANS. All other chemicals were analytical grade from Sigma.
Protein and Activity Assays--
Rhodanese concentrations were
determined using both A280
nm0.1% = 1.75 (16) and micro-bicinchoninic
acid (BCA) assays (Pierce). Activity was calculated from the absorbance
at 460 nm of the complex formed between the reaction product,
thiocyanate, and ferric ion (1). GroEL/ES concentrations were measured
using the BCA assay.
Crystallization of Wild Type and
NH2-terminal-truncated (
1-7) Rhodanese--
Single
crystals of the sulfur-containing form of both wild type and
NH2-terminal-truncated (
1-7) rhodanese were grown at 4 °C in sitting-drop vapor-diffusion experiments. Droplets (5 pl)
containing protein at a concentration of approximately 4 mg/ml in the
presence of 10% (w/v) PEG 6000, 50 mM Tris-HCl, 50 mM sodium acetate, 1 mM sodium thiosulfate, pH
7.3, were equilibrated with a reservoir solution (0.8 ml) containing
16% (w/v) PEG 6000, 50 mM Tris-HCl, 50 mM
sodium acetate, 1 mM sodium thiosulfate, pH 7.3.
X-ray Diffraction Data Collection and Processing--
The x-ray
source for data collection was a Siemens M18X-HF rotating anode
generator, operating at 50 kV and 90 mA, with an apparent focus size of
0.3 × 3 mm. Copper radiation was selected by a graphite crystal
monochromator. A .5-mm collimator was used. Data were measured on a
HIGH STAR area detector system, mounted on a three-axis goniometer. The
crystal was rotated 0.25° per frame. Two crystals for both wild type
and
1-7 mutant were necessary to collect an entire set of
diffraction data. Scaling and merging were performed with the SAINT
program (Siemens Industrial Automation). The wild type and mutant forms
of the protein are isomorphous: the space group is P2,2,2. For wild
type rhodanese the unit cell dimensions are: a = 89.74 Å,
b = 72.35 Å, and c = 39.07 Å; for the
1-7 mutant,
a = 90.69 Å, b = 73.03 Å, and c = 39.67 Å. Taking into account one molecule per asymmetric unit, this gives a
VM of 1.9 Å3/Da, which corresponds to a
solvent content of 37%. Table IV gives the statistics for data
collection and the percentage of reflections measured as a function of
resolution for the two data sets.
Structure Solution and Refinement--
The structure
determination for wild type rhodanese in the orthorhombic crystal form
was accomplished by means of the molecular replacement method, using
the high resolution structure of sulfur-substituted rhodanese in the
monoclinic crystal form as a starting model (13). The model, deprived
of all solvent molecules, was positioned in a triclinic cell with
100-Å edges and a Patterson map was calculated. Rotation and
translation functions were performed with the package AMORE, making use
of the reflections with I greater than 3
(I), between 8- and 3-Å
resolution. The highest peak of the rotation function was subjected to
translation, which gave rise to a solution with a crystallographic
R factor of 32.8. Cycles of rigid body refinement reduced
the R factor to 0.288 (Rfree = 0.36).
From this point onward, refinement was carried on with the package XPLOR: some cycles of minimization on atomic positions were alternated with the refinement of B factors. Five percent of the
reflections were excluded from the refinement and used for the
calculation of Rfree. Periodically, electron
density maps were calculated with coefficients | 2Fobs
Fcalc | or | Fobs
Fcalc | and the model manually adjusted. Solvent molecules were positioned inside peaks
close to the polar side chains. Water molecules with a B factor greater than 65 Å2 were excluded or their position
corrected. In a total of 26 cycles, the R factor was reduced
to the final value of 0.166 (Rfree = 0.215). The
final model consists of 2327 protein atoms and 63 solvent molecules.
For the mutant enzyme, whose crystals are isomorphous with the wild
type protein, the atomic coordinates of the model from the previous
refinement, deprived of solvent and of the first 7 amino acids,
represented the initial model. The initial R factor was
0.36, which dropped, after several cycles of minimization, to 0.248 (Rfree = 0.327). The map calculated with
coefficients | 2Fobs
Fcalc | clearly showed the absence of any
electron density corresponding to amino acids from 1 to 7. A refinement
procedure similar to that used for the wild type protein brought the
R factor to the final value of 0.162 (Rfree = 0.219). The final model contained 2264 protein atoms and 104 solvent molecules. The maps were displayed on an
IRIS 4D Graphics Workstation (SiliconGraphics) using the program TOM.
