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INTRODUCTION |
Despite their presence in all organisms examined to date, small
heat shock proteins (sHsps)1
remain one of the most disparate family of stress proteins. More than
25 different sHsps have been identified in the cytosol and organelles
of heat-shocked plants (1); mammalian and yeast cells encode fewer
sHsps (2). The overall homology between sHsps isolated from diverse
sources is not as pronounced as that found in other stress protein
families. In fact, sHsps are only grouped as a family of heat shock
proteins based on a low degree of homology in a core region of about 85 amino acids (the
-crystallin domain), their ability to be induced by
cellular stress, and their small protomer molecular mass, which usually
ranges between 15 and 30 kDa. Because of structural and functional
similarities, the major eye lens proteins
A- and
B-crystallin are
also included in the sHsp family.
sHsps are abundant in unstressed cells (3), and their synthesis is
up-regulated by cellular stress as well as a variety of additional
signals related to the growth state and oncogenic status of the cell
(e.g. Refs. 4-6). Most sHsps assemble into large oligomers
in a process that requires an intact NH2 terminus (7). In
mammalian cells, sHsps form complexes ranging from 300 to 800 kDa (2,
8).
-Crystallin, which is normally isolated as an 800-kDa oligomer,
has been reported to range in size from 280 kDa to 10 MDa (for a
review, see Ref. 9). Plant sHsps exhibit a more uniform size
distribution (
200-240 kDa); pea Hsp18.1 consists of 12 subunits
arranged in a globular structure, whereas pea Hsp17.7 is organized in
both round and triangular structures (10). The recently crystallized
Methanococcus jannaschii Hsp16.5 (MjHsp16.5) is organized as
a hollow sphere built from 24 subunits (11). Finally,
Mycobacterium tuberculosis Hsp16.3 forms another type of
oligomeric structure, a 150-kDa triangular trimer of trimers (12).
sHsps are believed to play a key role in thermoprotection because their
overexpression confers a significant degree of thermotolerance to host
cells (13-17). Mammalian sHsps appear to perform this function by
stabilizing cytoskeletal elements such as actin (7, 18, 19). In other
systems, sHsps may increase thermotolerance by preventing the
aggregation of cellular proteins accumulating as a result of cellular
stress. Indeed, a number of sHsps suppress the thermal aggregation of
model proteins in vitro (10, 12, 20-23). Furthermore,
murine Hsp25, human Hsp27, and pea Hsp18.1 and Hsp17.7 exhibit a
bona fide molecular chaperone function because they
partially improve the reactivation of certain chemically unfolded
proteins (10, 20). In the above cases, the addition of ATP to the
refolding buffer is not necessary to enhance refolding. Nevertheless,
the reactivation yields of citrate synthase (CS) by human
B-crystallin are increased 2-fold in the presence of ATP (17). The
formation of large oligomeric structures appears to be necessary for
molecular chaperone function because NH2-terminally truncated variants of Caenorhabditis elegans
Hsp16.2 and the only known monomeric sHsp, C. elegans
Hsp12.6, are unable to suppress the aggregation of test substrates (7,
24). Purified murine Hsp25 (23) and pea Hsp18.1 (22) have been shown to
bind thermally denatured proteins on their surface and maintain them in
a folding-competent state. This observation has led to the idea that
sHsps act as a reservoir of partially folded proteins that are
accessible for refolding by ATP-dependent molecular
chaperones once the effect of cellular stress has abated (23).
In contrast to the relatively large amount of information available on
eukaryotic sHsps and
-crystallins, little is known about their
Escherichia coli homologs. The approximately 16-kDa E. coli IbpA and IbpB polypeptides were first discovered as
contaminants tightly associated with recombinant protein inclusion
bodies (25). These inclusion-body-associated
proteins were later found to cosediment with host proteins
aggregated intracellularly by heat shock (26). The ibpA and
ibpB genes are part of an E
32-transcribed
operon that is the strongest heat-inducible operon of E. coli (27). The bacterial sHsps are about 50% identical to each
other at the amino acid level and share 20-27% identity with homologs
from plant chloroplasts, Drosophila, C. elegans, and Xenopus (25, 27). IbpAB are dispensable in E. coli, and deletion of the ibp operon only leads to
modest effects on growth and viability at high temperatures (28). The
fact that a
ibp mutation exerts a deleterious effect on
the growth of dnaK756 but not groES30 mutants at
high temperatures suggests that IbpAB cooperate with DnaK-DnaJ-GrpE but
not with GroEL-GroES in vivo (28). This result is in
agreement with a recent investigation of the interplay between IbpB and
the major chaperone systems showing that purified IbpB forms stable
complexes with denatured malate and lactate dehydrogenase and that the
bound proteins are specifically delivered to the DnaK-DnaJ-GrpE, but
not to the GroEL-GroES system in vitro (29).
To gain further information on the structure-function relationship of
bacterial sHsps, we have purified and refolded IbpB from a
ibp strain. We show that despite its pronounced size
heterogeneity, IbpB shares many of the traditional features of sHsps.
Our results indicate that reversible structural modifications induced
by temperature changes in the 32-50 °C range drive the exposure of
denaturant-sensitive hydrophobic regions that may constitute substrate
binding sites for nonnative proteins. Charge effects appear to be
important for the optimal exposure of hydrophobic domains to the solvent.
