We studied the oligomeric features of two classes of bacterial sHsps by size exclusion chromatography and nanoelectro-spray mass spectrometry.Proteins of both classes formed large comple
Trang 1Temperature and concentration-controlled dynamics of rhizobial
small heat shock proteins
Nicolas Lentze1, J Andrew Aquilina2, Mareike Lindbauer1, Carol V Robinson2and Franz Narberhaus1,3 1
Institut fu¨r Mikrobiologie, Eidgeno¨ssische Technische Hochschule, Zu¨rich, Switzerland;2Department of Chemistry, Cambridge University, Cambridge, UK and3Lehrstuhl fu¨r Biologie der Mikroorganismen, Ruhr-Universita¨t Bochum, Bochum, Germany
A hallmark of a-crystallin-type small heat shock proteins
(sHsps) is their highly dynamic oligomeric structure which
promotes intermolecular interactions involved in subunit
exchange and substrate binding (chaperone-like activity)
We studied the oligomeric features of two classes of bacterial
sHsps by size exclusion chromatography and
nanoelectro-spray mass spectrometry.Proteins of both classes formed
large complexes that rapidly dissociated upon dilution and
at physiologically relevant heat shock temperatures.As the
secondary structure was not perturbed, temperature- and
concentration-dependent dissociations were fully reversible
Complexes formed between sHsps and the model substrate citrate synthase were stable and exceeded the size of sHsp oligomers.Small Hsps, mutated in a highly conserved gly-cine residue at the C-terminal end of the a-crystallin domain, formed labile complexes that disassembled more readily than the corresponding wild-type proteins.Reduced com-plex stability coincided with reduced chaperone activity Keywords: a-crystallin; chaperone; oligomerization; sHsp; small heat shock protein
Small heat shock proteins (sHsps or a-Hsps) form a distinct
family of molecular chaperones.They are found in most
organisms and are typically induced upon stress [1–3].Most
sHsps tested, to date, prevent thermal- or chemical-induced
aggregation of a variety of model substrates in vitro by
binding to unfolding intermediates.The resulting sHsp–
substrate complexes are very large and stable [4–8].In
contrast to other chaperones, the chaperone activity of
sHsps is generally believed to be ATP-independent.As a
consequence, sHsps lack refolding activity.Upon binding
to sHsps, partially denatured proteins are maintained in a
refoldable state, promoting subsequent refolding in
cooper-ation with ATP-dependent chaperones [6–8].Recently, it
was shown that the release and subsequent refolding of
sHsp-bound substrates is efficiently mediated by ClpB/
DnaK [9]
sHsps exhibit a low molecular mass of 12–43 kDa.A
typical feature is the formation of large oligomeric
com-plexes, often exceeding 500 kDa [10].Both poly- and
monodisperse quaternary structures have been reported
for eukaryotic and prokaryotic sHsps [11–14].Two sHsp
structures were solved by X-ray crystallography:
Methano-coccus jannaschii Hsp16.5 was shown to form a hollow,
football-like 24-mer structure, whereas wheat Hsp16.9 is
organized as a dodecameric double disk [15,16].Despite the difference in the quaternary structure, the monomeric fold
of both sHsps is very similar and, in each case, dimers are probably the main building units of the complex.Several studies have shown that sHsp complexes are very dynamic
in terms of intersubunit exchange and dissociation/reasso-ciation processes [17–21].The dynamic behaviour is thought
to be interrelated to substrate interaction.Exposure of hydrophobic patches (which are putative substrate-binding sites) upon heating, was demonstrated for several sHsps [5,14,22]
Bacterial sHsps can be divided into two classes – A and B – according to their primary structure [23,24].Like other rhizobial species, the nitrogen-fixing soybean symbiont, Bradyrhizobiumjaponicum, contains both classes.The recently established total genome sequence revealed 11 sHsp genes, seven coding for class A and four coding for class B proteins [25].Members of both classes were shown
to form large complexes and to protect the model substrate citrate synthase (CS) from thermal aggregation in vitro [26] Formation of mixed oligomers is restricted to members
of the same class.Recently, we have shown that class A complexes of HspH are in a concentration-dependent equilibrium with smaller subspecies [27].HspH exchanged subunits at temperatures well below those needed for heat-induced sHsp expression in B japonicum.The oligomeric state of HspH did not seem to respond to heat treatment
as preincubation up to 55C did not change migration on a gel-filtration column when chromatography was performed
at room temperature
There is strong evidence that complex size and chaper-one activity are linked.Mutations, and N- and C-terminal truncations of B japonicum class A and class B proteins, which led to smaller complexes, were accompanied by decreased chaperone activity [27,28].The only exception was a mutation of a highly conserved glycine (G114) in the
Correspondence to F.Narberhaus, Ruhr-Universita¨t Bochum,
Lehrstuhl fu¨r Biologie der Mikroorganismen, Geba¨ude
NDEF 06/783, D-44780 Bochum, Germany.
