A Backbone-reversed Form of an All-
-Crystallin Domain from a Small Heat-shock Protein (Retro-HSP12.6) Folds and Assembles into Structured Multimers*,
Anshuman Shukla,
Manoj Raje and
Purnananda Guptasarma
From the
Institute of Microbial Technology, Sector 39-A, Chandigarh 160036,
India
Received for publication, March 26, 2003
, and in revised form, April 24, 2003.
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ABSTRACT
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The structural consequences of polypeptide backbone reversal
("retro" modification) remain largely unexplored, in particular,
for the retro forms of globular all-
-sheet proteins. To examine whether
the backbone-reversed form of a model all-
-sheet protein can fold and
adopt secondary and tertiary structure, we created and examined the
recombinant retro form of a 110-residue-long polypeptide, an
-crystallin-like small heat-shock protein, HSP12.6, from C.
elegans. Following intracellular overexpression in fusion with a
histidine affinity tag in Escherichia coli, purification under
denaturing conditions, and removal of denaturant through dialysis,
retro-HSP12.6 was found to fold to a soluble state. The folded protein was
examined using fluorescence and CD spectroscopy, gel filtration
chromatography, non-denaturing electrophoresis, differential scanning
calorimetry, and electron microscopy and confirmed to have adopted secondary
structure and assembled into a multimer. Interestingly, like its parent
polypeptide, retro-HSP12.6 did not aggregate upon heating; rather, heating led
to a dramatic increase in structural content and the adoption of what would
appear to be a very well folded state at high temperatures. However, this was
essentially reversed upon cooling with some hysteresis being observed
resulting in greater structural content in the heated-cooled protein than in
the unheated protein. The heated-cooled samples displayed CD spectra
indicative of structural content comparable to that of any naturally occurring
globular protein. Attempts are being made to refine crystallization conditions
for the folded protein.

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FIG. 1. A, fluorescence emission spectrum of RETHSP-2. B, gel
filtration chromatogram showing elution of RETHSP-2 from an analytical SMART
Superdex-200 column (calibration shown as inset).
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INTRODUCTION
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The structure of a naturally occurring globular protein is determined by
its amino acid sequence. The amino acid sequence has a definite polarity with
the C=O group of every residue forming a peptide bond with the NH group
of the next residue. Here, we explore the structural-biochemical consequences
of reversing the polarity of the polypeptide backbone through the creation of
a novel protein with an amino acid sequence that is the exact reverse of the
sequence of a naturally occurring protein, the
-crystallin-like small
heat-shock protein HSP12.6 from Caenorhabditis elegans
(1).
The consequences of effecting such a transformation have previously been
explored both in theory and in experiment. Among works dealing with
polypeptides that are large enough to be called proteins, the following
discussions are worthy of note. (a) Guptasarma
(2,
3) hypothesized that the retro
form of an all-
-sheet protein would fold into a topological mirror image
of the structure adopted by the parent sequence through mirror imaging of the
entire scheme of side chain-side chain interactions facilitating folding. The
dihedral angles characterizing each residue in the parent structure would thus
change both sign as well as definition in the mirror-imaged structure because
of the replacement of C=O by NH and vice versa. As a
consequence, every
would become
and every
would
become
. Notably, with
-sheets, such a transformation could
conceivably allow each residue to remain in a
-sheet configuration
(3). However, with
-helices, such a transformation would be expected to effect a change in
the handedness of the helix and so mirror imaging would not ordinarily occur
for single helices and all-helix protein structures, but rather, it would
occur only with isolated helices in predominantly
-sheet structural
contexts. In other words, only helices that could pay the energy penalty for
switching handedness through stabilization by packing contacts with other
mirror-imaged substructures would undergo the transformation. (b)
Skolnick and colleagues (4)
performed folding simulations with the retro form of an all-
protein,
the B domain of Staphylococcal protein A, and showed that 3 of 12
folding simulations led to mirror-imaged structures, whereas the remaining
nine simulations folded into
-helical structures. (c) Another
result with
-helical proteins was obtained experimentally by Grutter
and colleagues (5) who showed
that the retro form of a GCN4 leucine zipper folds into a structure
(determined crystallographically) that is almost identical to the parent
structure and not a mirror image of the parent structure. The similarity of
the parent and retro structures was explained on the basis of the fact that
there was a 2-fold palindrome in the hydrophobicity profile of the protein,
intersecting the central cavity in the structure of the protein. (d)
Importantly, whereas the retro forms of
-helical proteins did display a
tendency to fold, an experimental attempt to reverse the backbone of a protein
containing
-sheets produced a polypeptide that displayed no tendency to
fold. Serrano and colleagues
(6) demonstrated that the retro
form of an Src homology 3 domain does not fold. However, structural modeling
carried out by the same group established that a mirror image topology was
feasible, especially in combination with folding to a molten-globule state
rather than a rigid unique structure
(6). Since only
40% of an
Src homology 3 domain constitutes
-structure with the remaining
polypeptide being folded into rigid loop-like structures that do not qualify
to be called secondary structure (the 60-residue-long domain has only
25
residues of 60 forming
-strands
(7)), the question of what
would happen upon reversal of a larger protein with a much greater propensity
to form
-sheets has thus far remained open to question.