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EXPERIMENTAL PROCEDURES |
Strains and Plasmids--
Plasmid pTb-ibpAB and pTb-ibpA are
pT7blue (Novagen) derivatives carrying polymerase chain
reaction-amplified DNA fragments encoding the complete ibp
operon or the ibpA gene without their native
promoter.2 To place the
ibp operon (or the ibpA gene) under
transcriptional control of the T7 promoter, plasmid pTb-ibpAB (or
pTb-ibpA) was digested with XbaI and EcoRI, and
the small fragment was ligated into the same sites of pET-22b+
(Novagen) to yield pJSibpAB (or pJSibpA). Plasmid pJSibpAB was digested
with NdeI, and the backbone was religated. In the resulting
plasmid, pJSibpB, the T7 promoter is followed by the authentic
ibpB ribosome binding site, coding sequence, and
transcription terminator. All constructs were verified by restriction
analysis and DNA sequencing. The
ibp1::kan null mutation (28) was
moved to E. coli BL21(DE3) (Novagen) by P1 transduction to
yield JGT14. This strain was used as a host for recombinant protein production.
Purification of IbpB--
JGT14(pJSibpB) cultures were grown at
30 °C in 500 ml of LB medium supplemented with 0.2% glucose and 50 µg/ml carbenicillin. At A600
0.4, the
cultures were induced with 1 mM
isopropyl-
-D-thiogalactopyranoside and incubated at
30 °C for an additional 5 h. Cells were harvested by
centrifugation at 7,000 × g for 10 min, resuspended in
15 ml of 25 mM
NaH2PO4·H2O, pH 7.0 (buffer A),
and disrupted by chemical lysis (30). Briefly, the paste was incubated
on ice with 400 µl of 10 mg/ml lysozyme and 40 µl of 9 mg/ml
phenylmethylsulfonyl fluoride for 20 min. DNase (200 units) and
deoxycholic acid (20 mg) were added, and the mixture was incubated at
37 °C for 30 min. Cellular debris was removed by centrifugation at
17,000 × g for 30 min. The clarified lysate was
supplemented with 0.8 ml of 200 mM dithiothreitol and 0.4 ml of 5% polyethyleneimine and incubated at room temperature for 10 min to precipitate contaminating nucleotides. After centrifugation at
17,000 × g for 30 min, ammonium sulfate was added to
the supernatant to a concentration of 20% (w/v). Precipitated proteins
were sedimented by centrifugation at 10,000 × g for 15 min and discarded. Ammonium sulfate was added slowly to 40% (w/v), and
insoluble proteins were recovered by centrifugation after a 45-min
incubation with stirring on ice. The protein pellet was resuspended in
2 ml of 25 mM
NaH2PO4·H2O, pH 7.0, containing 5 M GdnHCl (buffer B) and incubated on ice for 2 h.
Denatured proteins were loaded onto a Superose 6 HR column (Amersham
Pharmacia Biotech) equilibrated and developed at 0.3 ml/min in buffer
B. Fractions eluting between 54.5 and 64.5 min from multiple runs were
pooled. Purified IbpB was refolded at 4 °C by dialysis against
buffer A for 16 h, with buffer exchange at 3 and 12 h, using
Spectra/Por dialysis tubing (VWR Scientific) of molecular mass cutoff
12-14 kDa. The protein was concentrated to 0.3-0.5 mg/ml using
Centriplus-100 concentrators (Amicon) of molecular mass cutoff 100 kDa.
Further concentration favored the formation of insoluble aggregates.
This operation also led to the removal of contaminating low molecular
mass proteins, and the resulting IbpB was
95% pure. IbpB samples
were centrifuged at 12,000 × g for 5 min before all
experiments, and concentrations were determined using the Bradford
assay (Bio-Rad). All concentrations are reported in terms of protomers.
Size Exclusion Chromatography--
For the experiment of Fig.
3A, 20 µg of IbpB was injected onto a BioSep-S4000 size
exclusion column (Phenomenex) equilibrated in 100 mM
KH2PO4, pH 7.0, 0.2 M NaCl. For the
experiment of Fig. 6A, 25-µl samples of 0.2 mg/ml IbpB
were incubated at the indicated temperatures for 1 h, centrifuged
at 12,000 × g for 5 min, and injected immediately onto
the BioSep-S4000 column equilibrated in buffer A. The column was
developed at 0.5 ml/min, and the absorbance was recorded at 280 nm. The
following standards were used for calibration:
-crystallin (20 kDa),
citrate synthase (100 kDa), aldolase (158 kDa), catalase (232 kDa),
ferritin (440 kDa), thyroglobulin (660 kDa), GroEL (812 kDa), and blue
dextran (2 MDa).
Aggregation Suppression Experiments--
Thermal aggregation
suppression experiments were performed using pig heart CS and equine
liver alcohol dehydrogenase (Sigma) essentially as described (21, 31).
CS monomer concentration was determined using an extinction coefficient
of 7.75 × 104 M
1
cm
1 (32), and the protein was diluted to a working
concentration of 3 µM in 40 mM HEPES, pH 7.8, 20 mM KOH, 50 mM KCl, 10 mM
(NH4)2SO4, 2 mM
CH3COOK (buffer C). Buffer C (700 µl) was supplemented
with 100 µl of CS (final concentration 300 nM) and 200 µl of IbpB diluted in buffer A to the indicated final concentrations.
