The Family of Cold Shock Proteins of Bacillus
subtilis
STABILITY AND DYNAMICS IN VITRO AND IN
VIVO*
Thomas
Schindler
§¶,
Peter L.
Graumann§
**,
Dieter
Perl
,
Saufung
Ma

,
Franz X.
Schmid
, and
Mohamed A.
Marahiel
From the
Laboratorium für Biochemie,
Universität Bayreuth, 95440 Bayreuth and the
Biochemie,
Fachbereich Chemie, Hans-Meerwein-Strasse, Philipps-Universität
Marburg, 35032 Marburg, Germany
 |
ABSTRACT |
Bacillus subtilis possesses three
homologous small cold shock proteins (CSPs; CspB, CspC, CspD, sequence
identity >72%). They share a similar
-sheet structure, as shown by
circular dichroism, and have a very low conformational stability, with
CspC being the least stable. Similar to CspB, CspC and CspD unfold and
refold extremely fast in a N
U
two-state reaction with average lifetimes of only 100-150 ms for the
native state and 1-6 ms for the unfolded states at 25 °C. As a
consequence of their low stability and low kinetic protection against
unfolding, all three cold shock proteins are rapidly degraded by
proteases in vitro. Analysis of the CSP stabilities
in vivo by pulse-chase experiments revealed that CspB and
CspD are stable during logarithmic growth at 37 °C as well as after
cold shock. The cellular half-life of CspC is shortened at 37 °C,
but under cold shock conditions CspC becomes stable. The proteolytic
susceptibility of the CSPs in vitro was strongly reduced in
the presence of a nucleic acid ligand, suggesting that the observed
stabilization of CSPs in vivo is mediated by binding to
their substrate mRNA at 37 °C and, in particular, under cold shock conditions.
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INTRODUCTION |
Cold shock proteins
(CSPs)1 are found in a wide
range of Gram-positive and Gram-negative bacteria, often in families of
three (as in Bacillus subtilis) to nine (as in
Escherichia coli) highly homologous members (identity
>70%) (for review see Ref. 1). Recently, CSPs were also found in
Aquifex aeolicus (2) and Thermotoga maritima (3),
indicating that CSPs were present at the origin of bacterial divergence
and therefore are presumably an evolutionarily old class of proteins. A
CSP-homologous domain (cold shock domain) is found in many eukaryotic
nucleic acid-binding proteins (for review see Ref. 4), where it confers
specific RNA binding (5, 6). CSPs bind to single-stranded DNA and RNA
in a cooperative manner and with low sequence specificity (7-9). As a
model for the cold shock domain, the structures of CspB (B. subtilis) and CspA (E. coli) were solved, revealing
similar, compact five-stranded
-barrel folds (10-13). CSPs possess
binding sites for single-stranded nucleic acids on their antiparallel three-stranded
-sheets, which involve basic and aromatic residues. These are the so-called RNA-binding ribonucleoprotein motifs (13, 14).
CSPs were discovered originally because they are strongly induced in
response to cold shock (15), and thus they were assumed to be important
for adaptation to low temperatures. The major cold shock protein, CspA
from E. coli, was in fact shown to increase the synthesis of
several cold stress-inducible proteins after a decrease in temperature
(16). However, different members of the E. coli CSP family
are regulated differently and appear to perform functions also during
cell division or during the stationary phase (17). Recent work shows
that in B. subtilis, CSPs are essential for protein
synthesis at low as well as at optimal temperature and also during the
stationary phase (8). Moreover, CspA from E. coli
destabilizes secondary and tertiary structures in RNA in
vitro (7), which led to the assumption that CSPs facilitate initiation of translation as "RNA chaperones" by preventing the formation of stable, nonproductive secondary structures in mRNA under various conditions.
Induction of CSPs after cold shock originates from an increase in
transcription of their genes (18) and, to a greater extent, from the
stabilization of their mRNAs (19, 20). In addition, the
cspA-mRNA seems to be translated more efficiently than
the mRNAs coding for proteins that are not induced by cold stress (21). Whether the concentration of CSPs is also regulated on the level
of protein stability is not yet known. CspB from B. subtilis
exhibits a very low conformational stability, and its native form
exists in an extremely dynamic equilibrium with the unfolded form. The
average lifetime of the folded conformation is only about 100 ms under
physiological conditions. Therefore, we proposed that CspB might be
subject to rapid degradation in vivo (22).
The three members of the CSP family from B. subtilis show
sequence identities of 72-80% (Table I) and can complement each other
in vivo (8). CspB is important both at low and at optimal temperatures. CspC functions mainly at low temperature and CspD mainly
at optimal temperature.