Fax: + 49 234 321 4620, Tel.: + 49 234 322 3100,
E-mail: franz.narberhaus@rub.de
Abbreviations: CS, citrate synthase; nanoESI-MS, nanoelectrospray
mass spectrometry; SEC, size exclusion chromatography;
sHsps, small heat shock proteins.
(Received 8 March 2004, revised 19 April 2004,
accepted 22 April 2004)
Trang 2central a-crystallin domain (named after the human lens
protein, a-crystallin) of HspH.The G114A protein was
wild-type-like in terms of complex formation and
hetero-oligomerization, but impaired in chaperone activity.Thus,
it was speculated that G114 might be involved in substrate
binding [27]
In the present study, we investigated the influence of
temperature and concentration on sHsp complexes by two
different techniques, namely size exclusion
chromatogra-phy (SEC) and electrospray ionization mass spectrometry
(ESI-MS).Several recent studies have demonstrated that
ESI-MS is a powerful technique to study large,
noncova-lent protein complexes [29,30].We show, in the present
study, that the size of B japonicum class A and class B
sHsp complexes is adjusted to the ambient temperature
and to protein concentration, and that mutant proteins
equivalent to HspH(G114A) form unstable complexes
Materials and methods
Bacterial strains and plasmids
Escherichia coli DH5a, grown in LB (Luria–Bertani)
medium, was used for recombinant DNA techniques,
according to standard protocols [31].Construction of
pET-derived plasmids for the production of B japonicum
HspBHis (pRJ5304), HspHHis (pRJ5307), HspCHis
(pRJ5305) and HspFHis (pRJ5306), has been described
previously [26].The HspBHisderivative, HspBHis(G116A)
(pRJ5691), was constructed by primer-based site-directed
mutagenesis (Quick-ChangeTM Site-Directed Mutagenesis
Kit; Stratagene), using pRJ5304 as the
template.Mutagen-esis was performed according to the manufacturer’s
instruc-tions.The correct nucleotide sequence was confirmed by
automated DNA sequencing.All sHsps used in this study
carry a C-terminal hexahistidine tag
Protein expression and purification
E coli BL21(DE3)pLysS was freshly transformed with
expression plasmids.Inoculated cultures were grown at
30C to an attenuance (D), at 600 nm, of 0.6, before
expression was induced by the addition of isopropyl
thio-b-D-galactoside (0.5 mM).After 2 h, cells were harvested
and resuspended in binding buffer [500 mM KCl, 20 mM
Tris/HCl, 5 mM imidazole, 10% (v/v) glycerol, pH 7.9]
containing 1 mM phenylmethanesulfonyl fluoride and
10 lgÆmL)1DNase I.Cells were lysed in a French pressure
cell at 1000 p.s.i Soluble crude extracts were obtained after
centrifugation at 12 000 g for 30 min at 4C.Protein
purification was carried out under native conditions by
affinity chromatography using Ni-nitrilotriacetic acid resin
(Qiagen), as described previously [26,27].Purified proteins,
not used within a few days, were stored at)20 C or )80 C
in elution buffer [500 mMKCl, 20 mMTris/HCl, 250 mM
imidazole, 10% (v/v) glycerol, pH 7.9]
Chaperone activity assay
The ability of HspB and HspB(G116A) to protect CS
from thermally induced aggregation was monitored over
a time-period of 31.5 min Different concentrations of
HspB and HspB(G116A), in 1 mL of 50 mM sodium phosphate buffer (pH 6.8), were preincubated for 10–15 min at 43C prior to the addition of CS to a final concentration of 600 nM.CS aggregation was measured as increased light scattering at 360 nm, in an Ultrospec 3000 spectrophotometer (Amersham Pharmacia Biotech).CS (Sigma) was dialyzed against Tris/EDTA buffer (10 mM Tris/HCl, 1 mM EDTA, pH 8.0) and stored at )20 C before use
Gel filtration Standard size-exclusion chromatography was performed on
a Superdex 200 HR 30/10 column (Amersham Pharmacia Biotech), as described previously [27,28].For gel filtration