Alcohol dehydrogenase (5 µM final concentration) in 300 µl of 100 mM
NaH2PO4·H2O, pH 7.0, 200 mM NaCl, 4 mM EDTA was supplemented with 100 µl of H2O and 200 µl of IbpB diluted in buffer A to the
indicated final concentrations. Right angle light scattering was
measured on a Hitachi F4500 fluorescence spectrophotometer at 45 °C
(CS) or 41.5 °C (alcohol dehydrogenase) with excitation and emission
wavelengths at 500 nm and slit widths set at 2.5 nm (33). In all cases, stock solutions were stored on ice until mixing and transferred rapidly
to thermostated fluorescence cuvettes.
Unfolding Experiments--
For chemical denaturation studies,
IbpB samples (1 µM) were incubated for 1 h at room
temperature in buffer A supplemented with the indicated concentrations
of GdnHCl. Equilibrium was reached during this time period. The
tryptophan red shift was measured in a Hitachi F4500 fluorescence
spectrophotometer at an excitation wavelength of 280 nm and slit widths
set at 2.5 nm. Emission spectra were plotted between 310 and 380 nm and
fitted with a ninth-order polynomial equation. Tryptophan maximum
emission wavelengths were obtained from the fit. Samples were next
supplemented with 10 µM bis-ANS (Molecular Probes), and
emission spectra were recorded after a 5-min incubation using an
excitation wavelength of 396 nm. Data were plotted between 450 and 550 nm, fitted with a ninth-order polynomial equation, and maximum bis-ANS
emission wavelengths and intensities were calculated from the fit. For
temperature denaturation studies, single protein samples (1 µM) were heated from low to high temperatures in a
thermostated spectrophotometer cell with a 1-h incubation at the
indicated points. This time period was sufficient to reach equilibrium.
Tryptophan
max were obtained as above. Reversibility was
assessed by cooling the sample down to the indicated temperatures and
measuring
max after 1 h. Five scans were averaged
for all experiments, and the background fluorescence of buffers alone
or buffers supplemented with bis-ANS was subtracted from sample spectra.
CD Spectroscopy--
IbpB samples at a 0.05 mg/ml (0.62 µM) final concentration were incubated at room
temperature for 1 h in the presence of the indicated
concentrations of GdnHCl or NaCl or held for 1 h at the indicated
temperatures in a thermostated cell of 1-mm path length. CD spectra
were recorded on a Jasco 720 spectropolarimeter. Spectra were smoothed
using the operating software noise reduction filter and represent the
average of 16 scans corrected for buffer absorbance.
Transmission Electron Microscopy--
IbpB (1 µM)
in buffer A was stained with 0.5% uranyl acetate. A 1-µl aliquot was
applied to a Formvar-coated copper grid, vacuum dried, and carbon
coated in a carbon evaporator. A Philips EM400 transmission electron
microscope operating at 100 keV was used to image the sample at
magnifications ranging between 6,000 and 280,000.
Dynamic Light Scattering--
IbpB (1 µM) in 6 ml
of buffer A was filtered through a 0.22-µm membrane directly into a
prewashed light scattering cell. The cell was inserted into the
goniometer of a Brookhaven laser spectrometer (Brookhaven Instruments,
Holtsville, NY) and data were collected at room temperature at a
scattering angle of 45° with sampling time of 5 min and response
times between 2 and 200 µs using a Brookhaven BI 9000AT
autocorrelator (34). Particle size distribution was calculated using
the CONTIN method (35).
Bis-ANS Photoincorporation--
IbpB (2.8 µM) in
22 µl of buffer A containing 100 µM bis-ANS and
supplemented or not with 1 M NaCl was incubated in
Eppendorf tubes held at 23, 37, or 45 °C for 1 h. Samples were
next irradiated for 20 min at the indicated temperatures by placing a
hand-held UV lamp (Mineralight UVGL-25 at 254 nm setting) on top of the open tubes. Reducing SDS loading buffer was added, and samples were
fractionated on a 15% SDS minigel after a 5-min incubation at
95 °C. The gel was photographed with a Kodak DC120 digital camera on
a UV transilluminator and later stained using a colloidal Coomassie
staining kit (Novex). The intensity of the fluorescent bands was
quantified using NIH Image 1.60 for PowerPC.
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RESULTS |
Expression and Purification of the Bacterial sHsps--
Two
pET-22b+ derivatives encoding the individual ibpA and
ibpB genes under transcriptional control of the
bacteriophage T7 promoter were constructed by polymerase chain reaction
as described under "Experimental Procedures." To avoid the
complication of mixed oligomer formation associated with the presence
of the chromosomal ibp operon, a BL21(DE3) derivative
bearing the
ibp1::kan null mutation (28) was
generated by P1 transduction and used as an expression host. Fig.
1A shows that each sHsp was
produced at moderate and comparable levels following
isopropyl-
-D-thiogalactopyranoside induction of
the transformants. However, whereas overproduced IbpA partitioned
approximately equally between soluble and insoluble cellular fractions,
IbpB accumulated mostly in a soluble form.

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Fig. 1.
Cellular localization of the bacterial sHsps
and purification of IbpB. Panel A,
fractionation of overexpressed IbpA and IbpB. JGT14 cells harboring the
control vector pET22b+ or plasmids encoding the ibpA
(pJSibpA) or ibpB gene (pJSibpB) under control of the T7
promoter were grown in supplemented LB medium at 37 °C. 3 h
postinduction, cultures were fractionated into whole cell
(w), soluble (s), and insoluble (i)
fractions, and samples corresponding to identical amounts of culture
were subjected to 15% SDS-polyacrylamide gel electrophoresis as
described (64). Panel B, refolded and
concentrated IbpB. Markers (lanes M) correspond to the
following molecular masses (from top to bottom):
104, 81, 47.7, 34.6, 28.3 and 19.2 kDa (Bio-Rad). The position of
IbpA/B is shown by the arrow.