Here we investigated the stability and the folding kinetics of CspC and
CspD of B. subtilis. These two cold shock proteins resemble
CspB in their rapid unfolding and refolding and in their low
thermodynamic stability. Marginal stability linked with high conformational dynamics might be an effective means for regulating the
cellular concentration of CSPs. To explore this possibility, we
investigated the proteolytic stabilities of CspB, CspC, and CspD
in vitro. The proteolytic susceptibility of all three
proteins is in fact high but decreases strongly in the presence of
substoichiometric amounts of single-stranded nucleic acids. In
vivo, CspB and CspD, but not CspC, were stable at 37 and at
15 °C. CspC was significantly stabilized after a cold shock. These
findings suggest that CSPs are complexed with a nucleic acid ligand in
the cell, under cold shock conditions as well as at 37 °C, and are
thereby protected against proteolytic attack.
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EXPERIMENTAL PROCEDURES |
Cloning and Heterologous Expression of cspC and cspD--
The
cspC and cspD structural genes were amplified
using polymerase chain reaction (95 °C for 30 s; 47 °C for 1 min during the first 5 cycles and 55 °C for 1 min during the
following 30 cycles; and 72 °C for 1 min) employing primers
5'-GGGGTACCCCAAGATAGTATATACTGTGTGG-3' and
5'-CCATCGATGGTTAAGCTTTTTGAACGTTAGCAGC-3' for cspC and
5'-GGGGTACCCCAGTACTAGGAGGAATTAAGC-3' and
5'-GCTCTAGAGCATCTCATCATCATGTATTGAG-3' for
cspD, respectively. The cspC fragment was cloned
into pBluescript SK(
)II vector (Stratagene) using KpnI and
ClaI restriction endonucleases; cspD was
blunt-ended using Klenow polymerase and cloned into
EcoRV-digested pBluescript SK(
)II vector. Correct
orientation of the cspC and cspD genes, respectively, was verified by sequencing. The resulting plasmids pcspC
and pcspD, respectively, were transformed into E. coli K38 pGP1-2 (23). Cells were grown in rich medium with 50 µg/ml ampicillin and 40 µg/ml kanamycin at 30 °C until reaching an optical density of 0.7, shifted to 42 °C, and further incubated for 2 h. Cells were centrifuged, resuspended in ice-cold buffer G50 (20 mM
Tris-HCl, pH 7.5, 2 mM EDTA, 2 mM
dithioerythritol), and subjected to sonication.
Purification of CspB, CspC, and CspD--
Cold shock proteins
were purified according to a general method described by
Schindelin et al. (24); however, the method was modified and
extended. Cellular extracts were applied to anion exchange
chromatography (Superperformance150-10 Fraktogel EMD TMAE-650 [S])
and separated by a 0-1 M NaCl gradient in Buffer A (6 mM Tris-HCl, pH 6.8, 2 mM dithioerythritol, 2 mM EDTA). CspB and CspC eluted at a concentration of 300 mM NaCl and CspD at 360 mM NaCl. Because at 400 mM NaCl several small proteins of E. coli
eluted, only the first fractions containing CspD were taken for further
purification. CSP-enriched fractions were pooled, freeze-dried, and
dissolved in Buffer C (50 mM Tris-HCl, pH 7.8, 100 mM KCl). Size exclusion chromatography on HiLoad 16/60
SuperdexTM 75 prep grade using Buffer C and a flow rate of
1 ml/min resulted in peak elution of CspB after 86 ml, of CspC after 87 ml, and of CspD after 83 ml. Fractions that were >95% pure according
to SDS-PAGE were pooled and subjected to hydrophobic interaction chromatography (butyl-Sepharose). After the addition of 0.67 volume of
saturated ammonium sulfate, CSPs were loaded onto the matrix equilibrated in 40% ammonium sulfate, 50 mM potassium
phosphate buffer, pH 7.0. The proteins were eluted with 50 mM potassium phosphate buffer, pH 7.0. The
protein-containing fractions were pooled, dialyzed against decreasing
concentrations of ammonium hydrogen carbonate, and lyophilized. Protein
concentrations were assayed by the method of Bradford (25) and by
absorption measurements. Molar absorption coefficients at 280 nm for
CspB, CspC, and CspD were calculated from their corresponding amino
acid composition to be 5690, 5690, and 6970 M
1 cm
1, respectively.