We decided to examine the consequences of reversing the sequence of a
larger all-
-sheet protein. We chose as a parent sequence for backbone
reversal the 110-residue-long HSP12.6 from C. elegans. This is the
smallest known homolog of all of the proteins belonging to the family of
-crystallin-like heat-shock domains
(1). Based on actual
determination of the structures of several members of this family
(8,
9),
-crystallin-like
domains are currently accepted to be proteins of a defined all-
-fold.
Our investigations reveal that the reversed sequence too folds and assembles
into a multimer like the parent.
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EXPERIMENTAL PROCEDURES
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Molecular Genetic Manipulations and Design of
ConstructsFrom the sequence of the gene encoding the parent
protein, Hsp12.6 (1), the
sequence of a novel gene encoding retro-HSP12.6
(RETHSP)1 was created
by reversing the sequence of codons used by the parent gene. The DNA encoding
the reversed sequence (retro-HSP12.6) was then synthesized through a
combination of contract synthesis of double-stranded oligonucleotides and our
own molecular genetic manipulations to derive constructs encoding
retro-HSP12.6 with a choice of restriction sites flanking the sequence for
insertion into the vector pQE30 (Qiagen) to facilitate expression in fusion
with a His6 affinity tag. Finally, two forms of retro-HSP12.6 were
created. (i) The first form, RETHSP-1, was a backbone-reversed form of the
110-residue-long parent sequence flanked by N- and C-terminal extensions. The
N-terminal extension consisted of 12 residues incorporating a 10-residue
affinity tag (MRGSHHHHHH) and an additional two residues contributed by the
cloning site (GS). The C-terminal extension of nine residues (VDLQPSLIS) was
entirely due to the choice of restriction sites at the multiple cloning site
of pQE-30, as the vector's own stop codon was used. (ii) To make the second
form, RETHSP-2, the C-terminal extension was removed through inclusion of a
stop codon immediately after the backbone-reversed HSP12.6 sequence.
Expression of proteins from both constructs was first checked in XL1Blue,
which was the cloning host. The sequence of the insert in the positive clone
was confirmed through automated DNA sequencing on an ABI 310 Prism sequencer,
and the plasmid was transformed into the expression host M15pREP4. The
sequences of HSP12.6, RETHSP-1, and RETHSP-2 are shown in
Table I for reference.