at different temperatures, a 10· 600 mm Superformance column (Merck), equipped with a temperature adjustable jacket connected to a water bath, was packed with Sephacryl S-300 HR (Amersham Pharmacia Biotech) The columns were equilibrated with elution buffer [500 mM KCl, 20 mM Tris/HCl, 250 mM imidazole, 10% (v/v) glycerol, pH 7.9] Sample aliquots of 200 lL were injected and separated on a BioCAD perfusion chromato-graphy system (PerSeptive Biosystems), at a flow rate of 1.0 mLÆmin)1on the Sephacryl column and at 0.6 mLÆmin)1
on the Superdex column.Absorbance was recorded at a wavelength of 280 nm.The systems were calibrated with the following proteins from Amersham Pharmacia Biotech: thyroglobulin (699 kDa), ferritin (440 kDa), aldolase (158 kDa), albumin (67 kDa) and ribonuclease A (13.7 kDa)
CD spectroscopy Far-UV spectra in the range of 200–260 nm were recorded
on a BioLogic scanning spectrometer MS450 equipped with
a water bath connected to the cuvette chamber.Proteins were suspended at 100 lgÆmL)1 in potassium phosphate,
KF buffer (50 mM potassium phosphate, 100 mM KF,
pH 7.0) The path length of the cuvette was 2 mm The reported spectra are the average of five scans fitted by the fifth order polynomial function.Blank corrections were performed at each temperature
MS Frozen aliquots of purified HspF and HspC were thawed at room temperature and concentrated using Millipore Ultra-free-0.5 Biomax 5 kDa devices The concentrated protein was buffer exchanged by loading 120 lL onto a Superdex
200 HR 10/30 size-exclusion column (Amersham Pharma-cia) and eluting at 0.4 mLÆmin)1with 200 mMammonium acetate, at 8C.Fractions corresponding to the major eluting peak were pooled and concentrated to 1.4 mgÆmL)1 using the Biomax centrifugal filters
Nanoelectrospray MS (nanoESI-MS) experiments were performed on an LCT mass spectrometer (Micromass UK Ltd).Typically, 2 lL of solution was electrosprayed from gold-coated glass capillaries prepared in-house.In order to preserve noncovalent interactions, the following instrument parameters were used: capillary voltage, 1.5 kV; cone gas,
100 LÆh)1; sample cone, 170 V; extractor cone, 8 V; ion
Trang 3transfer stage pressure, 8.0· 10)3mbar; and ToF analyzer
pressure, 1.4· 10)6mbar.Thermal dissociation
experi-ments were carried out using a thermocontrolled nanoESI
probe designed in-house [30].NanoESI capillaries,
contain-ing HspF, were pre-equilibrated at the temperatures indi-cated, for 60 s prior to data acquisition.All spectra were calibrated externally using a solution of cesium iodide, and processed usingMASSLYNXsoftware (Micromass UK Ltd) The peak series were identified manually.A mass was generated based on an algorithm built into theMASSLYNX software, where m/z-1 for two consecutive charge states was multiplied and then divided by the difference between the m/z values
Results
Temperature- and concentration-dependent oligomerization of class A proteins
HspH oligomers have been previously shown to dissociate into smaller particles upon dilution [27].Like all other
B japonicumsHsps investigated to date, they were found
to partition into two fractions during gel filtration on a Superdex 200 HR 30/10 column [27,28].One peak coincided with the void volume of the column, whereas the other represented a dynamic concentration-dependent fraction Two distinct peaks were also observed in the present study, during SEC on a temperature-adjustable Sephacryl S-300
HR column (Fig.1A, solid line) The second peak of a sample with a concentration of 1 mgÆmL)1 eluted at
200 kDa at room temperature.Preincubation of the protein at 40C did not result in a shift of the peak when subsequent gel filtration was performed at room tempera-ture (broken line).In contrast, the HspH complex dissoci-ated and eluted at 70 kDa when the column was run at
40C (dotted line)