Circular Dichroism Spectroscopy--
Circular dichroism in the
far- and near-UV range was measured at 25 °C in 20 mM
potassium phosphate buffer, pH 7.0, for the native protein or in 7.2 M urea in 0.1 M sodium cacodylate HCl, pH 7.0, for the unfolded protein. Data were collected with an 0.2-mm step
resolution, a time constant of 1 s, and a scan speed of 20 nm/min,
using a Jasco J600 spectropolarimeter and cuvettes of a path length of
0.05 cm for far-UV and 1 cm for near-UV range.
Urea-induced Unfolding Transitions--
Samples of CspB, CspC,
and CspD were incubated at a concentration of 1.5 µM for
1 h at 25 °C in the presence of 0.1 M sodium cacodylate HCl, pH 7.0, and varying concentrations of urea.
Fluorescence of samples was measured at 343 nm with a 5 nm bandwidth.
Excitation wavelength was 280 nm (3 nm bandwidth). Experimental data
were analyzed by assuming that the transition between the folded and unfolded conformation is a U
N two-state reaction and that the fluorescence emissions of the native and the unfolded protein depend
linearly on the urea concentrations (26). A nonlinear least squares fit
of the experimental data was used to obtain the Gibbs free energy of
stabilization,
Gstab, as a function of the
urea concentration (27). CspC and, to a lesser extent, CspD showed a
slight tendency to stick to the walls of quartz cuvettes at very low
concentrations of urea, leading to a higher error in the measured
fluorescence under those conditions. The first data point(s) were
therefore not used for the nonlinear least squares fit.
Stopped-flow Kinetic Experiments--
A DX17MV sequential mixing
stopped-flow spectrometer (Applied Photophysics, Leatherhead, U.K.)
was used for all kinetic measurements. The folding kinetics were
followed by the change in fluorescence above 300 nm after excitation at
280 nm (10 nm bandwidth). The zero time point and the dead time of
mixing were determined using the procedure suggested by Tonomura
et al. (28). All unfolding and refolding experiments were
carried out in 0.1 M sodium cacodylate HCl, pH 7.0. To
initiate unfolding, typically 16 µM native protein was
diluted 11-fold with buffers of varying urea concentrations to give
final urea concentrations between 2.5 and 8.0 M. To
initiate refolding, 16 µM unfolded protein in 5.9 M (CspC) or 7.6 M (CspB, CspD) urea was diluted
11-fold with aqueous buffer or with urea solutions of varying
concentrations to give the desired final urea concentration. Kinetics
were measured eight times under identical conditions, averaged, and
analyzed as monoexponential functions using software provided by
Applied Photophysics. Folding kinetics were analyzed on the basis of a
U
N two-state folding reaction, where the measured rate constant
is equal to the sum of the microscopic rate constants for refolding
(kUN) and unfolding
(kNU). Log kUN and log
kNU are assumed to vary linearly with the
concentration of urea. Values for the equilibrium constant
KStab = [N]/[U] as a function of urea
concentration were calculated from
kUN/kNU.
Protease Digestion Assay--
40 µM purified CspB,
CspC, CspD, or hen egg white lysozyme were incubated in buffer (20 mM Tris-HCl, 5 mM MgCl2, 50 mM NaCl, pH 8.6) containing 67 µg/ml trypsin at
25 °C in the presence or absence of 20 µM
54YB+ single-stranded DNA
(5'-GAATTCGCAGACGTGGGAATCCTACTGATTGGCCAAGGTGCTGGTGGTGTGTGG-3'). At various times (2, 4, 6, 10, 15, 30, 60, and 120 min), aliquots were withdrawn and subjected to SDS-PAGE. The Coomassie-stained gels
were analyzed using a digital video camera (Gelprint 2000i from
MWG-Biotech, Ebersberg, Germany), and the remaining amounts of
full-length CSPs were quantified by using ONE-Dscan, version 1.0 (Scanalytics, Billerica, MA).