Expression, Purification, and FoldingThe expression of both
proteins in the expression host Escherichia coli M15pREP4 was low but
this was compensated for by setting up larger culture volumes. For expression,
the cells were grown overnight and a 1% secondary innoculum was added to an
appropriate volume of LB. Cells were induced with 1 mM
isopropyl-1-thio-
-D-galactopyranoside at an optical density
of 0.6 and harvested 4 h after induction. Harvested cells were suspended in
lysis buffer containing denaturant (8 M urea, 0.1 M
NaH2PO4, 0.01 M Tris-Cl, pH 8.0) and lysed by
sonication. The lysate was centrifuged at 18,000 x g for 1 h,
and the supernatant thus obtained was loaded onto a nickel-nitrilotriacetic
acid column in the presence of the denaturant, urea. Washing (with 8
M urea, 0.1 M NaH2PO4, 0.01
M Tris-Cl, pH 6.3) and elution (with 8 M urea, 0.1
M NaH2PO4, 0.01 M Tris-Cl, pH 5.9
and pH 4.5) were also done under denaturing conditions. Dialysis of the eluted
protein to remove denaturant was done against 20 mM Tris. After
dialysis, concentration was effected through centrifugation under vacuum to
the point at which the protein started to precipitate after which the sample
was centrifuged and supernatant was taken for an estimation of concentration
of soluble protein. This was taken as the maximally concentrated solution of
the protein since upon further concentration the protein precipitated. The
change in molarity of buffer following such concentration was also estimated
by reckoning for the change in volume effected by centrifugal vacuum
concentration.
Spectroscopy and MicrocalorimetryProtein concentrations
were estimated through UV absorption measurements at 280 nm using a predicted
molar extinction coefficient of 12,780 for proteins encoded by both RETHSP-1
and RETHSP-2. Fluorescence spectra were collected on a PerkinElmer LS-50B
spectrofluorimeter with variable excitation and emission bandpasses as
appropriate using excitation with light of 280 nm and scanning protein
emission between 300 and 400 nm. CD spectra were collected at intervals of 1
nm on a Jasco J-810 spectropolarimeter through scanning of wavelengths from
250 to 200 nm using a protein concentration of 0.4 mg/ml and a cuvette path
length of 0.2 cm. CD signals below 200 nm could not be collected because the
spectra were noisy. Consequently, no attempt was made to estimate secondary
structural contents from this data. Calorimetry was carried out using a
Microcal MC-2-ultrasensitive microcalorimeter using a protein concentration of
80 µM and a scan rate of 60 K/h. Fourier Transform Infrared
spectra for the solid precipitate of RETHSP-1 obtained through concentration
beyond the solubility limit were collected on a PerkinElmer Spectrum BX
instrument with the protein sample placed between two calcium fluoride windows
at a resolution of 1 cm1, taking an average of 32
scans.
Chromatography and Non-denaturing Gel ElectrophoresisGel
filtration chromatography was performed on a Pharmacia SMART system using an
analytical Superdex-200 column (bed volume
2.4 ml, void volume 0.8 ml) and
a flow rate of 0.1 ml/min through use of 0.05-ml protein samples of
concentration at 0.4 mg/ml. The fractionation range of the column was 6,00,000
Da, and the exclusion limit was 16,00,000 Da. Non-denaturing gel
electrophoresis for the determination of the native molecular weight of
RETHSP-2 was carried out by the standard procedures involving: (i)
determination, plotting, and (linear) fitting of changes in the relative
mobilities of protein samples as a function of gel acrylamide percentage by a
least squares fitting method, followed by (ii) by plotting of the negative
value of the slope thus obtained for each standard protein against its known
native molecular weight (Ferguson plot), least squares fitting of this data to
a straight line, and interpolation of the value of the slope obtained for
RETHSP-2 into the plot.
Electron MicroscopyTransmission electron microscopy studies
were carried out through use of routine negative staining procedures employing
phosphotungstic acid and uranyl acetate on a JEOL 1200 EX-2 microscope.
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RESULTS AND DISCUSSION
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Solubility of RETHSP-1 and RETHSP-2No precipitation was
observed during dialysis-based removal of denaturant from solutions of either
RETHSP-1 or RETHSP-2 following His6 tag-based affinity purification
on nickel-nitrilotriacetic acid-agarose columns in the presence of urea. The
protein samples obtained were found to be soluble up to a concentration of
1.21.5 mg/ml for RETHSP-1 and
0.6 mg/ml for RETHSP-2.
Concentration beyond these values led to protein precipitation.