To examine, more closely, the temperature-dependent dissociation process, SEC of HspB, another class A protein, was performed at six different temperatures (Fig.1B) Almost the entire fraction eluted in the void volume (> 1 5 MDa) at 4C.Much smaller oligomers, with a peak of 300 kDa, were observed at 25 C.The equilibrium shifted towards smaller species at increasing temperatures, finally resulting in a retention time, corres-ponding to 40 kDa, at 45C.As for HspH, the disassembly process was fully reversible (Fig.1C) In contrast, the
Fig 1 Reversible, temperature-controlled dissociation of class A pro-teins Gel filtration was performed on a Sephacryl S-300 HR column and protein elution was monitored by absorption at 280 nm The protein concentration was 1 mgÆmL)1for HspH and 2 mgÆmL)1for HspB.(A) Elution profiles of ice-cooled HspH injected onto the col-umn equilibrated at 25 C (––) or 40 C (ÆÆÆÆ), and of a sample recorded
at 25 C after incubation for 15 min at 40 C and cooled on ice (- - -) The derived molecular mass values of the main peaks were 214 kDa,
75 kDa and 207 kDa, respectively.(B) Ice-cold HspB samples were injected onto the column equilibrated at the temperatures indicated The determined molecular masses (indicated from 4 C to 45 C) were
‡1.5 MDa, and 296, 229, 147, 75 and 43 kDa (C) Elution of an HspB sample incubated at 43 C for 15 min, cooled on ice and analyzed on the column equilibrated at 25 C.The elution times of peaks from samples run at 25 C and 43 C in (B) are indicated.(D) Gel filtration calibration curves at 25 C (m, ––) and 40 C (n, - - -) with standard proteins: thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), albumin (67 kDa) and ribonuclease A (13.7 kDa).
Trang 4elution of standard proteins was completely independent
of temperature (Fig.1D)
Temperature- and concentration-dependent
oligomerization of class B proteins
A temperature-controlled shift towards smaller species,
similar to that found with class A proteins, was observed
with HspF, a class B sHsp (Fig.2A) The protein was
previously shown to form oligomeric species of 400 kDa
[28].On the Sephacryl material, it eluted at 100 kDa, when
injected from a sample with a concentration of 3 mgÆmL)1,
at 25C.At 45 C, the protein eluted as a complex of
30–40 kDa.As with class A proteins, the oligomeric state
adjusted rapidly to the temperature, as shown by the
injection of ice-cooled samples onto the pre-equilibrated
column.The particle size also correlated with protein
concentration (Fig.2B).HspF eluted as a small oligomer
when it was injected from a diluted sample (0.5 mgÆmL)1)
NanoESI-MS was used to confirm the effects of concen-tration and temperature upon the oligomerization of the class B sHsps.When HspC (1.4 mgÆmL)1) was analyzed at a temperature of 30C, a spectrum containing a distribution
of oligomers from 12 to 24 subunits was obtained (Fig.3A)
In this case, the predominant species consisted of 18 subunits (18mer) with a molecular mass of 356 kDa, within
an oligomeric range of 238–476 kDa.When this sample was diluted fourfold and analyzed under identical MS condi-tions, the major species was found to be a 12mer protein This decrease in the relative population of species greater than 238 kDa is consistent with a reduction in the overall average molecular mass of the polydisperse assemblies observed by gel filtration
In order to assess the quaternary changes associated with temperature, HspF (0.7 mgÆmL)1) was equilibrated
at a range of temperatures prior to online analysis by nanoESI-MS.At 14C, the ratio of 12mer (the major oligomer at this concentration) to dissociated species (monomer and dimer) was 1 : 1, based upon peak areas in the spectrum (Fig.4) This indicated that a substantial proportion of the HspF monomers was involved in
Fig 2 Temperature and concentration-dependent dissociation of the
class B protein, HspF (A) Gel filtration profiles of ice-cold HspF
samples run on a Sephacryl S-300 HR column equilibrated at the
temperatures indicated.The protein concentration was 3 mgÆmL)1.