Pulse-Chase Labeling of Cellular Proteins and Two-dimensional Gel
Electrophoresis--
B. subtilis JH642 was grown in M9
minimal medium complemented with 0.01% yeast extract at 37 °C (29)
to an optical density (600 nm) of 0.45 and labeled with 20 µCi of
[35S]methionine for 10 min or shifted to 15 °C for 5 min and then labeled for 30 min. A fraction was mixed with 0.1 volume
of stop solution (10 mM Tris-HCl, pH 7.5, 1 mg/ml
chloramphenicol) and incubated on ice before centrifugation. After
washing with wash solution (10 mM Tris-HCl, pH 7.5, 0.1 mg/ml chloramphenicol), cells were frozen. The remaining culture was
chased with a 50,000-fold excess of cold methionine. Samples were
withdrawn after 10, 30, 60, 90, 120, and 180 min (37 °C) and after
1, 4, 8, 12, 24, and 36 h (15 °C). Cells were resuspended in
Buffer B (10 mM Tris-HCl, pH 7.4, 1 mg/ml MgCl2 × 6H2O; 50 µg/ml RNase A, 50 µg/ml DNase I, 100 µg/ml lysozyme; 243 µg/ml phenylmethylsulfonyl fluoride) and
disrupted by sonication. After centrifugation, the supernatants were
assayed for protein concentration according to Bradford (25), lyophilized, and resuspended in Buffer E (0.5 g of dithiothreitol, 2 g of CHAPS, 12.7 mg of phenylmethylsulfonyl fluoride, 27 g
of urea, 2.5 ml of ampholytes, pH 3-10, and 30 ml of distilled
H2O (52 ml, final volume)) to yield equal concentrations of
total proteins. Two-dimensional gel electrophoresis was performed as described by Graumann et al. (29).
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RESULTS |
Overproduction and Purification of CspC and CspD--
CspC and
CspD were overproduced employing the method of Tabor and Richardson
(23) and purified by a method based on the procedure of Schindelin
et al. (24) (see "Experimental Procedures"). It is
apparent that CspC (7.2 kDa) migrates at a molecular mass of 8 kDa
(Fig. 1, lane 5) and CspB
migrates at 9 kDa (not shown), whereas CspD (7.2 kDa) migrates at 13 kDa (Fig. 1, lane 9). This discrepancy is caused by the
effects of SDS, because in native PAGE, CspD (pI 4.32) and CspB (pI
4.31) comigrate, whereas CspC migrates more slowly because of the
reduced negative charge (pI 4.53) (data not shown). UV spectra of
purified protein fractions revealed absorbance maxima at 260 rather
than 280 nm, which indicates that nucleic acids were still present.
These residual, bound nucleic acids could be removed by hydrophobic
interaction chromatography, resulting in UV spectra with shapes as
expected from the chromophore composition of the CSPs and maxima at 280 nm (data not shown).

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Fig. 1.
A Coomassie-stained 10% SDS-PAGE.
Lanes 1 and 10, 10-kDa protein ladder (Bio-Rad);
lanes 2 and 6, whole cell extracts from K38
(pGP1-2 pcspC (lane 2) or pcspD (lane 6)) grown
at 30 °C; lanes 3 and 7, whole cell extract
from K38 (pGP1-2 pcspC (lane 3) or pcspD (lane
7)) shifted to 42 °C for 2 h; lanes 4 and
8, fractions containing peak elution of CspC and CspD,
respectively, after anion exchange chromatography; lanes 5 and 9, fractions of CspC and CspD, respectively, after
size exclusion chromatography.
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Structure and in Vitro Stability of CspC and CspD--
The
circular dichroism (CD) spectra of CspB, CspC, and CspD in the far- and
near-UV region (Fig. 2) are very similar,
indicating that the purified CSPs were properly folded and that, as
expected from the high sequence homology (Table
I), CspC and CspD resemble CspB in their
three-dimensional structure. The ellipticity in the far-UV range is low
because the CSPs lack
-helices. The CD maximum at 197 nm (Fig.
2A) originates from the antiparallel
-sheet structure of
the CSPs. This maximum is slightly reduced in the CD spectrum of CspC
because CspC is the least stable protein and is not completely folded
under the conditions of Fig. 2 (see below). The CD in this region is
extremely sensitive to non-native molecules because these molecules
possess a pronounced minimum at 200 nm. CspD has a Phe residue at
position 38 instead of a Tyr (as in CspB; see Table I). The minor
differences in the near-UV CD may originate from this difference in
sequence. Nevertheless, the close similarity among the CD spectra in
Fig. 2 indicates that the differences in sequence between CspB, CspC,
and CspD do not change the overall folded conformation.

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Fig. 2.
Circular dichroism spectra in far-UV
(A) and near-UV (B) range of CspB
( ), CspC (- - -), and CspD from B. subtilis (- -) recorded at 25 °C in 20 mM
potassium phosphate buffer. Dotted line shows control
spectrum of CspB denatured in 7.2 M urea. The protein
concentration was typically 40 µM.
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Table I
Sequence alignment of the CSP protein family in B. subtilis
CspB is the reference molecule for this sequence alignment. Colons mark
identical positions and points mark conservative exchanges.