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We first attempted to purify native IbpA and IbpB by standard
chromatography (e.g. DEAE, Mono Q and hydroxyapatite). Under these conditions IbpA, and to a lesser extent IbpB, smeared over many
fractions. The bacterial sHsps were therefore purified by ammonium
sulfate precipitation followed by denaturing gel filtration chromatography and refolded by dialysis. Although the refolding yield
of IbpB was about 60%, little IbpA (<5%) could be refolded, and the
purified protein had a high tendency to form insoluble aggregates upon
concentration and storage. The further characterization of IbpA was
therefore not pursued in the present study. Purified IbpB was
approximately 95% pure as judged by videodensitometric scanning of
overloaded gels (Fig. 1B).
Secondary and Quaternary Structure of IbpB--
At 20 °C, the
far UV CD spectrum of IbpB is virtually identical to that of
-crystallins (36-38) and very similar to that of other sHsps (38).
There is a single minimum at 213 ± 1 nm with mean residue
ellipticity at 213 nm, [
]213, of
3.3
millidegrees cm2 dmol
1 (Fig.
2, trace 1), and no fine
features are obvious between 200 and 245 nm. The spectrum is consistent
with of a mostly
-pleated structure that appears to be highly
conserved among sHsps (11, 39).

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Fig. 2.
Temperature-induced secondary structure
changes. The CD spectra of IbpB (0.62 µM final
protomer concentration) were recorded at 20 °C (trace 1)
or after a 1-h incubation at 37 °C (trace 2) or 55 °C
(trace 3). Trace 4 shows the CD spectrum of the
55 °C sample after cooling for 1 h at 20 °C.
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sHsps typically form large oligomers that range in size between 300 and
800 kDa. Because most IbpB (>85%) did not flow through the membrane
of 100-kDa molecular mass cutoff concentrators during the purification
process, the bacterial sHsps also appeared to assemble into an
oligomeric structure. Analysis by size exclusion chromatography was
performed on a calibrated BioSep-S4000 column (Fig.
3A). Although a large fraction
of the injected material remained irreversibly bound to the gel
filtration matrix, the majority of the eluted IbpB was found as a
rather broad peak above the 2-MDa marker and near the void volume of
the column. In agreement with this result, Veinger et al.
recently reported a molecular mass in excess of 2 MDa for IbpB purified
under native conditions (29). Inspection of negative stain electron
micrographs of IbpB at room temperature revealed pronounced size
heterogeneity (Fig. 3B). The smallest particles were roughly
spherical and 10-20 nm in diameter (Fig. 3B; data not
shown). These oligomers interacted to form larger particles in the
100-200 nm range (Fig. 3C), which themselves appeared to
aggregate into µm size structures of variable shape (Fig.
3B). The existence of IbpB populations of average sizes
13 ± 3 and 190 ± 24 nm was confirmed by dynamic light
scattering after filtration through 0.22-µm filters. Larger particles
ranging in size between 400 nm and 1.7 µm were detected after a 48-h
incubation at room temperature. Because IbpB was quantitatively
recovered upon filtration through 0.22-µm membranes (data not shown),
the amorphous, soluble aggregates appear to be loose structures that arise over time from the interaction of smaller particles.

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Fig. 3.
Quaternary structure of IbpB.
Panel A, analysis of purified IbpB by size
exclusion chromatography on a BioSep S-4000 column. Absorbance at 280 nm is shown versus elution volume. The elution positions of
calibration proteins are indicated by arrows.
Panel B, low magnification (× 6,000) TEM image
of negative stain IbpB showing the three different IbpB subpopulations.
Panel C, high magnification (× 280,000) TEM
image of an IbpB particle of intermediate size.
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IbpB Reduces the Thermal Aggregation of CS and Alcohol
Dehydrogenase--
One of the hallmarks of molecular chaperones is
their ability to suppress the aggregation of thermally or chemically
unfolded substrate polypeptides. Fig.
4A shows that purified IbpB
alleviated the thermal aggregation of CS at 45 °C in a
concentration-dependent manner, indicating that the
refolded protein is functional in a standard test of molecular
chaperone function. Aggregation was suppressed by about 50% in the
presence of 6 µM IbpB, a concentration corresponding to a
CS:IbpB ratio of 1:20 based on monomers. Half-suppression of CS thermal
aggregation is observed at comparable CS:sHsp ratios (
1:11-1:15)
with pea Hsp17.7 (10) and C. elegans Hsp16.2 (7), and the
same result is achieved at the much smaller ratio of 1:3 with pea
Hsp18.1 (10) and murine Hsp25 (23). Because we were unable to
demonstrate complete suppression of CS aggregation at higher IbpB
concentrations due to the tendency of the purified protein to aggregate
when stock solutions were concentrated above 0.5 mg/ml, similar
experiments were repeated with alcohol dehydrogenase at 41.5 °C.
Fig. 4B shows that IbpB prevented the thermal aggregation of
this enzyme much more efficiently, although residual aggregation was
still observed at late time points in the presence of 5 µM IbpB (a concentration corresponding to a alcohol
dehydrogenase:IbpB ratio of 1:1 based on monomers).

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Fig. 4.