Spectrofluorimetric CharacterizationThe two forms, RETHSP-1
and RETHSP-2, displayed wavelengths of maximal fluorescence emission
(
max) of 348 and 351, respectively, indicating only very
nominal burial of their aromatic residues (two tryptophans and one tyrosine).
The emission spectrum of RETHSP-2 is shown in
Fig. 1A.
RETHSP-1 and RETHSP-2 Appear to Be Trimers/Tetramers at Low
ConcentrationsBecause both proteins were soluble, we examined
their quaternary structural status using gel filtration chromatography. On an
analytical SMART Superdex-200 column, RETHSP-2 eluted at 1.47 ml
(Fig. 1B),
corresponding to a molecular mass of
5253 kDa. In some
preparations of the protein, a minor population was also found to elute close
to the void volume of the column (0.80.9 ml), which had a bed volume of
2.4 ml and a fractionation range of 10,000600,000 Da with an exclusion
limit of 1.3 x 106 Da. RETHSP-2 has a polypeptide molecular
mass of 13,970 Da, indicating that the majority population eluting at
1.47 ml falling within the optimal fractionation range for the column is
predominantly tetrameric. In comparison with the value of 5253 kDa that
was obtained through gel filtration, determination of the native molecular
mass at low protein concentration by non-denaturing gel electrophoresis (see
Fig. 6, C and
D) yielded an estimated molecular mass of 4546
kDa. Both of these estimates turn out to lie between the values expected for
trimeric (
42 kDa) and tetrameric (
56 kDa) states of a
14-kDa
polypeptide like RETHSP-2. Thus, three possibilities apply. (i) The molecule
is assembled into a tetramer that behaves as a smaller species on account of
compactness. (ii) The molecule is assembled into a trimer that behaves as a
larger species on account of being swollen, or (iii) the molecule forms a
mixed population of trimers and tetramers existing in equilibrium. Because
techniques used to determine multimeric molecular masses including
non-denaturing gel electrophoresis, gel filtration chromatography, dynamic
light scattering, and analytical ultracentrifugation are all influenced to
varying extents by molecular shape and effective hydrodynamic volume, which
need not correlate perfectly with size for non-spherical species, estimates of
native molecular mass do not always correspond to expected multiples of
subunit molecular weight. We emphasize that dynamic light scattering or
analytical ultracentrifugation data could provide more accurate information
concerning whether the population is mostly trimeric or tetrameric, and we are
organizing to perform these experiments. Meanwhile, we have obtained
preliminary plate-like crystals of the protein and attempts are being made to
refine crystallization conditions toward eventual structure determination,
which should resolve the issue.

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FIG. 6. Characteristics of RETHSP-2 prior to heating and following cooling to
room temperature after heating at 90 °C. Panel A, elution
from a Superdex-200 column of unheated and heated-cooled samples as indicated.
Panel B, Stern-Volmer plots of acrylamide quenching of protein
fluorescence for unheated (open triangles) and heated-cooled
(open circles) samples. Panel C, representative
non-denaturing PAGE (10% acrylamide, stacking/resolving gels of pH 8.8). The
first five lanes on the left show the markers, carbonic
anhydrase, chicken egg albumin, bovine serum albumin, urease, and
-lactalbumin, respectively, with isoforms visible where present. The
last two lanes, respectively, correspond to unheated and
heated-cooled samples of RETHSP-2. Panel D, Ferguson plot (both axes
in log10 scale) showing five standard protein molecular masses in
kDa plotted against the negative values of slopes of individual linear (least
square) fits obtained for each protein (Kr)
through initial plotting of relative mobility versus gel acrylamide
percentage. The interpolation of the value of the slope obtained for RETHSP-2
is shown by horizontal and vertical lines.
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Notably, the occasional observation of a soluble higher order multimer at
the void volume of the Superdex-200 column indicates that this assembly may
also be capable of associating further into larger multimers approaching sizes
of 600,000 or more, especially at high protein concentrations.