Ribonucleoprotein (RNP) motifs are in bold.
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The urea-induced equilibrium unfolding transitions of the three CSPs,
as monitored by the decrease in fluorescence emission of the single Trp
residue at position 8, are shown in Fig.
3. With increasing concentrations of
urea, the fluorescence intensities of all three CSPs decrease in single
cooperative transitions. CspB and CspD show very similar stabilities.
The midpoints of their unfolding transitions are at 3.9 M
urea (CspB) and 4.1 M urea (CspD) (Table
II). The conformational stability of CspC
is considerably lower. The midpoint of its transition is at 2 M urea (Fig. 3; Table II). For CspB, we have previously
shown that the change in fluorescence reflects global unfolding of the
-barrel (22). The transitions in Fig. 3 were analyzed according to
the linear two-state model (26). The resulting values for the Gibbs free energy of stabilization in the absence of a denaturing agent (
GStab(H2O)) are
11.4 kJ/mol
for CspB and
10.2 kJ/mol for CspD (Table II), although the midpoint
of the unfolding transition is slightly higher for CspD. This is a
consequence of the small difference in cooperativity (m = 
GStab/
[urea]) between the transition of CspD (m = 2.5 kJ/mol) and CspB
(m = 2.9 kJ/mol). CspC is significantly less
stable than CspB and CspD (
GStab(H2O) = 6.0 kJ/mol; Table
II). Thus, under native conditions, 8% of all CspC molecules are
expected to be in an unfolded state, compared with 1% for CspB. The
high percentage of unfolded CspC molecules was also apparent in its
far-UV CD spectrum (see above).

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Fig. 3.
Urea-induced unfolding transitions of CspB
( ), CspC ( ), and CspD ( ) in 0.1 M sodium
cacodylate, pH 7.0, at 25 °C followed by change in fluorescence
emission at 343 nm after excitation at 280 nm (CspC, CspD) or 295 nm
(CspB). The protein concentrations were 1.5 µM for
CspC and CspD and 13.5 µM for CspB. The continuous
lines represent least squares fit analyses of the experimental
data based on a two-state U N unfolding mechanism. For resulting
values for cooperativity (m) and Gibbs free energy
( GStab), see Table II. The data points of
measured fluorescence of CspC and CspD at 0 M urea
concentrations were not included in the fit (see "Experimental
Procedures"). rel., relative.
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Table II
Thermodynamic parameters for folding of CspB, CspC, and CspD
All data were determined at 25 °C in the presence of 0.1 M sodium cacodylate HCl, pH 7.0.
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Conserved Two-state Folding Mechanism for CSPs--
The unfolding
and refolding kinetics of CspC and CspD were measured after rapid
11-fold dilutions of the native and unfolded proteins, respectively, to
various final concentrations of urea in a stopped-flow apparatus. As in
the equilibrium unfolding experiments (Fig. 3), the folding kinetics
were monitored by fluorescence. All kinetic curves could be well
described by monoexponential functions. As observed previously for
CspB, the unfolding of CspC and the unfolding of CspD are reversible
two-state processes under all conditions, and identical values were
obtained for the measured rate constants
in unfolding
and refolding experiments performed at the same concentration of
denaturant in the transition regions (Fig.
4, A and C).
Moreover, identical final fluorescence values were reached,
irrespective of whether the kinetics started from the totally unfolded
or the native protein, and these final values followed the equilibrium
unfolding transitions (cf. Figs. 3 and 4, B and
D). The initial fluorescence values of unfolding trace the
base line for the native protein, and the initial values of refolding
trace the base line of the unfolded protein (Fig. 4, B and
D). There is no indication for rapid changes in fluorescence during the experimental dead time, and therefore partly folded intermediates seem not to accumulate before the rate-limiting event of
folding. As found previously for CspB (22), the urea dependence of the
apparent rate constants of folding (
) of both CspC and
CspD, shown in Fig. 4, A and C, are very well
described by the two-state model (U
N), where
equals
the sum of the microscopic rate constant of unfolding
(kNU) and refolding (kUN) (
= kUN
+kNU). The refolding kinetics of CspD almost
coincide with those of CspB (the dotted line in Fig.
4C), and the extrapolated rate constant of folding in the
absence of urea (1080 s
1) is virtually identical for the
two proteins (Table II). CspC refolds more slowly than CspB and CspD
(Fig. 4C). The rate constant of its refolding at 0 M urea is 6.5-fold decreased to 170 s
1.

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Fig. 4.