Gel Filtration Chromatography Analysis--
Each rhodanese
species was separately loaded on a Superose 12 high performance gel
filtration column connected to a fast protein liquid chromatography
system (Pharmacia). The protein sample for each run was at a
concentration of 0.2 mg/ml in 50 mM
Na2HPO4, pH 7.6. A 200-µl aliquot was
injected onto the column equilibrated with 200 mM NaCl, 1 mM EDTA, and 10 mM Tris-HCl, pH 8.0. The flow rate was kept constant at 0.3 ml/min. The effluent was analyzed by
enzymatic activity and absorbance at 280 nm.
Sedimentation Velocity Analysis--
Each rhodanese species was
examined separately by sedimentation velocity ultracentrifugation using
a Beckman XLA analytical centrifuge. The rhodanese concentration was
0.5 mg/ml for each analysis. The temperature was maintained at
25 °C. The rotor speed was 55,000 rpm and absorbance at 280 nm was
monitored during at least 20 scans of the sedimenting boundary. Data
were collected and analyzed with the UltraScan program developed by B. Demeler, Department of Biochemistry, University of Texas Health Science Center at San Antonio. This analysis is based on the method of van
Holde and Weishet (17). All data were corrected for buffer density and viscosity.
Urea Unfolding of Rhodanese Proteins Monitored by Tryptophan
Fluorescence and bis-ANS Fluorescence--
Wild type rhodanese or
mutant protein (50 µg/ml) was denatured at 0-8 M urea in
a buffer containing 50 mM
Na2S2O3, 50 mM
Tris-HCl, and 200 mM
-mercaptoethanol, pH 7.8, for
24 h at 25 °C. Samples were then excited at 280 nm and emission
spectra were scanned from 300 to 400 nm using an SLM 48000C
spectrofluorometer. Fluorescence is a sensitive measure of rhodanese
conformation, and all the species retained the 8 tryptophans found in
the wild type protein (Trp-14, Trp-35, Trp-112, Trp-113, Trp-133,
Trp-275, Trp-278, and Trp-287). A Perkin-Elmer LS-50B
spectrofluorometer was used to measure fluorescence intensities at 500 nm after excitation at 399 nm for bis-ANS fluorescence (18, 19). 30 µM bis-ANS was added to each sample immediately prior to
excitation. Intensities were corrected with buffer blanks without
protein. All analyses were performed at 25 °C.
Urea denaturation was performed under conditions are known to produce
transitions reflecting reversible unfolding (3). The intrinsic
tryptophan fluorescence intensities as a function of urea concentration
were fit to a model for a two-state transition, [N (native)
[dharrow] D (denatured)]. Values for the free energy of unfolding,
GD, at a given urea concentration were calculated
from the equilibrium constant, KD, using,
|
(Eq. 1)
|
where f is fraction of protein and y is
measured fluorescent emission intensities. Linear extrapolations of
these values to [urea] = 0 by the method of Pace were used to
estimate the conformational stability of the protein,
GDH2O (20).
Circular Dichroism--
CD spectra were scanned at 25 °C in a
0.1-cm path length cell from 260 to 340 nm for near UV data and from
180 to 280 nm for far UV data using a Jasco J500C spectropolarimeter.
All samples were in 50 mM Na2HPO4,
pH 7.6, and were corrected using buffer blanks. Protein concentrations
for far UV-CD measurements were at 7-8 µM, and 50 to 75 µM for near UV-CD measurements. CD data were calculated
in terms of mean residue ellipticity (
) at a specified
wavelength using a mean residue weight of 115 (21, 22).
Capture/Release of Native Rhodanese Species by
GroEL/ES--
Rhodanese is enzymatically inactive when it interacts
with GroEL, and capture of rhodanese is defined in this study as the loss of enzymatic activity that occurs in the presence of GroEL compared with parallel controls without GroEL. Each rhodanese species,
at a final concentration of 150 nM, was added to a buffer containing 2.5 µM GroEL, 10 mM
MgCl2, 10 mM KCl, 2 mM ADP, 200 mM
-mercaptoethanol, 50 mM
Na2S2O3, 50 mM
Tris-HCl, pH 7.8, at 23 °C. Rhodanese activities for each sample
were recorded hourly using 30-µl aliquots assayed for 10 min. These
activities were normalized to parallel control samples under the same
conditions, but without GroEL. To release bound rhodanese, 5 µM GroES and 5 mM ATP were added to all
samples after incubation for 90 h in the above buffer conditions.
After an additional 25 h, 2 mM more fresh ATP was
added to all samples. Release of rhodanese was monitored by measuring
regain of activity. To prevent microbial growth over the long
incubation times, all buffers were filtered through 0.22-µm filters
and/or by autoclaving. The samples were incubated in sterile microcentrifuge tubes.