IbpB suppresses the thermal aggregation of CS
and alcohol dehydrogenase. The ability of increasing
concentrations of IbpB to suppress the aggregation of CS at 45 °C
(panel A) and alcohol dehydrogenase at 41.5 °C
(panel B) was determined by light scattering as
described under "Experimental Procedures." All concentrations are
expressed on a protomer basis.
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Temperature Affects IbpB
Conformation--
Temperature-dependent structural changes
have been proposed to play a key role in the mechanism of action of
sHsps (22). To determine how an increase in temperature would affect
the secondary structure of IbpB, far UV CD spectra were recorded after
incubating the protein for 1 h at 37 or 55 °C. Fig. 2 shows
that both [
]213 and
[
]220 decreased in a linear fashion upon
temperature increase, suggesting a progressive loss of secondary
structure and/or extensive structural rearrangements. Despite the
appearance of a shoulder centered at
230 nm in the sample incubated
at 55 °C (Fig. 2, trace 3), IbpB retained a significant
amount of
-structure at this temperature, as evidenced by the
persistence of a trough at 213 nm and the lack of the strong minimum at
200-204 nm which is characteristic of completely denatured proteins
(40). Upon cooling to 20 °C, the CD spectrum of IbpB that had been
held at 55 °C became virtually identical to that recorded at
37 °C (Fig. 2, trace 4). Thus, temperature-driven changes
in IbpB secondary structure are either only partially reversible, or
reversibility is limited to an upper range of temperature.
IbpB contains two tryptophan residues at positions 13 and 83 (25) which
are suitable to probe the conformation of the protein by intrinsic
fluorescence spectroscopy. To gain further information on
temperature-induced structural rearrangements, the maximum emission
wavelength of the tryptophan residues (
max) after
excitation at 280 nm was measured using a protein sample that had been
allowed to equilibrate for 1 h at temperatures ranging between 5 and 80 °C. Below 20 °C,
max was approximately
338.5 nm, indicative of the fact that tryptophan residues experience a
hydrophobic environment at low temperatures (Fig.
5A). Between 22 and 50 °C,
a single cooperative transition to longer wavelengths was observed with midpoint at about 32 °C. Further heating to temperatures as high as
80 °C did not cause any significant change in
max,
which remained approximately equal to 347 nm (Fig. 5A). The
reversibility of the thermally induced conformational change was next
studied by heating samples to 32, 50, or 60 °C and bringing them to
lower temperatures in a successive series of cooling steps. Fig.
5B shows that the structural modifications experienced by
IbpB between 10 and 32 °C are irreversible because the
max of the cooled samples is red shifted by about 5 nm
relative to the unheated control. However, structural changes appear to
be perfectly reversible between 32 and 50 °C (Fig. 5B,
circles) and reversible between 32 and 60 °C
(squares) despite a more pronounced hysteresis. These results are in good agreement with the CD data of Fig. 2 and suggest that reversible conformational changes only occur in an upper range of
temperatures.

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Fig. 5.
Effect of temperature on IbpB intrinsic
tryptophan fluorescence. Panel A, melting of
IbpB. Single IbpB samples (1 µM final protomer
concentration) were incubated at progressively higher temperatures for
1 h before intrinsic fluorescence spectra were recorded. The
change in tryptophan maximum emission wavelength ( max)
is plotted versus the incubation temperature.
Panel B, reversibility of thermally induced
conformational changes between 32 and 60 °C. Single IbpB samples
were heated progressively to 32 ( ), 50 ( ), or 60 °C ( )
before being cooled to lower temperatures (open symbols).
Samples were incubated for 1 h at the indicated temperatures
before each data point was collected.
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To investigate the effect of temperature increases on the quaternary
structure of IbpB, ice-cold samples of the purified protein were
injected on a calibrated BioSep-S4000 size exclusion column immediately
before or after a 1-h incubation at either 30 or 50 °C, and the
column was developed at room temperature. Fig.
6A shows that incubation at
high temperatures led to a progressive decrease in the intensity of the
high molecular mass peak and the concomitant appearance of a new
species comparable in size to typical sHsp oligomers (approximate
molecular mass, 605 kDa). To confirm these results, aliquots of
ice-cold IbpB were transferred rapidly to buffer held at various
temperatures, and the change in right angle scattering intensity was
recorded over time (Fig. 6B). In agreement with size
exclusion chromatography data, incubation between 23 and 42 °C
resulted in a progressive decrease in scattering intensity, as would be
expected if large aggregates were resolved into smaller particles. At
48 °C, however, the initial decrease in light scattering was
followed by a slow rise in intensity, and a plateau value was reached
after about 1,000 s. Because the magnitude of the light scattering
increase was relatively modest (
20%) and did not change at an IbpB
concentration of 4 µM (data not shown), this result is
best explained by an increase in the number of scattering centers at
high temperatures. Finally, we did not observe significant changes in
light scattering intensity when IbpB samples heated to a steady-state
scattering plateau at 50 °C were cooled to 15 °C over 45 min
(data not shown), indicating that IbpB does not undergo extensive
aggregation upon temperature downshift.

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Fig. 6.
Effect of temperature on IbpB quaternary
structure. Panel A, high temperature
incubation favors the dissociation of IbpB into smaller oligomers.
Ice-cold IbpB was injected onto a calibrated BioSep S-4000 column
directly or after a 1-h incubation at 30 or 50 °C. The column was
developed at room temperature. A280 nm is shown
versus elution volume. The elution positions of calibration
proteins are indicated by arrows. Panel
B, influence of the incubation temperature on IbpB light
scattering. IbpB samples (1 µM) were transferred to
thermostated cuvettes held at the indicated temperatures. The change in
light scattering intensity at 500 nm was monitored over time. Smoothed
spectra are shown.