Dependence of unfolding ( , ) and
refolding ( , ) kinetics of CspC (A, B) and CspD
(C, D) on concentration of urea in 0.1 M
sodium cacodylate, pH 7.0, at 25 °C. In A and
C, the apparent rate constant is plotted as the function of
urea concentration. Continuous lines in
A and C represent the fit of the data according
to a kinetic two-state mechanism, and broken lines give the
calculated dependence of microscopic constants of velocity
(V) of unfolding (kUN) and refolding
(kUN) on concentration of urea (Table II).
Kinetic analysis of CspB, taken from Ref. 27, is shown by dotted
lines. B and D are plots of start ( , )
and end points ( , ) of kinetics. All points are averages of at
least eight measurements. For CspD (panel D), the start and
end points were also analyzed according to a two-state model, resulting
in values for GStab(H2O) and
m of 11.8 kJ/mol and 2.77 kJ/(mol·M),
respectively. At 0 M urea, already 8% of the CspC
molecules were unfolded, such that values for unfolding of CspC do not
reflect the base line of the native protein.
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For all three proteins, the microscopic rate constants of unfolding are
only marginally dependent on urea concentration, and they all
extrapolate to a common value of 12 s
1 under native
conditions in the absence of urea (Table II). This shows that the
remarkably high frequency of unfolding under physiological conditions
that was first noted for CspB is a conserved property of all three cold
shock proteins.
The kinetic and equilibrium data (compared in Table II) are mutually
consistent. This consistency indicates that the linear two-state model
is an excellent representation for the folding transitions of all three
cold shock proteins and that the fluorescence change in the equilibrium
unfolding (Fig. 3) indeed reflects global unfolding.
Proteolytic Susceptibility of CSPs in Vitro--
The low
thermodynamic stability combined with the high conformational dynamics
of all three CSPs should render them very sensitive to proteolytic
digestion. To examine this possibility, we exposed CspB, CspC, CspD,
and hen egg white lysozyme in a control experiment to 67 µg/ml
trypsin at 25 °C. After various time intervals, samples were assayed
by SDS-PAGE. The decrease of the intensity of the band for the intact
protein with time is shown in Fig.
5A for CspB. All three CSPs
were rapidly cleaved by trypsin, with half-times shorter than 5 min
(Fig. 6). The half-time of degradation of
lysozyme exceeded 2 h under the same conditions (Fig.
6A, inset). The protein with the lowest
thermodynamic stability, CspC, is degraded most rapidly (Fig.
6C), as expected.

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Fig. 5.
Tryptic digest of CspB (25 °C in 20 mM Tris-HCl, 5 mM MgCl2, 6%
glycerol, pH 8.6, 40 µM CspB,
67 µg/ml trypsin) in the absence
(A) or the presence (B) of the
single-stranded DNA 54YB+ ligand (half the amount of CspB)
followed by Coomassie-stained SDS-PAGE. Aliquots were withdrawn at
indicated times. The reaction was stopped by the addition of SDS sample
buffer and immediate transfer to 95 °C. K, control (40 µM CspB, no trypsin).
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Fig. 6.
Time course of tryptic digest of CSPs
(40 µM), CspB (A),
CspD (B), and CspC (C) in the absence
( ) or the presence ( ) of 20 µM 54YB+ single-stranded
DNA. Aliquots were withdrawn at the indicated time points and
analyzed by SDS-PAGE (see Fig. 5). The kinetics of hen egg white
lysozyme are shown as inset in panel A.
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The cold shock proteins bind with high affinity to single-stranded DNA
molecules containing ATTGG sequences, such as the 54YB+
oligonucleotide (30). This DNA ligand protected the CSPs strongly against cleavage by trypsin (Figs. 5B and 6) although it was
present only in a substoichiometric amount (20 µM)
relative to the CSPs (40 µM) during the proteolysis. The
half-lives of CspB and CspD increased more than 10-fold (Fig. 6,
A and B). The stabilization of CspC was less
pronounced but still significant (Fig. 6C), which is in
agreement with earlier findings that CspC has a markedly reduced
affinity for single-stranded DNA and RNA in vitro (8). The
half-life of lysozyme did not change in the presence of
54YB+ (inset in Fig. 6A).
Stability of CSPs in Vivo--
The stabilities of CspB, CspC, and
CspD in vivo in B. subtilis were determined by
pulse-chase experiments. In minimal medium at 37 °C, the doubling
time of JH642 was 108 min, which, after a cold shock to 15 °C,
increased to 24 h (data not shown). From Fig.