GroEL/ES-assisted Refolding of Urea-unfolded Rhodanese--
All
protein samples were denatured for 4 h at 25 °C at 90 µg/ml
in 8 M urea containing 50 mM
Na2S2O3, 50 mM
Tris-HCl, and 200 mM
-mercaptoethanol, pH 7.8. Refolding
was performed by diluting to a protein concentration of 3.6 µg/ml
into the buffer above without urea, additionally containing 10 mM MgCl2, 10 mM KCl, 2 mM ATP, 2.5 µM GroEL, and 5 µM
GroES. Rhodanese assays were performed at specified time points during
refolding by diluting 25 µl of the sample into the assay reaction
mixture and stopping the reaction after 15 min.
 |
RESULTS |
Wild Type and Mutant Forms of Rhodanese Are Homogenous and
Monomeric
Rhodanese species were analyzed for aggregation that may occur due
to truncation of the NH2 terminus. Gel filtration behavior of mutants and wild type were identical (not shown). A single peak with
the same shape and same enclosed area eluted at the same volume
expected for monomeric rhodanese, independent of the species used.
Mutant and wild type proteins gave virtually identical sedimentation
velocity results. Each protein behaved as a single, pure component when
the data were analyzed by the method of van Holde and Weichert (17), in
that there was little variation in the integral distribution of
s20,w values at all boundary fractions
scanned. The s20,w values for the native
proteins were as follows: wild type = 2.97 ± 0.09 S,
1-7 = 2.88 ± 0.10 S, and
1-9 = 2.83 ± 0.07 S. No evidence of large aggregates or oligomers (dimer, trimer, etc.) was detected for any species.
Conformational Stability,
GDH2O, Is Diminished in
Rhodanese Mutants
We investigated the conformational properties of
1-3,
1-7, and
1-9. The kinetic properties of these species
were reported previously and found to be very similar (7). The
stability of each species,
GDH2O was derived from
denaturation curves by extrapolating the measured
G
values to [urea] = 0. Tryptophan fluorescence was used to monitor unfolding as described under "Experimental Proedures" (23-25), and
these data give a good fit to a two-state transition.
Fig. 1a shows that increasing
NH2-terminal truncation destabilizes rhodanese, and Fig.
1b shows these data within the transition regions as plots
of calculated
GD versus [urea] for each sample. Table I presents the
conformational stabilities (
GDH2O), the
transition midpoints (U1/2), and the wavelengths of
the fluorescence spectral maxima for the native forms of the
mutants. The U1/2 relates the slope of the
lines (m) in Fig. 1b and the
GDH2O by the relation
U1/2 =
GDH2O/m.
Deletion of the first 3 NH2-terminal residues
from wild type rhodanese had no significant effect on any of
these parameters (both
GDH2O
6.25 ± 0.10 kcal/mol and both U1/2
4.0 ± 0.02 M urea). However,
1-7
was destabilized measurably (
GDH2O = 5.2 ± 0.4 kcal/mol, U1/2 = 3.27 ± 0.02 M urea), and there was a significant
destabilization for
1-9
(
GDH2O = 4.2 ± 0.2 kcal/mol, U1/2 = 2.74 ± 0.03 M
urea).

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Fig. 1.
a, intrinsic tryptophan fluorescence in
urea-denatured rhodanese NH2-terminal truncation mutants.
Transitions from native to denatured protein are shown for each mutant
and compared with a wild type control. Native proteins were denatured
in increasing concentrations of urea. Fluorescence wavelength ( )
maxima were calculated by taking the derivative of spectrofluorometric
scans ranging from 300 to 400 nm at each urea concentration. All points
are averages of three determinations without error bars for clarity.
Curves generated from compiled data are fit to the points for each
sample (SigmaPlot). b, extrapolations to 0 M
urea to estimate conformational stability
( GDH2O) of
rhodanese NH2-terminal truncation mutants in native states.
The free energy of unfolding ( GD) for each mutant
and wild type from a was calculated at each urea
concentration in the transition curve region as described under
"Experimental Procedures." GD is then plotted
against urea concentration. Linear regression extrapolation of the
plots to 0 M urea estimates conformational stability of the
native structure (20).
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Table I
Biophysical characteristics of purified rhodanese mutants
Conformational stabilities were extrapolated for native proteins, see
Fig. 1B and detailed description under "Experimental
Procedures" and Ref. 20. The standard deviations for the
conformational stabilities at 0 M urea were given as 95%
confidence interval data plotted for the linear regression
extrapolations. Urea denaturation transition midpoints were calculated
from the nonlinear curve fit function: y = (a d)/[1 + (x/c)b] + d; where c = U1/2 (urea denaturation transition midpoint),
x = [urea], y = GD, a = asymptotic maximum,
b = slope function, and d = asymptotic
minimum. Standard deviations for transition midpoints are from at least
20 iterations of the fit function. Spectral maxima for the native forms
of the proteins are taken from the original three trials ± standard deviations.