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High Temperatures Trigger the Exposure of Hidden Hydrophobic
Domains--
The fluorescent probe bis-ANS exhibits little intrinsic
fluorescence but becomes highly fluorescent when bound to hydrophobic domains in proteins. Because molecular chaperones rely on the transient
exposure of hydrophobic patches to capture partially folded substrate
proteins, bis-ANS has been used extensively in the study of their mode
of action. Because the bis-ANS quantum yield varies with temperature,
the intensity of emission spectra collected at different temperatures
cannot be directly compared. It has, however, been shown that bis-ANS
can be incorporated in the vicinity of its binding site by exposing
protein samples to shortwave UV light (41, 42) and that the efficiency
of this process is independent of the temperature (22). To determine if
an increase in temperature would lead to the exposure of hidden hydrophobic regions, aliquots of IbpB were incubated in the presence of
an excess of bis-ANS at 23, 37, or 45 °C, and the probe was photoincorporated into the sHsp by UV irradiation. Fig.
7A shows that approximately
50% more bis-ANS was incorporated into IbpB at 37 °C relative to
23 °C and that this value raised to
80% at 45 °C. The
reversibility of the process was tested by incubating IbpB to various
temperatures for 1 h, transferring the samples to room temperature
for 5 h, and recording the bis-ANS emission spectra after the
addition of 10 µM probe. There was essentially no
variation in bis-ANS emission intensity or maximum wavelength below
55 °C. However, a progressive loss in emission intensity and a
slight red shift in
max were observed above 55 °C
(Fig. 7B). Taken together, the above results indicate that
the temperature-driven exposure of buried hydrophobic regions in IbpB
is reversible in the physiological temperature range for E. coli.

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Fig. 7.
High temperatures promote the reversible
exposure of bis-ANS binding sites to the solvent. Panel
A, increased bis-ANS photoincorporation at high
temperatures. IbpB samples (2.8 µM final protomer
concentration) were incubated for 1 h at the indicated
temperatures in the presence of 100 µM bis-ANS. The probe
was photoincorporated by exposing the samples to 254 nm UV light for 20 min. Labeled IbpB was fractionated by 15% SDS-polyacrylamide gel
electrophoresis, and the gel was photographed on a UV transilluminator
(right panel) or after staining with a Coomassie colloidal
staining kit (left panel). The fluorescence intensity of
IbpB bands normalized to the 23 °C control sample is shown below the
right panel. Lane M contains the 19.2-kDa marker.
Panel B, exposure of hydrophobic sites is
reversible. IbpB (1 µM) was incubated at the indicated
temperatures for 1 h and allowed to cool at room temperature for
5 h. Samples were supplemented with 10 µM bis-ANS
and maximum emission intensity ( ) and wavelength ( ) were
determined.
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|
Chemical Unfolding Studies--
To compare the effects of
temperature and chatropes on IbpB conformation, intrinsic tryptophan
fluorescence experiments were repeated after incubation in the presence
of up to 5 M GdnHCl. IbpB exhibited a clearly biphasic
unfolding behavior (Fig. 8A,
) and was highly sensitive to the presence of small concentrations of the denaturant. The tryptophan
max changed smoothly
from 339 to 348 nm between 0 and 1 M GdnHCl with transition
midpoint at
300 mM GdnHCl. The shape and magnitude of
the tryptophan red shift were highly similar to that observed in the
melting experiments of Fig. 5 and most likely correspond to the
exposure of one of the two tryptophan residues to the solvent. A second
cooperative transition centered at 2.3 M was detected
between 1.8 and 3 M GdnHCl. Thereafter,
max
remained unchanged, suggesting that IbpB is denatured completely
above 3 M GdnHCl and that both tryptophan residues are
solvent-exposed. The CD spectra of IbpB incubated in the presence of
1.2 M GdnHCl (e.g. after the first transition) and 4 M GdnHCl (after the second transition) were recorded
down to 215 nm because of the strong absorption of these denaturants below this wavelength. Traces 1-3 in Fig. 8B
indicate that major changes in secondary structure occur between 0 and
1.2 M GdnHCl. At the latter denaturant concentration
(trace 2), no feature is apparent between 215 and 245 nm,
and the mean ellipticity at 220 nm is
1.1 millidegrees
cm2 dmol
1. This value falls between those
obtained when IbpB is incubated at 37 and 55 °C (Fig. 2;
[
]220 =
1.6 and
0.8 millidegree
cm2 dmol
1, respectively). Assuming that
[
]220 varies linearly with the incubation
temperature between 20 and 55 °C (as suggested by the data of Fig.
2), the overall secondary structure of IbpB in the presence of 1.2 M GdnHCl should be similar to that it displays at
48.5 °C. Again, this result is in very good agreement with the
intrinsic tryptophan fluorescence results of Figs. 5 and 8A. In the presence of 4 M GdnHCl (Fig. 8B,
trace 3), the IbpB spectrum exhibits a shoulder at 223 nm
and a [
]220 value of
0.25 millidegree cm2 dmol
1 which are consistent with a
denatured conformation containing residual secondary structure.

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Fig. 8.
Chemical unfolding of IbpB.
Panel A, GdnHCl denaturation. IbpB samples (1 µM final protomer concentration) were incubated with the
indicated concentrations of GdnHCl at 23 °C for 1 h. Tryptophan
max ( ) were recorded. Samples were next supplemented
with 10 µM bis-ANS, and bis-ANS maximum emission
intensity ( ) and wavelength ( ) were determined after 5 min.
