Structure and properties of chimeric small heat shock proteins containing yellow fluorescent protein attached to their C-terminal ends

Recombinant chimeras of small heat shock proteins (sHsp) HspB1, HspB5, and HspB6 containing enhanced yellow fluorescent protein (EYFP) attached to their C-terminal ends were constructed and purified. Some properties of these chimeras were compared with the corresponding properties of the same chimeras containing EYFP attached to the N-terminal end of sHsp. The C-terminal fluorescent chimeras of HspB1 and HspB5 tend to aggregate and form a heterogeneous mixture of oligomers. The apparent molecular weight of the largest C-terminal chimeric oligomers was higher than that of the corresponding N-terminal chimeras or of the wild-type proteins; however, both homooligomers of N-terminal chimeras and homooligomers of C-terminal chimeras contained fewer subunits than the wild-type HspB1 or HspB5. Both N-terminal and C-terminal chimeras of HspB6 form small oligomers with an apparent molecular weight of 73–84 kDa. The C-terminal chimeras exchange their subunits with homologous wild-type proteins. Heterooligomers formed by the wild-type HspB1 (or HspB5) and the C-terminal chimeras of HspB6 differ in size and composition from heterooligomers formed by the corresponding wild-type proteins. As a rule, the N-terminal chimeras possess similar or slightly higher chaperone-like activity than the corresponding wild-type proteins, whereas the C-terminal chimeras always have a lower chaperone-like activity than the wild-type proteins. It is concluded that attachment of EYFP to either N-terminal or C-terminal ends of sHsp affects their oligomeric structure, their ability to form heterooligomers, and their chaperone-like activity. Therefore, the data obtained with fluorescent chimeras of sHsp expressed in the cell should be interpreted with caution.     

Utilization of fluorescent chimeras for investigation of heterooligomeric complexes formed by human small heat shock proteins

Fluorescent chimeras composed of enhanced cyan (or enhanced yellow) fluorescent proteins (ECFP or EYFP) and one of the four human small heat shock proteins (HspB1, HspB5, HspB6 or HspB8) were expressed in E. coli and purified. Fluorescent chimeras were used for investigation of heterooligomeric complexes formed by different small heat shock proteins (sHsp) and for analysis of their subunit exchange. EYFP-HspB1 and ECFP-HspB6 form heterooligomeric complex with apparent molecular weight of ∼280 kDa containing equimolar quantities of both sHsp. EYFP-HspB5 and ECFP-HspB6 formed heterogeneous oligomeric complexes. Fluorescent proteins inside heterooligomeric complexes formed by HspB1/HspB6 and HspB5/HspB6 chimeras are closely located, making possible effective fluorescence resonance energy transfer (FRET). Neither the wild type HspB8 nor its fluorescent chimeras were able to form stable heterooligomeric complexes with the wild type HspB1 and HspB5. Homo- and hetero-FRET was used for analysis of subunit exchange of small heat shock proteins. The apparent rate constant of subunit exchange was temperature-dependent and was higher for HspB6 forming small oligomers than for HspB1 forming large oligomers. Replacement induced by homologous subunits was more rapid than the replacement induced by heterologous subunits of small heat shock proteins. Fusion of fluorescent proteins might affect oligomeric structure of small heat shock proteins, however fluorescent chimeras can be useful for investigation of heterooligomeric complexes formed by sHsp and for analysis of kinetics of their subunit exchange.     

The chaperone-like activity of rat HspB8/Hsp22 and dynamic molecular transition related to oligomeric architectures in vitro

HspB8/Hsp22 is a functionally distinct small heat shock proteins (sHsp) and is preferentially expressed in brain, heart, skeletal, and smooth muscle. HspB8 is also associated with neuromuscular function and protein quality control by proteasomes in cardiac hypertrophy. However, the molecular properties in vitro and molecular oligomerization remain uncertain. In this investigation, the rat HspB8 gene was expressed in E.coli cells, and mature HspB8 protein was efficiently prepared. The chaperone-like activity of HspB8 in vitro was quantitatively analyzed by model substrates. Size exclusion chromatography revealed that HspB8 had polydisperse oligomers and underwent dynamic molecular transition in solution, existing in a dynamic equilibrium between various oligomers. In a nonphysiological solution, HspB8 was predominantly octamers. In a physiological solution (pH 7.4), HspB8 mainly formed tetramers. The dynamic interactive transition maybe was helpful to maintain its molecular complxes in solution. In a FRET assay, subunit exchange occurred frequently between the various oligomers with a rate of 0.12, 0.089, and 0.064 min(-1) at 50°C, 43°C, and 37°C, respectively. It also demonstrated the dynamic molecular properties of HspB8 in solution.     