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As seen in Fig. 1a and Table I, there were measurable red
shifts of the fluorescence maxima for
1-7 in the absence of urea (336-337 nm) compared with the maximum for wild type or
1-3 (334 nm) suggesting increased solvent exposure of tryptophans in the native
structures of the
1-7 mutant.
Hydrophobic Surfaces Are More Easily Exposed in the Rhodanese
Truncation Mutants Compared with Wild Type
The fluorescence enhancement when the molecule bis-ANS binds to
hydrophobic surfaces was used to compare hydrophobic exposure in the
truncation mutants during urea-induced unfolding.
Fig. 2 shows the bis-ANS fluorescence as
a function of the urea concentration. The increase in intensity
starting at low urea concentrations has been suggested to reflect the
formation of folding intermediates with exposed hydrophobic surfaces.
At higher urea concentrations, the protein begins to unfold further,
and the organized surfaces are disrupted, accompanied by a decrease in
fluorescence intensity. The fluorescence intensity reaches a maximum at
an intermediate point between the opposing tendencies of formation of
the folding intermediates and their subsequent unfolding. Fig. 2 shows
that increased truncation facilitates hydrophobic surface exposure at
low urea concentrations. The parameters characterizing these
transitions are given in Table II.
1-7 and
1-9 showed more bis-ANS fluorescence in the absence of
urea, suggesting greater hydrophobic exposure in the unperturbed
structures of these mutants; the results are in keeping with increasing
hydrophobic exposure with increasing truncation.

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Fig. 2.
Bis-ANS fluorescence in urea-denatured
rhodanese NH2-terminal truncation mutants. Normalized
intensities of the emission wavelength of bis-ANS fluorescence (500 nm)
were measured for each sample at increasing concentrations of urea.
Averages of at least 3 intensity determinations for each urea
concentration for separate protein samples are plotted without error
bars for clarity. No significant shift in the wavelength for the
emission peak (500 nm) was detected when scanned from 450 to 550 nm
during urea denaturation experiments.
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Table II
Urea denaturation transition parameters for rhodanese mutants monitored
by bis-ANS fluorescence
Parameters are measured from plots in Fig. 2.
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1-9 Rhodanese Exhibits an Altered Near UV-CD Signal
The mutants and wild type proteins were analyzed by UV-CD. Spectra
of the wild type protein were in agreement with those described previously (26). The spectra for the mutants and wild type were virtually identical in the far UV. Table
III compares the ellipticities for the
proteins at 210 and 292 nm. Far UV-CD molar ellipticities at 210 nm for
all mutants were not significantly different from wild type, ~7 × 10
3 deg·cm2·dmol
1,
suggesting that the secondary structures are not significantly different.
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Table III
Molar ellipticities of rhodanese mutants at far (210 nm) and near (292 nm) UV-CD wavelengths
The specified wavelengths were chosen based on characteristic maxima
for elliptically polarized light seen for all samples scanned in the UV
spectrum. All samples are corrected for the buffer, 50 mM
sodium phosphate, pH 7.6. Protein concentrations were determined using
A2800.1% readings and BCA assays and ranged
from 7 to 8 µM in the 210 readings and 50-75
µM in the 292 readings. See "Experimental
Procedures" for details.
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The molar ellipticities at 292 nm,
292, were used to
compare the tertiary structures in the vicinity of the Trp residues
(Table III). Molar ellipticities were the same for wild type,
1-3,
and
1-7 (55 to 60 deg·cm2·dmol
1).
However,
292, for
1-9 was considerably smaller than
for the other species. This smaller value of
292 = 39.7 deg·cm2·dmol
1 is similar to that seen for
structurally perturbed wild type protein. Thus, wild type in 3 M urea had a
292 of 48.5 deg·cm2·dmol
1, in 4 M urea,
292 = 33.4 deg·cm2·dmol
1,
and in 6 M urea,
292 = 9.2 deg·cm2·dmol
1. The decreased molar
ellipticity seen for
1-9 suggests an altered tertiary structure
similar to urea-perturbed wild type.
X-ray Structures of Wild Type and
1-7 Show Virtually Identical
Structures That Are Not Perturbed by the Crystal Environments
Overall Structures for Wild Type Rhodanese and
1-7 Crystallized
in the Orthorhombic Crystal Form--
A schematic representation of
the two domain structure of rhodanese is shown in Fig.
