Temperature and concentration-controlled dynamics of rhizobial small heat shock proteins Nicolas Lentze 1 , J. Andrew Aquilina 2 , Mareike Lindbauer 1 , Carol V. Robinson 2 and Franz Narberhaus 1,3 1 Institut fu ¨ r Mikrobiologie, Eidgeno ¨ ssische Technische Hochschule, Zu ¨ rich, Switzerland; 2 Department of Chemistry, Cambridge University, Cambridge, UK and 3 Lehrstuhl 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 citratesynthasewerestableandexceededthesizeofsHsp 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, Bradyrhizobium japonicum, 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 55 °C 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 ¨ tBochum, 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) Eur. J. Biochem. 271, 2494–2503 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04180.x central 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 HspB His (pRJ5304), HspH His (pRJ5307), HspC His (pRJ5305) and HspF His (pRJ5306), has been described previously [26]. The HspB His derivative, HspB His (G116A) (pRJ5691), was constructed by primer-based site-directed mutagenesis (Quick-Change TM 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 30 °C 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 m M ). After 2 h, cells were harvested and resuspended in binding buffer [500 m M KCl, 20 m M Tris/HCl, 5 m M imidazole, 10% (v/v) glycerol, pH 7.9] containing 1 m M phenylmethanesulfonyl fluoride and 10 lgÆmL )1 DNase 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 4 °C. 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 °Cor)80 °C in elution buffer [500 m M KCl, 20 m M Tris/HCl, 250 m M 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 m M sodium phosphate buffer (pH 6.8), were preincubated for 10–15 min at 43 °C prior to the addition of CS to a final concentration of 600 n M . 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 m M Tris/HCl, 1 m M EDTA, pH 8.0) and stored at )20 °Cbeforeuse. 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 m M KCl, 20 m M Tris/HCl, 250 m M imidazole, 10% (v/v) glycerol, pH 7.9]. Sample aliquots of 200 lLwere injected and separated on a BioCAD perfusion chromato- graphy system (PerSeptive Biosystems), at a flow rate of 1.0 mLÆmin )1 on 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 m M potassium phosphate, 100 m M 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 )1 with 200 m M ammonium acetate, at 8 °C. 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 Ó FEBS 2004 Dynamics of bacterial sHsps (Eur. J. Biochem. 271) 2495 transfer stage pressure, 8.0 · 10 )3 mbar; and ToF analyzer pressure, 1.4 · 10 )6 mbar. 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 using MASSLYNX software (Micromass UK Ltd). The peak series were identified manually. A mass was generated based on an algorithm built into the MASSLYNX 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. japonicum sHsps 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 40 °C 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 40 °C (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 (>1.5MDa)at4°C. 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 45 °C. 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 andproteinelutionwasmonitoredbyabsorptionat280nm.The protein concentration was 1 mgÆmL )1 for HspH and 2 mgÆmL )1 for 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 °Cto45°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 °Cand43°C in (B) are indicated. (D) Gel filtration calibration curves at 25 °C(m, ––) and 40 °C(n, )withstandard proteins: thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), albumin (67 kDa) and ribonuclease A (13.7 kDa). 2496 N. Lentze et al. (Eur. J. Biochem. 271) Ó FEBS 2004 elution 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 25 °C. 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 30 °C, 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 14 °C, 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 °Cto45 °C) were 338, 110, 80, 61, 45 and 38 kDa. (B) Elution profile at 25 °Cofasample 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 )in200m 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 (Xmer Y+ ) are shown. Ó FEBS 2004 Dynamics of bacterial sHsps (Eur. J. Biochem. 271) 2497 higher-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 14 °Cismuchgreater than monomers (dimers) at this temperature. At 20 °C, a similar ratio was observed. When HspF was heated to 31 °C, 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 42 °C 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 25 °C 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 25 °C, 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 23 °C, 33 °Cor43 °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 43 °C 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 °Cand31°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. 2498 N. Lentze et al. (Eur. J. Biochem. 271) Ó FEBS 2004 assemblies. 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)and1.2l M (j)ofHspBpro- 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 °Cto45°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. Ó FEBS 2004 Dynamics of bacterial sHsps (Eur. J. Biochem. 271) 2499 contrast, 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 )1 protein. The path length was 2 mm. The mean residue ellipticity was calculated from the following equation: ðhÁM MRW Þ=ð10ÁcÁlÞ; 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 )andHspH(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. 2500 N. Lentze et al. (Eur. J. Biochem. 271) Ó FEBS 2004 complexes that are very sensitive to dilution and tem- perature. Even moderate temperature changes, in the range between 25 °Cand35°C,ledtoasignificant 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 25 °C and 35 °C 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 60 °C[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 Hsp16.5arelocatedinashort connecting loop, between b-strands 8 and 9, which is involved in dimer interaction [15,16]. At 45 °C, 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.Amutationin Mycobacterium tuberculosis 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. Ó FEBS 2004 Dynamics of bacterial sHsps (Eur. J. Biochem. 271) 2501 too 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. References 1. Narberhaus, F. 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