Evidence of Formation of a Higher Order Multimer upon
ConcentrationAs already mentioned, RETHSP-1 has a solubility limit
of
1.21.5 mg/ml. Beyond this protein concentration, protein
precipitates are obtained. Whereas the gel filtration studies reported above
used the sample remaining in the supernatant after concentration, examination
of the precipitated protein using transmission electron microscopy and
negative staining showed the presence of a globular, bead-like form with a
diameter of roughly 1820 nm (Fig.
2, panels A and B). We are in the process of
analyzing multiple images of these beads to attempt partial three-dimensional
reconstruction. Fourier Transform Infrared spectra of these beaded samples
clearly show the presence of secondary structure
(Fig. 2C) with C=O
stretch band maxima at 1629 and 1651 cm1,
indicative of predominantly
-sheet with some helical content.

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FIG. 2. Panels A and B, negatively stained transmission electron
micrographs of RETHSP-1 precipitates obtained through concentration beyond
1.01.2 mg/ml. RETHSP-2 showed similar precipitates. The white
bar in both panels corresponds to a length of 50 nanometers
(nm). Panel C, FTIR spectrum of the above precipitate. The C=O
stretch bands in the amide I region show a dominance of the 1629
cm1 absorption characteristic of -sheet
structure.
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Evidence of Secondary Structure FormationThe CD spectrum of
any protein is a linear combination of the contributions of peptide bonds in
various secondary structures. The CD spectrum of RETHSP-1
(Fig. 3A) indicates
/
-secondary structural content together with a substantial
component of randomly coiled structure. It may be noted that for completely
randomly coiled polypeptides, no negative ellipticity signal is observed
between 250 and 210 nm. Only at wavelengths below 210 nm are spectra observed
to show a negative band that dips to display a minimum at
198 nm. Since
out of a total of 131 residues RETHSP-1 contains 21 residues that comprise N-
and C-terminal extensions that are in any case not expected to participate in
structure formation (16% of the chain) and, furthermore, because any negative
CD signal resulting from random coil is nearly 45 times stronger than
that due to a
-sheet configuration for any peptide bond (i.e.
in a plot of mean-residue ellipticity), the CD spectrum of RETHSP-1 might
arguably be expected to be dominated by a negative band at
198 nm,
however, with significant negative mean residue ellipticity being visible even
at wavelengths above 210 nm. Gratifyingly, this is what is seen.

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FIG. 3. Far-UV CD spectra of retro-HSP12.6 constructs. Panel A, the
retroprotein, RETHSP-1, at room temperature. Panel B, the
retroprotein, RETHSP-2, at room temperature. Panel C, the
retroprotein, RETHSP-2, following heating to 90 °C and cooling to room
temperature.
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When the C-terminal extension is removed as in RETHSP-2, the length of the
chain reduces to 122 residues, bringing down the number of extra residues to
12 (only 9% of the chain) from 21. This would be expected to reduce the
contribution of residues in a randomly coiled configuration to the CD spectrum
and thus lead to a shift of the band minimum to a longer wavelength. As shown
by the CD spectrum of RETHSP-2, such a shift is exactly what is seen. RETHSP-2
shows a band minimum at 210 nm together with another band minimum at
230
nm (Fig. 3B). Notably,
the mean residue ellipticity of the entire spectral range is enhanced by a
factor of almost 2.5 over that seen with RETHSP-1, indicating that this
polypeptide is significantly more structured even though there is still some
random coil component.
Heating Causes Structural Consolidation Rather than
UnfoldingTo examine whether heating of the protein is
characterized by an endothermic reaction resulting in a change in enthalpy
associated with unfolding, differential scanning calorimetry was carried out
with RETHSP-1. The scan showed a small, almost indiscernible endothermic
reaction followed by a dramatic and unexpected exothermic reaction at high
temperatures (Fig. 4). Such an
exothermic reaction could only be due to the formation rather than destruction
of non-covalent contacts. Such contacts could be because of either aggregation
or further structura1 consolidation within the polypeptide. To examine whether
any aggregation occurs upon heating, we heated the proteins at 90 °C for
15 min and found no visible sign of aggregation. Gel filtration of cooled
samples also showed no signs of high molecular weight species (see
Fig. 6A). One further
test excluding any possibility of aggregation was a monitoring of the HT
voltage associated with the transmission of light through the sample in a CD
spectrometer during heating of the sample. The HT voltage in a
spectropolarimeter rises to compensate for reductions in light intensity not
associated with differential absorption of left and right circularly polarized
light when the detector is starved for light through absorption or scattering.