Small Heat Shock Proteins and Distal Hereditary Neuropathies

Classification of small heat shock proteins (sHsp) is presented and processes regulated by sHsp are described. Symptoms of hereditary distal neuropathy are described and the genes whose mutations are associated with development of this congenital disease are listed. The literature data and our own results concerning physicochemical properties of HspB1 mutants associated with Charcot–Marie–Tooth disease are analyzed. Mutations of HspB1, associated with hereditary motor neuron disease, can be accompanied by change of the size of HspB1 oligomers, by decreased stability under unfavorable conditions, by changes in the interaction with protein partners, and as a rule by decrease of chaperone-like activity. The largest part of these mutations is accompanied by change of oligomer stability (that can be either increased or decreased) or by change of intermonomer interaction inside an oligomer. Data on point mutation of HspB3 associated with axonal neuropathy are presented. Data concerning point mutations of Lys141 of HspB8 and those associated with hereditary neuropathy and different forms of Charcot–Marie–Tooth disease are analyzed. It is supposed that point mutations of sHsp associated with distal neuropathies lead either to loss of function (for instance, decrease of chaperone-like activity) or to gain of harmful functions (for instance, increase of interaction with certain protein partners).     

The pivotal role of the β7 strand in the intersubunit contacts of different human small heat shock proteins

Human αB-crystallin and small heat shock proteins HspB6 and HspB8 were mutated so that all endogenous Cys residues were replaced by Ser and the single Cys residue was inserted in a position homologous to that of Cys137 of human HspB1, i.e. in a position presumably located in the central part of β7 strand of the α-crystallin domain. The secondary, tertiary, and quaternary structures of thus obtained Cys-mutants as well as their chaperone-like activity were similar to those of their wild-type counterparts. Mild oxidation of Cys-mutants leads to formation of disulfide bond crosslinking neighboring monomers thus indicating participation of the β7 strand in intersubunit interaction. Oxidation weakly affects the secondary and tertiary structure, does not affect the quaternary structure of αB-crystallin and HspB6, and shifts equilibrium between monomer and dimer of HspB8 towards dimer formation. It is concluded that the β7 strand participates in the intersubunit interaction of four human small heat shock proteins (αB-crystallin, HspB1, HspB6, HspB8) having different structure of β2 strand of α-crystallin domain and different length and composition of variable N- and C-terminal tails.     

Mutations of small heat shock proteins and human congenital diseases

The structure and properties of different members of a large family of small heat shock proteins (sHsp) playing an important role in cell homeostasis are described. Participation of the N-terminal domain in formation of large oligomers and chaperone activity of sHsp is analyzed. The structure of the α-crystallin domain of sHsp is characterized and the role of this domain in sHsp dimerization and chaperone activity is discussed. The properties of the C-terminal region of sHsp are described, and its participation in formation of large oligomers and chaperone activity are analyzed. The data from the literature on HspB1 and HspB3 mutations are presented, and involvement of these mutations in development of certain neurodegenerative diseases is discussed. Mutations of HspB4 are described and data on involvement of these mutations in development of cataract are presented. Multiple effects of HspB5 mutations are analyzed, and data are presented indicating that mutations of this protein are accompanied by development of different congenital diseases, such as cataract and different types of myopathies. The data on HspB6 and HspB8 mutations are presented, and feasible effects of these mutations on proteins structure are analyzed. Probable mechanisms underlying sHsp mutation-induced development of different congenital diseases are discussed.     

Heterooligomeric complexes formed by human small heat shock proteins HspB1 (Hsp27) and HspB6 (Hsp20)

Formation of heterooligomeric complexes of human small heat shock proteins (sHsp) HspB6 (Hsp20) and HspB1 (Hsp27) was analyzed by means of native gel electrophoresis, analytical ultracentrifugation, chemical cross-linking and size-exclusion chromatography. HspB6 and HspB1 form at least two different complexes with apparent molecular masses 100–150 and 250–300 kDa, and formation of heterooligomeric complexes is temperature dependent. These complexes are highly mobile, easily exchange their subunits and are interconvertible. The stoichiometry of HspB1 and HspB6 in both complexes is close to 1/1 and smaller complexes are predominantly formed at low, whereas larger complexes are predominantly formed at high protein concentration. Formation of heterooligomeric complexes does not affect the chaperone-like activity of HspB1 and HspB6 if insulin or skeletal muscle F-actin was used as model protein substrates. After formation of heterooligomeric complexes the wild type HspB1 inhibits the rate of phosphorylation of HspB6 by cAMP-dependent protein kinase. The 3D mutant mimicking phosphorylation of HspB1 also forms heterooligomeric complexes with HspB6, but is ineffective in inhibition of HspB6 phosphorylation. Inside of heterooligomeric complexes HspB6 inhibits phosphorylation of HspB1 by MAPKAP2 kinase. Thus, in heterooligomeric complexes HspB6 and HspB1 mutually affect the structure of each other and formation of heterooligomeric complexes might influence diverse processes depending on small heat shock proteins.     