3. The overall structure of the wild type
enzyme, crystallized in the orthorhombic crystal form using PEG 6000 as
precipitant, is very similar to the structure of the monoclinic form
obtained in the presence of ammonium sulfate (data not shown): the root
mean square deviation between the C
positions of the two models is
0.47 Å (see Table IV for x-ray
statistics). Root mean square differences greater than 1 Å can be
observed only at positions 3 and 292, i.e. at the
NH2- and COOH-terminal ends, which are usually flexible
areas in proteins, and in the region from amino acid residue 194 to
199, a loop exposed to the solvent and involved in intermolecular
contacts in the orthorhombic crystal. Finally, helix B (residues
42-50) is also slightly shifted as compared with its position in the
monoclinic crystal form. Thus, drastically different crystallization
conditions and a different crystal packing do not affect significantly
the protein structure. The molecular model of the
1-7 mutant, which
crystallized isomorphously with the wild type enzyme in the presence of
PEG 6000, compares very well with that of the wild type protein (Fig.
4): excluding the first seven amino
acids, the C
's of the remaining portion of the two molecules
superimpose with a root mean square deviation of 0.23 Å. Appreciable
differences can be observed only at the COOH terminus. These findings
clearly indicate that the lack of the enzyme NH2 terminus
up to residue 7 is not critical for protein folding, in the sense that
native conformation can be achieved by the NH2-terminal
truncated enzyme. A detailed inspection of the region of the
NH2 terminus in the wild type enzyme shows that the short
sequence 1-7, which is located at the protein surface, establishes
several interactions: O4-NH2 Arg-121, 2.63 Å; N6-OH Tyr-261, 3.14 Å; OH Tyr-6-N
His-94, 3.37 Å; O7-O-
Ser-124, 2.63 Å. The presence of such interactions in the wild type enzyme must account for the higher stability of the wild type protein to
perturbations by urea relative to the
1-7 mutant (see above and
Ref. 7). The space previously occupied by residues 1-7 is not filled
with ordered solvent molecules in the crystal of the mutant.

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Fig. 3.
Schematic drawing of the folding of
rhodanese. Corresponding elements of secondary structure in
domains I and II are denoted by capital letters. The sulfur
atoms of the persulfide group at the active site are shown as
black spheres. The drawing was obtained using the program
MOLSCRIPT.
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Fig. 4.
Stereo drawing of the
-carbon trace for the model of the
sulfur-containing orthorhombic crystal form of rhodanese (dashed
line) superimposed on that of the
1-7 mutant (continuous
line).
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The Active Site--
The active sites for the monoclinic and
orthorhombic crystal forms of wild type sulfur-substituted rhodanese
and for the orthorhombic form of the
1-7 sulfur-substituted mutant
are highly preserved, especially with regard to protein atoms. The
persulfide group at Cys-247 in the sulfur-substituted form of the
enzyme is substantially stabilized by a number of hydrogen bonding
interactions between both sulfur atoms and neighboring groups at the
active site, as established for the monoclinic crystal form (4).
Virtually the same hydrogen bonding interactions involving the
persulfide group are present in the orthorhombic form of wild type and
1-7 mutant rhodanese. Despite the two drastically different
crystallization media for the monoclinic and orthorhombic crystal
forms, solvent molecules at the active site also show a remarkable
conservation. In a previous crystallographic study with monoclinic
crystals, the presence of a sulfate ion in close proximity to the
positive charges of Arg-186 and Lys-249 residues at the enzyme active
site was demonstrated (27). When the mother liquid of the monoclinic crystals of rhodanese, containing ammonium sulfate, was exchanged with
a cryo-protectant solution devoid of ammonium sulfate and containing
PEG 6000 as precipitant (13), the density accounting for the sulfate
ion at the active site was replaced by a peak of lower intensity,
attributable to a water molecule. This observation is consistent with
the finding of a substantial reactivation of the crystalline enzyme
inhibited by sulfate, upon replacement of ammonium sulfate with PEG
6000 as precipitant in the crystal mother
liquid.2 Solvent molecules
lying in a sphere of radius 10 Å from the S
247 bound to the
catalytic cysteine are represented in Fig.
5 and Table
V which compares wild type rhodanese and
1-7 mutant crystallized in the orthorhombic form. A solvent
molecule (O362) is present, at a short distance from S
, in both wild
type and mutant proteins and is also present in the monoclinic form
(Trp-581 in Fig. 2 in Ref. 13). Such a conserved water molecule might
be involved in the catalytic reaction. Six additional water molecules
are present in similar positions in both orthorhombic wild type and mutant rhodanese (see the Table V and Fig. 5), forming a network of
hydrogen bonds among them and with protein atoms. A very similar situation is observed for the monoclinic crystal form transferred to a
cryo-protectant solution devoid of ammonium sulfate (13): only one
additional solvent molecule is visible at the active site, despite the
significantly higher resolution of that data.