The HT voltage, therefore, is extremely sensitive to changes in the level of
scattering of transmitted light. We found no significant changes in HT voltage
associated with heating, signaling a lack of aggregation during heating.

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FIG. 4. DSC-scanning profile of RETHSP-1. An exothermic reaction suggestive
of the formation of non-covalent contacts is discerned. Details of the scan
are as mentioned within the panel.
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Because there was no evidence of aggregation and suspecting that structural
consolidation could indeed have occurred in the sample, we examined the CD
spectrum of RETHSP-2 at high temperature
(Fig. 5A). We also
examined the nature of changes in ellipticity associated with heating and
cooling of RETHSP-2 (Fig.
5B) to further confirm the changes in CD signal
associated with heating and investigate the extent to which such changes are
reversible for this protein. As is evident from
Fig. 5A, the protein
is significantly more structured at high temperatures than at room
temperature. Fig. 5B
reinforces this conclusion, demonstrating that there is a gradual increase in
the negative ellipticity at 218 nm during heating (effected at a rate of 5
°C/min) as well as a clear reduction in negative ellipticity associated
with cooling (effected at the same rate). This finding shows that the
structural consolidation effected through heating is largely reversed upon
cooling. However, some hysteresis is also clearly seen to be associated with
the process (Fig. 5B)
with the signal at 218 nm not returning to its original value upon cooling.
Thus, the structural gains effected through heating are not entirely lost upon
cooling, and as a result, the heated and cooled protein
(Fig. 3C) shows a
negative band maximum at 215216 nm with a second band at
230 nm,
indicating a clear predominance of
-sheet configuration and a well
folded state of the polypeptide even upon return to room temperature. It may
be emphasized once again that perhaps because of the fact that the
heat-induced changes do not reverse completely, the spectrum of the
heated-cooled protein (Fig.
3C) is different from that of the unheated protein
(Fig. 3B), which
displays a band minimum at
210 nm as well as from spectrum of the protein
at the high temperature of 92 °C (Fig.
5A), which most resembles the CD spectrum of a well
folded naturally occurring protein. The differences in signal intensities
among the various spectra, especially at lower wavelengths, conform to what is
expected for enhancement of
-sheet content at the expense of
unstructured content. As is well known, the negative ellipticity associated
with a peptide bond in a
-sheet configuration is much lower than that
associated with a peptide bond in a random coil.

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FIG. 5. Structural consolidation associated with heating of RETHSP-2.
Panel A, far-UV CD spectrum of RETHSP-2 collected at 92 °C.
Panel B, changes recorded in the mean residue ellipticity of RETHSP-2
at 218 nm in course of heating from a temperature of 20 °C to a
temperature of 90 °C at a rate of 5 °C/min (upper curve) and
in course of cooling from 90 to 20 °C (lower curve), following
incubation for 1 min at 90 °C.
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The intriguing semi-reversible thermally induced consolidation of structure
described above caused us to carry out a further comparative examination of
RETHSP-2 in the unheated, heated state, and cooled states. The unheated sample
and the heated-cooled sample were chromatographed through gel filtration
(Fig. 6A) and seen to
contain the same dominant trimeric/tetrameric population eluting at 1.47 ml on
a Superdex-200 column. As can be seen, there was no evidence of any additional
species eluting at the column's void volume (0.80.9 ml), indicating
that there was no generation of high molecular weight aggregates through the
process of heating and cooling. However, a slight difference can be seen in
the width-at-half-height of the elution as well as in the volume at which the
elution begins for the heated-cooled sample. Thus, the gross quaternary
structural status of RETHSP-2 is not changed through heating and cooling,
although as already pointed out clearly some of the structural gains effected
through heating are retained by the molecule after cooling, evident from the
hysteresis seen in the ellipticity signal as a function of temperature
(Fig. 5B) as well as
from differences in the CD spectral shapes of unheated, heated, and
heated-cooled samples (Figs.