7 it is apparent that at 37 °C, the
amount of CspB and CspD was approximately 50% after 120 min compared
with 0 min after pulse labeling (panels A and B).
Thus, the half-lives of CspB and CspD correspond to the doubling time
of logarithmically growing cells, similar to the majority of proteins
synthesized during this period of growth (not shown). Representative
for this group of proteins that are not subject to detectable
degradation are GsiB, GroES, and spot 4b. Only a few proteins, such as
PPiB, Hpr, and CheY, showed slight degradation with a half-life of
approximately 90 min (Fig. 7, A and B). In
contrast, CspC, Csi5, and spot 7b were no longer detectable after 120 min of chase (contrarily to 60 min after chase, not shown) and showed a
half-life of approximately 75 min. Therefore, these proteins are
detectably degraded during logarithmic growth.

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Fig. 7.
Lower acidic part of autoradiograms from
13.5% second dimension (SDS-PAGE) two-dimensional gels
containing cellular extracts from
[35S]methionine-labeled B. subtilis
JH642. A and B, grown at 37 °C until
midexponential phase (optical density at 600 nm
(A600) = 0.45). A, pulsed for 10 min;
B, chased for 2 h. C and D,
shifted at A600 = 0.45 from 37 to 15 °C.
C, pulsed for 30 min; D, chased for 24 h.
Equal amounts of proteins were subjected to two-dimensional gel
electrophoresis. Identification of spots is taken from Refs. 29 and 34.
CspB is present as formylated (more acidic spot) and deformylated
protein under cold shock conditions (C and
D).
|
|
After cold shock, synthesis of CspB, CspC, CspD, Csi5, CheY, and Hpr
increased, whereas that of GsiB, GroES, and spot 7b decreased (Fig. 7,
A and C) as reported previously (29). Twenty-four
hours after the chase (corresponding to one doubling time), the levels of all cold stress-induced proteins (CIPs, including CSPs) and of most
other proteins were about 50% of the levels after the pulse (Fig. 7,
C and D). Degradation of CSPs was still not
detectable 36 h after the pulse (not shown). These findings show
that with the exception of a few proteins (such as CheY, Fig. 7,
C and D), general protein degradation is low
after cold shock to 15 °C.
The results show that in B. subtilis, CspB and CspD are
stable at 37 °C as well as under cold shock conditions. CspC,
however, is completely stable only after a drop in temperature to
15 °C. Thus, in contrast to their low barrier against unfolding and
their pronounced protease sensitivity in vitro, CspB and
CspD are stable molecules in vivo, even in the absence of a
cold shock. This agrees with the finding that CSPs are essential at low
temperatures as well as at 37 °C (8). We showed that CSPs are
stabilized in the presence of a limiting amount of a nucleic acid
ligand in vitro, and therefore, we propose that in
vivo CSPs exist in a tight complex with their biological ligand
(probably mRNA) and are thereby stabilized. This hypothesis is
possible because mRNA is highly abundant in the bacterial cell and
because CSPs bind cooperatively and rather nonspecifically to RNA
in vitro (7, 8).
 |
DISCUSSION |
The CSPs of B. subtilis (CspB, CspC, CspD, sequence
identity 71-78%) share a common three-dimensional structure (10-13).
CspB folds extremely rapidly and reversibly without any intermediate steps and has a very low kinetic barrier to unfolding (22). Similarly
rapid two-state unfolding and refolding kinetics were found also for
CspC and CspD in this work. These properties are likely to be general
features of CSPs, because recently they were also found for CSPs from
other mesophilic, thermophilic, and hyperthermophilic bacteria (3,
31).
CspB and CspD of B. subtilis show virtually identical low
thermodynamic stabilities of
10 to
11 kJ/mol, and the stability of
CspC is further reduced to only
6 kJ/mol. As a result, 8% of CspC
molecules are expected to be denatured even under native conditions,
compared with 1% for CspB. Interestingly, in CspC, Pro-58 of CspB and
CspD is replaced by alanine (Table I). This substitution probably
contributes to the reduced conformational stability of CspC because Pro
residues reduce the entropy of the unfolded protein. All three CSPs
feature a highly dynamic native conformation. CspB and CspD show almost
identical rates of unfolding (12 s
1) and refolding (1000 s
1). Refolding of CspC is decelerated 8-fold, which
reflects its lowered stability, but it also unfolds at a rate of 12 s
1 under native conditions.
Consistent with their low thermodynamic stability and high frequency of
unfolding, we found that all three CSPs of B. subtilis are
excellent substrates for proteases in vitro. Their
susceptibilities parallel the rank order of conformational stability;
CspD is slightly more resistant to proteolytic degradation than CspB,
whereas CspC with its particularly low thermodynamic stability is
degraded most rapidly.