Panel B, GdnHCl-induced secondary structure
changes. The CD spectra of IbpB (0.62 µM final protomer
concentration) supplemented with no additive (trace 1), 1.2 M GdnHCl (trace 2), 4 M GdnHCl
(trace 3), or 1 M NaCl (trace 4) were
recorded after a 1-h incubation at 23 °C.
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|
To determine if hydrophobic domains become solvent-exposed during
GdnHCl denaturation (and in particular during the first transition),
IbpB treated with increasingly high concentrations of GdnHCl was
supplemented with a saturating amount of bis-ANS, and emission spectra
were recorded after excitation at 396 nm. Although the bis-ANS
fluorescence intensity (Fig. 8A,
) increased by about
25% in the presence of 300 mM GdnHCl, this change was transient, and only a slight increase in intensity was detected at
similar urea concentrations (data not shown). As the GdnHCl concentration was raised to
2 M, the bis-ANS intensity
decreased while its maximum emission wavelength (
) remained
approximately constant. These results indicate that bis-ANS binding
sites are progressively lost but that the hydrophobic character of the
remaining sites is unchanged. Above 2.5 M GdnHCl, the
further decrease in bis-ANS intensity was accompanied by a rise in
emission wavelength, a situation consistent with a loss of organized
hydrophobic surfaces as IbpB protomers unfold. Thus, although low
concentrations of GdnHCl and incubation at high temperatures appear to
exert similar effects on the secondary and tertiary structure of IbpB,
only temperature increases trigger the progressive exposure of
structured hydrophobic regions in the bacterial sHsp.
Ionic Effects--
A possible explanation for the fact that low
concentrations of GdnHCl but not urea lead to a slight increase in
bis-ANS intensity (Fig. 8A; data not shown) is that the
ionic character of GdnHCl, rather than its denaturing ability, is
responsible for the transient exposure of hydrophobic domains in the
sHsp. To investigate ionic effects in more detail, bis-ANS binding
experiments were repeated using IbpB that had been incubated with up to
1 M NaCl at room temperature. Under these conditions, the
bis-ANS emission maximum remained constant (Fig.
9,
), whereas the emission intensity increased linearly with the salt concentration (
). Thus, bis-ANS binding sites of comparable hydrophobic character are progressively gained upon salt addition. Because the tryptophan emission
max is unaffected by NaCl (Fig. 9,
) and the CD
spectrum of IbpB recorded in the presence of 1 M NaCl is
comparable to that obtained in the absence of salt (traces 1 and 4 in Fig. 8B), high salt concentrations do
not appear to induce major secondary or tertiary structural
changes.

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Fig. 9.
NaCl triggers the exposure of hidden
hydrophobic domains in IbpB. IbpB samples (1 µM
final protomer concentration) were incubated with the indicated
concentrations of NaCl for 1 h at 23 °C. Tryptophan
max ( ) were recorded. Samples were next supplemented
with 10 µM bis-ANS, and bis-ANS maximum emission
intensity ( ) and wavelength ( ) were determined after 5 min.
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To investigate possible synergism between temperature and ionic
effects, the photoincorporation experiment of Fig. 7A was repeated in the presence of 1 M NaCl. Under these
conditions, bis-ANS photoincorporation was reduced by 30-40% relative
to unsupplemented samples at all temperatures tested (data not shown).
The most likely explanation for this result is that high salt
concentrations interfere with the photooxidation process. Nevertheless,
the increase in bis-ANS photoincorporation with the incubation
temperature was comparable whether the buffer was supplemented or not
with NaCl (data not shown), suggesting that buried hydrophobic domains are not fully exposed by ionic perturbations.
 |
DISCUSSION |
Despite a low degree of homology restricted to a core region known
as the
-crystallin domain, all sHsps characterized to date share
common structural and functional features: (i) their protomers are
mostly
-pleated with little
-helical content, as judged from far
UV CD spectroscopy and crystallographic data (11); (ii) they form large
oligomers ranging in molecular mass between 200 and 800 kDa; and (iii)
they can suppress the aggregation of thermally or chemically denatured
model proteins by capturing folding intermediates on their surface (22,
23). Although E. coli IbpB behaves as a typical sHsp in
terms of function and protomer secondary structure (Figs. 2 and 4), it
exhibits uncommon size heterogeneity (Fig. 3). The basic oligomer
appears to be a roughly spherical particle approximately 15 nm in
diameter and 600 kDa in apparent molecular mass, a size and morphology
comparable to those of other sHsps (22, 23, 43, 44). Under our
experimental conditions, these oligomers interacted to form larger
structures in the 100-200-nm range, which themselves loosely
associated to yield amorphous, micrometer size clusters. The eye lens
protein
-crystallin, which has a dynamic oligomeric structure (44, 45), has also been isolated as distinct populations in the
600-900-kDa, 900-kDa-4-MDa, and greater than 10-MDa ranges (46).
However, because concentration, pH, ionic strength, and temperature of isolation have all been reported to affect the oligomeric state of
-crystallins (47, 48) and because IbpA may modulate the quaternary
structure of IbpB, it is not clear whether large IbpB oligomers also
exist in vivo.
Temperature increases have profound effects on IbpB conformation which
are characterized by: (i) a partial loss of secondary structure (Fig.