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Fig. 5.
View of a portion of the active site of wild
type rhodanese (a) and 1-7 mutant
(b), with the solvent molecules in the active site
cavity. Symbols for atoms are: C, O, N, and S.
|
|
The Cation-binding Site--
The presence of a cation-binding site
was postulated some time ago (28) and observed in a previous
crystallographic study (27) on the basis of a difference Fourier map
from rhodanese crystals soaked in a medium in which ammonium
sulfate was replaced by sodium thiosulfate. The cluster of peaks
and holes close to Asp-272 were interpreted as due to a Na+
ion, which substituted for an ammonium ion previously present. In both
structures of wild type and
1-7 mutant a solvent molecule in this
position shows a six-donor coordination: five oxygens and a sulfur atom
surround the central atom with a bipyramidal geometry, as shown in Fig.
6 and Table
VI. Interatomic distances between oxygens
and the cation are in the range 2.48-2.65 Å, expected for this kind
of coordination, while distances between cation and the sulfur atom of
Met-73 are 3.30-3.17 Å, respectively, for wild type and
1-7
mutant, accounting for the larger radius of the sulfur atom. The peak
in the electron density map, although higher than that expected for a
water molecule, is not very high (4
in 2 | Fobs
Fcalc | map and
10
in the | Fobs
Fcalc | map): it could correspond to a small
cation, like Na+ or K+, present in the buffers used for
protein purification and crystallization. The cation site is about 10 Å from the transferred sulfur atom that is bound to Cys-247 and not
expected to affect the catalytic activity, in accordance with the
finding that cations are not required for catalytic activity (29).

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Fig. 6.
View of the cation-binding site in the
crystal of wild type rhodanese, showing the distorted bipyramidal
coordination of the cation. Atom symbols as in Fig. 5 and Table V.
A very similar situation is present in the crystal of 1-7
mutant.
|
|
The Molecular Chaperone GroEL Can Slowly Capture
1-9
Rhodanese
GroEL associates with non-native protein conformations by
hydrophobic interactions to inhibit off-pathway misfolding of proteins. It does not interact with native proteins (30). Rhodanese mutants were
captured by GroEL (17-fold molar excess) in the presence of ADP at room
temperature (~23 °C) over long periods of time (up to 67 h
with precautions to maintain sterile conditions as described under
"Experimental Procedures"). Fig. 7
shows a gradual loss of rhodanese activity that is taken to represent
binding of the protein to GroEL. This binding correlates with the
length of truncation.
1-9 displayed almost complete inactivation at 67 h. The percent inactivations of the samples at 67 h were
as follows: wild type, 8.1 ± 3.4%;
1-3, 26.7 ± 4.6%;
1-7, 39.4 ± 1.7%; and
1-9, 86.3 ± 3.3%. Loss of
activity in identical controls without GroEL over the same time period
was wild type = 4.3 ± 6.9%,
1-3 = 5.3 ± 6.1%,
1-7 = 6.8 ± 0.5%, and
1-9 = 10.8 ± 7.4%. These slight decreases in activity due to factors other than
GroEL capture do not substantially alter the conclusions that can be
drawn from the data in Fig. 7.

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Fig. 7.
GroEL slow capture of rhodanese mutant
proteins. Interaction of GroEL with rhodanese mutants led to
inactivation of enzymatic catalysis followed over time. Activity is
given as percent of an identical parallel control sample containing no
GroEL. All data points are averages of three separate determinations
with standard deviations. Loss of activity in control samples without
GroEL for the entire time span of the experiment was not
significant.
|
|
Rhodanese-GroEL complexes formed by addition of denatured wild type
enzyme can be dissociated with regain of activity by addition of GroES
and ATP (31). In Fig. 8, the slow capture
experiments were extended to 90 h followed by addition of GroES
and ATP. The 0-h time point is the activity of each sample after the
90-h capture period compared with controls without GroEL. Wild type
activity increased 26% from 65.8 ± 1.5% to 92.1 ± 3.1%
after 1 h of incubation with GroES and ATP. The 92.1 ± 3.1%
of wild type activity was near the averaged maximum activity recovered,
94.4 ± 2.6% at 42 h. At the same time
1-3 also had a
26% recovery (53.7 ± 0.6% to 80.4 ± 4.1%), but
1-7
and
1-9 each had only a 10% recovery of activity (49.0 ± 0.3 to 60.1 ± 4.0% and 9.8 ± 1.1 to 20.9 ± 1.6%,
respectively). 24 h later
1-3 released another 10% (80.4 ± 4.1% to 90.8 ± 3.8%) to near its averaged maximum
recoverable activity of 92.1 ± 3.6%, and
1-7 and
1-9
each recovered another ~15% activity (60.1 ± 4.0 to 75.5 ± 3.0% and 20.9 ± 1.6% to 38.4 ± 3.5%, respectively).