The derived molecular masses (indicated from 4 C to 45 C) were 338,
110, 80, 61, 45 and 38 kDa.(B) Elution profile at 25 C of a sample
diluted from 3 mgÆmL)1(arrow) to 0.5 mgÆmL)1.
Fig 3 Concentration-dependent dissociation of HspC monitored by nanoelectrospray MS (nanoESI-MS) (A) NanoESI mass spectrum of HspC (1.4 mgÆmL)1) in 200 m M ammonium acetate, pH 7.0 Charge state series from a range of oligomers were observed between m/z 6500 and m/z 10 000.These charge states were used to assign the peaks to oligomers ranging from 12 to 24 subunits in size, centred around a major species containing 18 subunits.(B) Reanalysis, under identical conditions, after dilution of the sample to 0.35 mgÆmL)1.The spectrum contained ions within the same m/z range; however, a shift to lower molecular mass species was observed, such that ions arising from a dodecamer represented the major species in the spectrum Representative charge states of the associated oligomers (XmerY+) are shown.
Trang 5higher-order oligomerization at this temperature.It is
important to emphasize that the monomer signal arises
from a single molecule, whereas the dodecamer signal is
obtained from 12 subunits.Thus, the actual number of
subunits involved in dodecamers at 14C is much greater
than monomers (dimers) at this temperature.At 20C,
a similar ratio was observed.When HspF was heated to
31C, however, a dramatic shift in the ratio of 12mer to
dissociated species was observed, such that ions arising
from the dodecamer were reduced to 40% of those
arising from the dissociated species (inset).A sample
equilibrated at 42C produced a further (slight) decrease
in this oligomeric ratio.The peaks around m/z 2000 arise
from truncated species of 14.2, 15.7 and 16.6 kDa that
were not associated with the dodecamer under MS
conditions
Instability of HspB mutated in a highly conserved
G residue
Previously we have shown that the class A HspH(G114A)
mutant is a defective chaperone, despite wild-type-like
oligomerization [27].G114 corresponds to one of the most
highly conserved residues of sHsps [1].To further investigate
this site, a corresponding mutation was constructed in
another class A protein.HspB(G116A) eluted as a 360 kDa
complex when analyzed at 25C on a Superdex 200 HR 10/
30 column.At 2 mgÆmL)1, HspB formed a larger complex
of 600 kDa (Fig.5A).Like the equivalent HspH mutant
HspB(G116A) was a defective chaperone unable to prevent
temperature-induced aggregation of the model substrate,
CS (Fig.5B) To test the thermal stability of the sHsp
complexes, SEC was performed at different temperatures
At 25C, wild-type HspB eluted as a 370 kDa complex on
the Sephacryl material (Fig.5C), whereas HspB(G116A)
complexes were clearly reduced in size.A peak in the range
of 140 kDa was observed (Fig.5D).Likewise, the HspB
peak, at 40 kDa at 45 C (Fig.5C), was shifted to
20 kDa in the case of the mutated protein (Fig.5D) Temperature-induced dissociation was fully reversible in either case (data not shown, Fig.1C)
Temperature-resistant secondary structure of class A and class B proteins
Far-UV CD spectroscopy was performed in order to determine whether the temperature-dependent changes in oligomerization were accompanied by alterations in the secondary structure.The traces recorded are typical of b-strand proteins and show a single minimum elipticity in the range of 217 nm, similar to that observed for other sHsps [14,18,32].The spectra of HspH (Fig.6A) or HspF (Fig.6B), taken at 23C, 33 C or 43 C, were very similar, indicating that significant changes in the secondary structure
do not occur in the physiological temperature range Formation of large sHsp–substrate complexes
B japonicum sHsps have been shown to prevent heat-induced aggregation of the model substrate, CS [26].The formation of sHsp–CS complexes was examined by gel filtration.When HspH and CS were incubated together on ice for 50 min prior to injection, three peaks were detected
by SEC (Fig.7A, solid line).To analyze the composition of these peaks, fractions were collected and separated by SDS-PAGE (Fig.7B).CS eluted with a retention time consistent with its molecular mass (dimer, 96 kDa).Some HspH was found in the void volume fraction, but the majority resided
in a broad peak, reflecting its concentration-dependent dissociation.Co-incubation of HspH and CS at 43C before SEC resulted in a significant increase of the void volume fraction and a decrease of dimeric CS and the second HspH peak (Fig.7A, broken line), indicative of the formation of large and stable chaperone–substrate
Fig 4 Temperature-dependent dissociation of HspF monitored by nanoelectrospray MS (nanoESI-MS) Spectra were acquired at the temperatures indicated after pre-equilibration in the nanoelectrospray capillary.Charge states arising from a dodecamer were observed between m/z 6200 and m/z
7000, whereas those arising from monomers and dimers (dissociated species) were observed in the range m/z 1980 and m/z 3400.A substantial decrease in peak intensities of the 12mer charge state series was observed with increased temperature, particularly between 20 C and 31 C.The most abundant species was adjusted to 100% peak intensity.(Inset) Graph of the ratio of summed peak areas for charge state series of the 12mer relative to the dissociated species.Hsp70/DnaK was a co-purified contaminant from the Escherichia coli expression system.