2); (ii) the progressive exposure of tryptophan residues to the solvent
(Fig. 5A); (iii) the dissociation of large aggregates into
constituent oligomers (Fig. 6); and (iv) an increase in protein surface
hydrophobicity (Fig. 7). Several lines of evidence suggest that these
temperature-driven structural modifications have a functional role.
First, IbpB is remarkably thermostable and retains a significant amount
of secondary and tertiary structure up to 55 °C (Figs. 2 and 5).
Second, conformational changes appear to be reversible between the
physiological (37 °C) and maximum growth temperature (50 °C) for
E. coli (Figs. 2 and 5B). Finally, after
incubation to temperatures as high as 55 °C, solvent-exposed hydrophobic patches are reburied upon cooling (Fig. 7B). An
increase in bis-ANS labeling with the incubation temperature has been
reported previously for
-crystallin (49, 50) and pea Hsp18.1 (22), leading to the idea that temperature-induced structural rearrangements promote the exposure of normally hidden hydrophobic regions that allow
sHsps to bind and stabilize partially folded proteins as they denature.
Our results are fully consistent with this hypothesis. Such a
"molecular sponge" function would prevent saturation of the
DnaK-DnaJ-GrpE and GroEL-GroES major chaperone systems at high
temperatures. As the temperature returns to physiological values, IbpB
would act as a "reservoir" of partially folded proteins for the
DnaK-DnaJ-GrpE system (22, 23, 29), thereby eliminating the need for
de novo synthesis of thermolabile proteins. Alternatively, bound proteins could be presented to the cellular degradation machinery
for recycling.
Although high temperatures and incubation with up to 1.2 M
GdnHCl appear to induce comparable structural changes in IbpB (Figs. 2,
5, and 8), bis-ANS binding decreases above 0.3 M GdnHCl
while it increases progressively upon temperature upshift (Fig.
7A). This result suggests that efficient exposure of
structured hydrophobic patches to the solvent involves fine structural
features that are readily disrupted at low denaturant concentrations.
The bis-ANS binding sites of pea Hsp18.1 (22) and
-crystallin (51)
have recently been identified and are located at the NH2
terminus of the
-crystallin domain which is well conserved among all
sHsps (39). Based on the observations that bis-ANS photoincorporation decreases when these sHsps are preincubated with denatured proteins (22, 51) and that the bis-ANS binding sites of
-crystallin correspond to sequences involved in substrate binding as determined by
cross-linking experiments (51), it has been proposed that the binding
sites for bis-ANS and nonnative proteins are identical in sHsps (51).
Pairwise alignments between IbpB and Hsp18.1 indicate that the
homologous IbpB domain encompasses residues 50-67 and has as its
sequence ALAGFRQEDLEIQLEGTR. The Hsp18.1 region that binds bis-ANS maps
in a surface-exposed loop that links the third and fourth
-strands
of the composite
-sandwich in each folding unit of the MjHsp16.5
crystal structure (11). Whether low concentrations of GdnHCl disrupt an
organized hydrophobic site involving this loop or affects IbpB
hydrophobicity in a different fashion remains to be determined.
An interesting feature of the melting study of Fig. 5 is that the
structural changes that take place between 10 and 32 °C are not
reversible. Although the physiological significance of this phenomenon
remains unclear, it is possible that the cell needs to maintain any
IbpB synthesized at low temperatures (e.g. during the Hsp
up-regulation period that accompanies recovery from cold shock (52,
53)) in a minimally active form. Around 32 °C, a thermally induced
rearrangement may activate the protein into a fully functional form. In
support of this hypothesis, evidence for thermal switching events have
been reported between 25 and 30 °C in the case of GroEL (54, 55) and
around 30 °C for
-crystallin (56).
Our observation that IbpB hydrophobicity increases with NaCl
concentration is consistent with the hypothesis of ionic triggering of hydrophobic surface exposure proposed by Horowitz and co-workers for
GroEL (57). In this model, hydrophobic stretches in partially folded
protein substrates are presented to the chaperonin in a charged context
to stimulate and/or lead to the optimal exposure of hidden hydrophobic
binding sites to the solvent. In agreement with this idea, we noted
that although 1 M NaCl raises the hydrophobicity of IbpB by
about 40% at 23 °C (Fig. 9), the increase in bis-ANS photoincorporation with the incubation temperature remained comparable whether or not NaCl was present in the buffer (Fig. 7 and data not
shown). NMR solution studies have revealed the presence of flexible
COOH-terminal extensions 8-18 residues in length in both
-crystallins and mouse Hsp25 (58, 59). In MjHsp16.5, the ultimate 6 COOH-terminal residues in one subunit reach out to interact with
4
and
8 of a neighboring subunit (11), a location that is in close
proximity to the bis-ANS photoincorporation site. Because both the
COOH-terminal end and putative bis-ANS/substrate binding site of IbpB
are rich in charged residues, it is tempting to hypothesize that the
bacterial sHsp possesses a flexible COOH terminus that participates in
substrate binding through ionic triggering.
It has been shown that the portion of human granulocyte ribonuclease
that binds GroEL consists of 9 hydrophobic residues, 4 positively
charged arginines and no negatively charged amino acids (60).
Similarly, binding of substrates to DnaK is determined by 4-5
hydrophobic residues flanked by basic residues, and negatively charged
residues are excluded throughout (61-63). It therefore appears that
despite large differences in structure, several families of molecular
chaperones have conserved a combination of electrostatic and
hydrophobic effects as the most effective means to recognize, bind, and
possibly exchange partially folded proteins.