Addition of fresh ATP released another 10% of
1-7 activity and
30% of
1-9 activity to their respective maximum recoverable
activities of 85.7 ± 4.1 and 72.9 ± 3.4%.

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Fig. 8.
GroES slow release of GroEL-captured
rhodanese mutant proteins. Release of GroEL-captured rhodanese
mutants by addition of GroES and ATP resulted in reactivation followed
over time. Activity is given as percent of an identical parallel
control sample containing no GroEL as detailed under "Experimental
Procedures." The 0-h time point gives percent activity after a 90-h
incubation at 23 °C compared with parallel controls without GroEL.
All data points are averages of three separate determinations with
standard deviations. The experiments were stopped before loss of
activity in control samples without GroEL became significant.
|
|
Although the binding of
1-9 is quite slow under native conditions,
Fig. 9 shows that GroEL can assist
similarly the refolding of denatured wild type rhodanese and the
truncation mutants
1-3,
1-7, and
1-9 in the standard assay
for GroEL function. The extent of refolding is approximately the same
for all species, and, although the rates of refolding appear to be
somewhat slower the longer the truncation, the process occurs within a
similar time. Thus, while the wild type protein completed folding
within 5 min, the mutant
1-9 was only 50% folded at that time.

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Fig. 9.
GroEL/ES-assisted refolding of urea-unfolded
rhodanese. Rhodanese samples were first unfolded in urea and then
refolded by a 1:25 dilution into a buffer containing GroEL/ES (see
"Experimental Procedures"). Activity was calculated relative to
parallel control samples that were not subjected to urea denaturation.
These values were plotted as a function of the time after dilution at
which the activity assays were performed. At least three separate
trials were performed. Average activity points are given with standard
deviations. Curves were fit to the data using SigmaPlot.
|
|
 |
DISCUSSION |
Rhodanese truncation mutants lacking the first 7 or 9 NH2-terminal residues were previously shown to have
virtually indistinguishable catalytic constants, but changes in
structural integrity were suggested by subtle differences in the
reactivities of the active site Cys-247 (7). The present studies show
that these species are less stable to urea-induced unfolding than the
wild type enzyme. Extrapolation of urea unfolding data show decreases
in the free energies of stabilization for
1-7 and
1-9 relative
to wild type rhodanese of 1 and 2 kcal/mol, respectively. Even in the
absence of perturbation, the intrinsic fluorescence wavelength maxima of these mutants were slightly red-shifted. This increased tryptophan exposure may be due to partial uncovering of Trp-112 and Trp-113, which
are located just below the sequence Arg-7 to Leu-9 in the wild type protein.
These suggestions are supported by the x-ray structure of
1-7,
which is virtually identical to that of the wild type protein with the
exception of the missing residues. The crystal data suggest a high
degree of rigidity of sulfur-substituted rhodanese in its native
conformation. In particular, the two-domain structure of rhodanese is
not significantly affected by drastically different crystallization
conditions and crystal packing or by the lack of seven
NH2-terminal residues. The extent of conservation is very
high for the active site region, with regard to both protein atoms and
solvent molecules surrounding the active center. It is likely that the
initial structural changes that occur on urea perturbation involve
perturbation of the interdomain interface and separation of the
domains. This would be inhibited in the crystal. This interpretation
would reconcile the x-ray information and the numerous solution
studies of rhodanese.
The results with bis-ANS further support these conclusions. The urea
dependence of the bis-ANS fluorescence with wild type rhodanese has
been reported (19). Initial intensities are related to the hydrophobic
exposure on the unperturbed enzyme. The increasing intensities at the
lower urea concentrations have been ascribed to the formation of a
folding intermediate. The decreasing intensities as the urea is
increased even further represent the unfolding of these intermediates.
These opposing phenomena give rise to transitions of the type observed
in Fig. 2. The present results can be interpreted in terms of this
model. Even in the absence of perturbation, there is slightly more
hydrophobic surface accessible on
1-7 and
1-9 than on the wild
type protein. Fig. 2 shows that there are greater differences in the
rising phases of the intensities than in the falling phases. This would
indicate that the longer truncations make it easier to form
intermediates that bind bis-ANS. This latter conclusion is supported by
the observation that the easier it is to form the intermediates that
have the characteristics of molten globule states (increasing intensity
phases), the more difficult it is to fully denature them (decreasing
intensity phases). This result would be compatible with the formation
of intermediates that preferentially bind bis-ANS.