3B,
5A, and
3C, respectively).
Fluorescence emission spectroscopy at different temperatures did not shed
light on structural transitions. The 351-nm emission (
max)
of the protein alluded to earlier (Fig.
1A) remained at 351 nm, even at 9092 °C as
well as upon cooling to room temperature, displaying only a reversible
reduction in intensity with increased temperature but no change in other
spectral characteristics (Supplemental Fig.
1). Therefore, it would appear that the exposed tryptophan
residues of the protein remains largely exposed to the solvent, even in course
of the heat-induced structural consolidation and resettlement into a more
structured state upon cooling. Fluorescence quenching carried out for unheated
and heated-cooled samples reinforce this conclusion. Stern-Volmer plots
(Fig. 6B) show that
the accessibility of the fluorescing aromatic residues is virtually unaltered
between the unheated and the heated-cooled samples. To further investigate the
conclusion from gel filtration data, which indicated that the unheated and
heated-cooled samples have similar sizes despite their structural differences,
non-denaturing gel electrophoresis was carried out. The gels of four different
percentages of acrylamide were run. Variations in the relative mobilities of
five different protein standards as a function of varying gel density were
analyzed and used to construct a Ferguson plot. A representative gel (10%
acrylamide) is shown (Fig.
6C) with unheated and heated-cooled samples run in the
last two lanes, establishing that the hydrodynamic volumes of these two forms
are entirely similar. The Ferguson plot
(Fig. 6D) shows that
both forms correspond to a molecular mass of 4546 kDa as already
mentioned earlier.
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CONCLUSIONS
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Retro-HSP12.6 appears to fold and assemble into multimeric states that
further associate to form large globular structures. At low protein
concentrations, the polypeptide displays secondary structural content and no
tendency to aggregate, despite possessing solvent-exposed aromatic residues.
Secondary structural content is enhanced through heating and lost through
cooling as evidenced by CD spectroscopy with calorimetry showing an exothermic
reaction to be occurring upon raising of temperature without attendant
molecular aggregation. Thus, heating of this backbone-reversed all-
heat-shock protein results in enhancement of structural content, perhaps
because of improved hydrophobic interactions among residues at high
temperatures facilitating further hydrogen-bonding interactions and greater
structure and stability. At the high temperature of 92 °C, the protein
shows a CD spectrum not unlike that of any folded naturally occurring protein.
Upon cooling, there is loss of most of the structural content gained through
heating, but nevertheless some hysteresis is seen and the process is not fully
reversible, such that the heated-cooled protein shows greater structural
content than the unheated protein and shows a CD spectrum indicative of a
considerably better folded state. Most intriguingly, heating is not associated
with any aggregation. Independently, concentration of the protein was
observed, to lead to assembly of the molecule into larger bead-like
structures, which are precipitation-prone and show high secondary structural
content. Preliminary plate-like crystals of the protein have been obtained and
attempts are being made to refine crystallization conditions to carry out
further structural analysis.
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FOOTNOTES
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* The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
The on-line version of this article (available at
http://www.jbc.org)
contains Supplemental Fig.
1. 
To whom correspondence should be addressed. Tel.: 91-172-695225 (ext. 444);
Fax: 91-172-690585; E-mail:
pg{at}imtech.res.in.
1 The abbreviations used is: RETHSP, retro-heat-shock protein. 
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ACKNOWLEDGMENTS
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We thank Swati Sharma for carrying out the non-denaturing gel
electrophoresis experiments, Drs. Avadesha Surolia and Aditi Gupta for
carrying out the Differential Scanning Colorimetry scan, and Dr. K.V. Radha
Kishan for advice concerning crystallization. We also thank Anil Theophilus
for help with electron microscopy. A. Shukla thanks the CSIR (New Delhi,
India) for a doctoral research fellowship, and P. Guptasarma thanks CSIR and
INSA (New Delhi, India) and TWAS (Trieste, Italy) for research funding to
study protein folding and aggregation.
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REFERENCES
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