Interaction of small heat shock proteins with light component of neurofilaments (NFL)

The interaction of human small heat shock protein HspB1, its point mutants associated with distal hereditary motor neuropathy, and three other small heat shock proteins (HspB5, HspB6, HspB8) with the light component of neurofilaments (NFL) was analyzed by differential centrifugation, analytical ultracentrifugation, and fluorescent spectroscopy. The wild-type HspB1 decreased the quantity of NFL in pellets obtained after low- and high-speed centrifugation and increased the quantity of NFL remaining in the supernatant after high-speed centrifugation. Part of HspB1 was detected in the pellet of NFL after high-speed centrifugation, and at saturation, 1 mol of HspB1 monomer was bound per 2 mol of NFL. Point mutants of HspB1 associated with distal hereditary motor neuropathy (G84R, L99M, R140G, K141Q, and P182S) were almost as effective as the wild-type HspB1 in modulation of NFL assembly. At low ionic strength, HspB1 weakly interacted with NFL tetramers, and this interaction was increased upon salt-induced polymerization of NFL. HspB1 and HspB5 (αB-crystallin) decreased the rate of NFL polymerization measured by fluorescent spectroscopy. HspB6 (Hsp20) and HspB8 (Hsp22) were less effective than HspB1 (or HspB5) in modulation of NFL assembly. The data presented indicate that the small heat shock proteins affect NFL transition from tetramers to filaments, hydrodynamic properties of filaments, and their bundling and therefore probably modulate the formation of intermediate filament networks in neurons.     

Chaperone activity of human small heat shock protein-GST fusion proteins

Small heat shock proteins (sHsps) are a ubiquitous part of the machinery that maintains cellular protein homeostasis by acting as molecular chaperones. sHsps bind to and prevent the aggregation of partially folded substrate proteins in an ATP-independent manner. sHsps are dynamic, forming an ensemble of structures from dimers to large oligomers through concentration-dependent equilibrium dissociation. Based on structural studies and mutagenesis experiments, it is proposed that the dimer is the smallest active chaperone unit, while larger oligomers may act as storage depots for sHsps or play additional roles in chaperone function. The complexity and dynamic nature of their structural organization has made elucidation of their chaperone function challenging. HspB1 and HspB5 are two canonical human sHsps that vary in sequence and are expressed in a wide variety of tissues. In order to determine the role of the dimer in chaperone activity, glutathione-S-transferase (GST) was genetically linked as a fusion protein to the N-terminus regions of both HspB1 and HspB5 (also known as Hsp27 and αB-crystallin, respectively) proteins in order to constrain oligomer formation of HspB1 and HspB5, by using GST, since it readily forms a dimeric structure. We monitored the chaperone activity of these fusion proteins, which suggest they primarily form dimers and monomers and function as active molecular chaperones. Furthermore, the two different fusion proteins exhibit different chaperone activity for two model substrate proteins, citrate synthase (CS) and malate dehydrogenase (MDH). GST-HspB1 prevents more aggregation of MDH compared to GST-HspB5 and wild type HspB1. However, when CS is the substrate, both GST-HspB1 and GST-HspB5 are equally effective chaperones. Furthermore, wild type proteins do not display equal activity toward the substrates, suggesting that each sHsp exhibits different substrate specificity. Thus, substrate specificity, as described here for full-length GST fusion proteins with MDH and CS, is modulated by both sHsp oligomeric conformation and by variations of sHsp sequences.     

Expression of small heat shock proteins HspB2, HspB8, Hsp20 and cvHsp in different tissues of the perinatal developing pig

Recently, we have described the developmental expression of the small heat shock proteins (sHsps) Hsp27/HspB1 and alphaB-crystallin/HspB5 in different tissues of pigs from almost full-term foetuses to three years old adults (P. Tallot, J. F. Grongnet, J. C. David, Biol. Neonate, 83, 281-288, 2003). The data described in this report extends this study to four other members of the sHsp family (Hsp20/HspB6, cvHsp/HspB7, MKBP/HspB2 and HspB8). We studied expression of these proteins in porcine lens, brain, heart, liver, kidney, lung, skeletal muscle, stomach, and colon, and found a ubiquitous expression of Hsp20 and HspB8 as earlier reported for Hsp27 and alphaB-crystallin. In contrast, cvHsp and HspB2 expression is essentially restricted to heart and muscle. During development, the sHsps tend to (temporarily) increase in stomach, liver, lung, kidney, hippocampus, and striatum, while expression in heart is more or less constant, and a large variation is found in sHsp expression patterns in skeletal muscle. In cerebellum and cortex a temporary decrease of Hsp20 and HspB8 is observed directly after birth. The major impact of this study is that each tissue seems to have a unique profile of sHsp expression, which varies during development and may reflect the need of a particular tissue to maintain at all stages an optimal chaperoning machinery to protect against physiological stress.     