Trang 6assemblies.A transfer of CS and HspH into the void
volume, at the expense of the other fractions, was confirmed
by SDS-PAGE (Fig.7C).Neither HspH nor CS shifted into
the void volume when they were heat-treated separately
before gel filtration (data not shown)
Discussion
Oligomerization of sHsps responds to external conditions
Most sHsps are highly dynamic proteins which form flexible
oligomeric complexes that frequently exchange subunits by
dissociation and reassembly.Although we are far from a
consistent picture, it is well documented that at least two
parameters have a strong impact on the composition of
sHsp complexes.The average complex size often depends
on protein concentration and
temperature.Dilution-induced disassembly is usually reflected by broad and
tailing peaks when sHsps are run on a gel-filtration column
During recent studies on sHsp mutants, oligomers of the
class A protein, HspH, were found to be in a
concentration-dependent equilibrium with smaller subspecies [27].In the
present study, we demonstrate that
concentration-depend-ent dissociation is a general property of B japonicum sHsps,
as the class A protein, HspB, and the class B proteins, HspC
and HspF, behaved in an identical manner.Closer
inspec-tion, by ESI-MS, revealed that the predominant species in a
concentrated sample of HspC were 18mers.The presence of
larger (20, 22 and 24mer) and smaller (16 and 12mer)
particles indicates a dynamic composition from which
dimeric particles are released upon dilution.The appearance
of glutaraldehyde cross-linked sHsp dimers on SDS-PAGE
gels (data not shown) supports the notion that they might be
the main building units of class A and B proteins.Dimers
are also the basic building blocks of M jannaschii Hsp16.5
and wheat Hsp16.9 The dimerization interface was shown
to form the most extensive contacts in the oligomeric
complex [15,16].Likewise, unfolding of a yeast Hsp26 dimer
required much higher energy, as compared to the relatively
low energy needed for dissociation of the 24mer into dimers
[33].Real-time subunit exchange, monitored by ESI-MS, demonstrated that the closely related pea Hsp18.1 and wheat Hsp16.9 exchanged dimers as the main unit [17] In
Fig 5 Temperature-dependent dissociation and chaperone activity of
wild-type HspB compared to HspB(G116A) (A) Gel filtration profiles
of HspB (––) and HspB(G116A) (- - -).The molecular masses were
determined as 600 and 360 kDa, respectively.The protein
concentra-tion was 2 mgÆmL)1.Size exclusion chromatography was carried out
on a Superdex 200 HR 10/30 column at 25 C.(B) Thermal
aggre-gation of citrate synthase (CS) at 43 C in the presence of different
concentrations of HspB or HspB(G116A) was recorded at 360 nm.
The CS assay was performed in the absence (r) and presence of
150 n M (n), 300 n M (m), 600 n M (h) and 1 2 l M (j) of HspB
pro-teins.The CS concentration was 600 n M HspB alone was measured at
1.2 l M (e).Ice-cold HspB (C) or HspB(G116A) (D) samples were
separated on the Sephacryl S-300 HR column equilibrated at the
indicated temperatures.The protein concentration was 2 mgÆmL)1.