The pulse-chase experiments in Fig. 7 show that, despite this rapid
proteolysis of the purified proteins in vitro, CspB and CspD
are not detectably degraded during logarithmic growth of B. subtilis at 37 °C. This result is in agreement with the finding that the CSPs have an essential function at optimal temperature (8).
CspC is degraded at 37 °C with a half-life of about 75 min. CspB and
CspD are readily detectable in Coomassie stained SDS-PAGE from cells
grown at 37 °C, whereas CspC is only faintly visible by Western
blotting at this temperature (data not shown). After cold shock, all
three CSPs were stable for at least 36 h (>1 doubling time).
Thus, in the absence of a detectable turnover, enhanced synthesis
following cold shock (29) leads to an increase in the intracellular
concentrations of CSPs. The different in vivo CSP
stabilities agree with earlier findings that the function of CspC is
more important at low temperatures, whereas CspB performs its function,
which is not transient, at optimal growth temperature as well as under
cold shock conditions (8).
CspA of E. coli has also been reported to be stable after
cold shock at 10 °C. For its stability at 37 °C, contradictory
results have been obtained. Pulse labeling of cold-shocked cells
followed by a chase at low temperature and subsequent shift to optimal growth temperature was reported to produce CspA that could still be
detected after several hours at 37 °C (32). On the other hand,
induction of cspA from a heterologous promoter and
pulse-chase of CspA at 37 °C resulted in protein with a rather short
half-life (7 min under the experimental conditions employed) (19).
Induction of CSPs by cold shock originates from increased transcription
of csp genes and from stabilization of csp
mRNAs (18-20). Our data show that the increase in the CspC
concentration following a decrease in temperature is at least partly
mediated by enhanced protein stability, revealing that CSP induction
can also be achieved at the level of protein stability. The
conformational stability of CspC is considerably lower than that of
CspB and CspD and may account for efficient degradation of CspC at
37 °C. Following cold shock, the half-life of CspC may be prolonged
by a reduction of the proteolytic activity in the cell and/or a higher
conformational stability of CspC at low temperatures.
The CSPs are probably stabilized in vivo by the cooperative
binding to their mRNA substrates (7, 8). Indeed, CspB and CspD were
strongly stabilized against proteolysis in vitro by a
single-stranded 54-mer DNA ligand. It is remarkable that the rates of
proteolysis of CspB and CspD decreased more than 10-fold when the
concentration of the DNA ligand was only half the CSP concentration.
This finding suggests that several CSP molecules bind with strong
positive cooperativity to a single-stranded DNA molecule and thus
become protected against proteolytic cleavage. This binding by CSPs
destabilizes double-stranded regions of RNA and thus increases their
susceptibility to hydrolysis (7). Our results strongly suggest that
even in the absence of a cold shock, CSPs are permanently complexed
with nucleic acids, most likely with mRNA. On the other hand, the
cellular concentration of CSPs can be efficiently controlled by the
degradation of excess CSPs that are not bound to mRNA. A tight
regulation of CSP levels appears to be important in bacteria, because
heterologous induction of CspB in E. coli profoundly alters
the pattern of protein synthesis and leads to a marked decrease in
growth rate (33).
 |
ACKNOWLEDGEMENTS |
We thank Gabriele Schimpf-Weihland for
continuous technical support and the members of the Schmid and Marahiel
laboratories for many discussions. T. S. thanks the Jamaican national
soccer team for determining the first authorship in the game against the Japanese national team.
 |
FOOTNOTES |
*
This work was supported by the Deutsche
Forschungsgemeinschaft (SFB 395 and Schm 444/12-1), the Human Frontier
Science Program, and the Fonds der Chemischen Industrie.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.
§
These authors contributed equally to this work.
¶
Present address: The Rockefeller University, P. O. Box 3, Laboratories of Molecular Biophysics, 1230 York Ave., New York, NY 10021.
**
To whom correspondence should be addressed: Biological
Laboratories, Harvard University, Cambridge, MA 02138. Tel.:
617-495-0532; Fax: 617-495-9300; E-mail: graumann{at}fas.harvard.edu.

Present address: Imperial College, Wolfson Laboratories, Dept.
of Biochemistry, London SW7 2AZ, Great Britain.
The abbreviations used are:
CSP, cold shock
protein; PAGE, polyacrylamide gel electrophoresis; CD, circular
dichroism; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
 |
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