These observations are consistent with the x-ray structure that shows
the truncations uncover a hydrophobic cluster that lies below residues
Arg-7 to Leu-9. The small increases in hydrophobic accessibility on the
unperturbed truncation mutants are not sufficient to cause association
of rhodanese molecules as demonstrated by ultracentrifugation and gel
filtration, which demonstrate that each of the proteins used in this
study is homogeneous and monomeric.
Circular dichroism studies provide additional evidence for changes in
the tertiary structures induced by the truncations. The near UV-CD
molar ellipticities (Table III) show that the tertiary interactions
around the tryptophan residues of
1-9 are similar to a partially
unfolded wild type structure observed in a urea concentration between 3 and 4 M. This substantial decrease in molar ellipticity for
1-9 in the near UV reports a significant alteration in the
environments formed by the tertiary fold of the protein around at least
some of the tryptophans. These changes occurred while there was no
noticeable change in the secondary structures as reported by the far
UV-CD. Again, although these are characteristics of molten globule
states, some of the change may be due to alterations in the environment
of Trp in the vicinity of the truncations. The x-ray shows a complex
set of interactions among the aromatic residues in rhodanese, a number
of which could be expected to respond to the truncations described
here. Thus, Tyr-6 is close to Tyr-261, which in turn is close to
Trp-112 and Trp-113. In addition, Arg-6 is close to Trp-14. Thus, these
interactions are expected to be altered in addition to uncovering
Trp-112 and -113 by the truncations described here.
The molecular chaperone GroEL binds tightly to folding intermediates of
rhodanese, but it does not interact with native wild type. Differences
in stability of the species can be translated into differences in the
equilibrium, in the absence of perturbation, between native and
partially folded or denatured protein conformers. Thus, the ability of
GroEL to capture rhodanese was used to detect differences in the
ability of native rhodanese structures to adopt conformations that
could be recognized. More than 85% of
1-9 was captured by GroEL
under conditions where less than 11% of wild type was captured. The
1-7 mutant could also be bound by GroEL, but it is less able to be
bound than
1-9. The slow rates of association presumably follow
from the fact that, even though the intermediates are more accessible
from the native state, they are present at low concentrations at
equilibrium. These findings lend additional support to the view that
the NH2-terminal sequence of rhodanese makes a measurable
contribution to the conformational stability of the entire protein. The
results with GroEL refolding of rhodanese show that the yields are
equal when the various mutants are used, but the rates are somewhat
slower. This would follow, because the less stable mutants which more
easily form intermediates that can bind to GroEL would be expected to
have lower off-rates from the complex and faster recapture rates. The
suggestion that rhodanese bound to GroEL has been studied and suggested
to be in the form of a molten globule state in which the individual domains are folded but not properly associated, to give a structure with the characteristics of a molten globule (32). This is consistent with the suggestions above that perturbations induce separation of
domains that remain largely folded.
The present results provide evidence that NH2-terminal
truncation in rhodanese leads to decreased conformational stability. This structural lability extends previous findings that truncated rhodanese species can fold to fully active enzymes that have altered in vivo survivability after expression and differences in
active site reactivities. All the results are consistent with a model in which a folding intermediate of rhodanese consists of independently folded domains that are not optimally associated leaving easily exposed
hydrophobic surfaces. The formation of this intermediate would be
favored by NH2-terminal truncation. The separation of these
domains requires perturbation under normal solution conditions, and it
would be inhibited by contacts in the crystal. As an example of the
biological consequences of the effects we have described, the
labilization of the rhodanese structure by removal of the influence of
its NH2-terminal sequence suggests that the interaction of
this sequence with the mitochondrial import machinery could similarly
loosen the protein and help maintain the protein in an import competent
conformation required for the successful compartmentation of this enzyme.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant GM25177 and Robert A. Welch Foundation Grant AQ723 (to P. M. H.) and grants from the "Consiglio Nazionale delle
Ricerche" and "Ministero dell'Universita e della Ricerca
Scientifica e Tecnologica", Rome, Italy (to F. G., R. B.,
M. C., and G. Z.).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.
The atomic coordinates and structure factors (codes lBOH and
lBOI) of the two protein models have been deposited in the Protein Data
Bank, Brookhaven National Laboratory, Upton, NY.

To whom correspondence should be addressed: Dept. of
Biochemistry, University of Texas Health Science Center, 7703 Floyd
Curl Dr., San Antonio, Texas 78284-7760. Tel.: 210-567-3737; Fax:
210-567-6595; E-mail: horowitz{at}bioc02.uthscsa.edu.
2
R. Berni, unpublished observation.
 |
ABBREVIATIONS |
The abbreviation used is:
bis-ANS, 1,1'-bis(4-anilino)naphthalene-5,5'-disulfonic acid.
 |
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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.