The molecular mass values were (indicated from 25 C to 45 C) 370,
210, 110, 65 and 40 kDa for HspB and 140, 65, 40, 25 and 20 kDa for
HspB(G116A).As protein eluting in the void volume does not
con-tribute to the equilibrium (data not shown), only the dynamic fractions
were compared at similar concentrations, as indicated by the dotted
lines.
Trang 7contrast, the hexadecameric murine Hsp25 was shown to be
in concentration-dependent equilibrium with tetramers [34]
Concentration-dependent oligomerization is not a feature
common to all sHsps.Temperature-controlled dissociation
seems to be more common.However, again it is not
a unifying feature of all sHsps.Neither a-crystallin nor
Hsp42 are temperature-responsive [5,35].This reflects the
physiological conditions under which these proteins are
active.The a-crystallins serve two important functions in
the eye lens: a structural role; and a protective,
chaperone-like function [36].As the mammalian eye rarely encounters
major temperature fluctuations, thermal control of these
functions is not applicable.Similarly, Hsp42 is an abundant
sHsp in yeast, serving its important chaperoning task at
room temperature [35].Hence, its oligomeric state is not influenced by temperature.Hsp26, the second sHsp in yeast,
is, however, a temperature-controlled chaperone that dissociates at heat shock temperatures [5].At low temper-atures it associates into an inactive storage form
Our SEC and ESI-MS experiments consistently dem-onstrate that both class A and class B sHsps form large
Fig 6 Far-UV CD spectroscopy of HspH (A) and HspF (B) Spectra
were recorded at the indicated temperatures.The data represent the
average of five scans from a sample containing 100 lgÆmL)1protein.
The path length was 2 mm.The mean residue ellipticity was calculated
from the following equation:
ðhM MRW Þ=ð10clÞ;
where MRW ¼ mean residue molecular mass; c ¼ concentration
(mgÆmL)1); and l ¼ path length (cm).
Fig 7 Complex formation between HspH and citrate synthase (CS) (A) Gel filtration on a Superdex 200 HR 10/30 column of 20 l M HspH that was preincubated for 15 min at 43 C before the addition of 2 l M
CS and incubation for an additional 50 min at the same temperature (- - -).As a control, a sample containing CS (2 l M ) and HspH (20 l M ), which was incubated on ice for 50 min prior to size exclusion chro-matography, is shown (––).Protein from the control sample (B) and the heat-treated sample (C) was collected, precipitated, separated by SDS-PAGE and stained with Coomassie blue.Lanes 1 and 2 corres-pond to the peak that eluted at around 12.5 min (void volume) Lanes 3–6 represent the two peaks that eluted between 19 and 26 min.
Trang 8complexes that are very sensitive to dilution and
tem-perature.Even moderate temperature changes, in the
range between 25C and 35 C, led to a significant
decrease in complex size.The monomers observed in the
ESI-MS probably do not represent the actual in vivo state,
but rather are a result of dimer dissociation in the MS
process.The sHsps are presumably held together largely
by hydrophobic interactions, the effects of which are
greatly reduced during the electrospray process where the
solvent is removed from the gas-phase droplets.The
interactions of the dodecamer are presumably stronger
than the dimer owing to a greater unexposed surface area,
leading to the observation of more dodecamer at lower
temperatures.The dimer, however, dissociates to
mono-mer to the same extent, as this is an MS phenomenon,
not a temperature-related transition.Taken together, this
would explain why the distribution differs between MS
and SEC
The significant decrease in complex size between 25C
and 35C agrees well with the physiological demands
Growth of B japonicum is optimal at 30 C.At higher
temperatures, heat shock proteins are induced, in
partic-ular sHsps [37].It appears that the equilibrium is shifted
towards small species under these conditions.Complex
dissociation is often associated with increased chaperone
activity [5,16].The current opinion is that
substrate-binding sites, hidden in the complex, are liberated by
dissociation at elevated temperatures.Several studies show
that exposure of hydrophobic patches correlates with the
disassembly process.It was shown that temperature-driven
dissociation can lead to the exposure of hydrophobic sites
in yeast Hsp26 [5].For pea Hsp18.1, it was reported that
photo-incorporation of the hydrophobic dye, bis-ANS,
increased with increasing temperature [8].Similarly, high
temperature promoted the reversible exposure of
hydro-phobic bis-ANS-binding sites in E coli IbpB [14].The fact
that M jannaschii Hsp16.5 exchanged subunits only at
physiologically relevant temperatures above 60C [18],
underlines that the dynamic properties of sHsps are
perfectly adapted to their respective environmental niche
and cellular compartment
The temperature- and concentration-dependent
disas-sembly of class A and B protein was fully reversible.Free
dissociation and reassembly of suboligomeric particles is
possible, probably because the secondary structure of the
sHsps is not changed during this process.Only after
substrate binding does sHsps seem to be trapped in large
complexes (> 2 MDa).The formation of large and stable
chaperone–substrate aggregates was demonstrated for
other sHsps with a broad range of different substrates
[4,5,7,8,38].How bound substrates are passed on to the
resolubilizing chaperone machinery is not clear.Whether
this mechanism involves physical contact between sHsps
and its partner, Hsp70, is unknown.However, the
co-purification of E coli Hsp70/DnaK with HspF,
observed in our ESI-MS experiment, suggests that this
might be the case
Proper complex stability is crucial for chaperone activity
Only a few single amino acid-exchange mutants are
known to have a severe effect on the oligomerization and
chaperone activity of sHsps.There is a clear correlation between complex size and activity, as sHsps, unable to reach the fully assembled state, were defective chaperones [27].HspH(G114A), carrying a mutation in the highly conserved G-x-L motif of the a-crystallin domain, showed interesting properties in that its chaperone activity was abolished, although oligomerization was barely effected.A closer inspection of the dynamic properties of this protein and an equivalent mutant in HspB, revealed that these variants form labile complexes that dissociate much more readily than the wild-type proteins.The corresponding glycine residues in wheat Hsp16.9 and M jannaschii Hsp16 5 are located in a short connecting loop, between b-strands 8 and 9, which is involved in dimer interaction [15,16].At 45C, HspH(G114A) and HspB(G116A) eluted as apparent monomers, indicating that the dimer interface might indeed be destabilized in these variants.Similar, reduced oligomeric stability was described for an unrelated mutation in Synechocystis Hsp16.6 (L66A) that did not seem to alter quaternary structure but destabilized the complex and reduced chaperone activity [39].Intragenic suppressors that stabilized the complex restored chaper-one function.Interestingly, over-stabilizing mutations were counter-productive because they slowed down the rate of luciferase refolding in vivo A mutation in Mycobacteriumtuberculosis Hsp16.3, at amino acid L122, also resulted in normally sized, but unstable, complexes with reduced activity [40,41].The correspond-ing mutation in B japonicum HspH, at position L116, led
to reduced complex size and impaired chaperone activity [27].Altogether, it is quite puzzling that disassembly of sHsp oligomers is needed for chaperone activity, on the one hand, but that certain complex stability is required
on the other.In analogy to the stepwise unfolding of substrate proteins [42], we propose that dissociation of the chaperone creates various intermediates, one (or more) of which are capable of substrate binding (Fig.8) The model explains the chaperone defect of sHsps with
Fig 8 Hypothetical model on chaperone dynamics and chaperone sub-strate interaction of small heat shock proteins (sHsps) For details, see the text.
Trang 9too low or too high complex stability.Destabilizing
mutations may either pass through the active
intermedi-ate(s) too rapidly, or bypass it altogether.Strongly
stabilized variants are probably delayed in reaching the
active state.Stable substrate–chaperone complexes are
assembled from the intermediates in the
substrate-unfold-ing and chaperone-dissociation pathways
Acknowledgements
F.N.and N.L.thank Hauke Hennecke for continuous support.
J.A.A.is a Royal Society Howard Florey Fellow.Wolf-Diedrich
Hardt is acknowledged for providing the CD equipment.Thanks
are due to Justin Benesch who designed the thermocontrolled
nanoESI probe.M.L.is a student at the Karl-Franzens-University
of Graz and was supported by an ERASMUS fellowship.Funding
by the Swiss Federal Institute of Technology, Zu¨rich, is gratefully
acknowledged.
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