Physico-chemical properties of R140G and K141Q mutants of human small heat shock protein HspB1 associated with hereditary peripheral neuropathies

Some physico-chemical properties of R140G and K141Q mutants of human small heat shock protein HspB1 associated with hereditary peripheral neuropathy were analyzed. Mutation K141Q did not affect intrinsic Trp fluorescence and interaction with hydrophobic probe bis-ANS, whereas mutation R140G decreased both intrinsic fluorescence and fluorescence of bis-ANS bound to HspB1. Both mutations decreased thermal stability of HspB1. Mutation R140G increased, whereas mutation K141Q decreased the rate of trypsinolysis of the central part (residues 5–188) of HspB1. Both the wild type HspB1 and its K141Q mutant formed large oligomers with apparent molecular weight ∼560 kDa. The R140G mutant formed two types of oligomers, i.e. large oligomers tending to aggregate and small oligomers with apparent molecular weight ∼70 kDa. The wild type HspB1 formed mixed homooligomers with R140G mutant with apparent molecular weight ∼610 kDa. The R140G mutant was unable to form high molecular weight heterooligomers with HspB6, whereas the K141Q mutant formed two types of heterooligomers with HspB6. In vitro measured chaperone-like activity of the wild type HspB1 was comparable with that of K141Q mutant and was much higher than that of R140G mutant. Mutations of homologous hot-spot Arg (R140G of HspB1 and R120G of αB-crystallin) induced similar changes in the properties of two small heat shock proteins, whereas mutations of two neighboring residues (R140 and K141) induced different changes in the properties of HspB1.     

Agonist-independent and -dependent oligomerization of dopamine D(2) receptors by fusion to fluorescent proteins.

Oligomerization of the short (D(2S)) and long (D(2L)) isoforms of the dopamine D(2) receptor was explored in transfected Cos-7 cells by their C-terminal fusion to either an enhanced cyan or enhanced yellow fluorescent protein (ECFP or EYFP) and the fluorescent fusion protein interaction was monitored by a fluorescence resonance energy transfer (FRET) assay. The pharmacological properties of the fluorescent fusion proteins, as measured by both displacement of [(3)H]nemonapride binding and agonist-mediated stimulation of [(35)S]GTPgammaS binding upon co-expression with a G(alphao)Cys(351)Ile protein, were not different from the respective wild-type D(2S) and D(2L) receptors. Co-expression of D2S:ECFP+D2S:EYFP in a 1:1 ratio and D2L:ECFP+D2L:EYFP in a 27:1 ratio resulted, respectively, in an increase of 26% and 16% in the EYFP-specific fluorescent signal. These data are consistent with a close proximity of both D(2S) and D(2L) receptor pairs of fluorescent fusion proteins in the absence of ligand. The agonist-independent D(2S) receptor oligomerization could be attenuated by co-expression with either a wild-type, non-fluorescent D(2S) or D(2L) receptor subtype, but not with a distinct beta(2)-adrenoceptor. Incubation with the agonist (-)-norpropylapomorphine dose-dependently (EC(50): 0.23+/-0.06 nM) increased the FRET signal for the co-expression of D2S:ECFP and D2S:EYFP, in support of agonist-dependent D(2S) receptor oligomerization. In conclusion, our data strongly suggest the occurrence of dopamine D(2) receptor oligomers in intact Cos-7 cells.     

Domain swapping in human alpha A and alpha B crystallins affects oligomerization and enhances chaperone-like activity

alphaA and alphaB crystallins, members of the small heat shock protein family, prevent aggregation of proteins by their chaperone-like activity. These two proteins, although very homologous, particularly in the C-terminal region, which contains the highly conserved "alpha-crystallin domain," show differences in their protective ability toward aggregation-prone target proteins. In order to investigate the differences between alphaA and alphaB crystallins, we engineered two chimeric proteins, alphaANBC and alphaBNAC, by swapping the N-terminal domains of alphaA and alphaB crystallins. The chimeras were cloned and expressed in Escherichia coli. The purified recombinant wild-type and chimeric proteins were characterized by fluorescence and circular dichroism spectroscopy and gel permeation chromatography to study the changes in secondary, tertiary, and quaternary structure. Circular dichroism studies show structural changes in the chimeric proteins. alphaBNAC binds more 8-anilinonaphthalene-1-sulfonic acid than the alphaANBC and the wild-type proteins, indicating increased accessible hydrophobic regions. The oligomeric state of alphaANBC is comparable to wild-type alphaB homoaggregate. However, there is a large increase in the oligomer size of the alphaBNAC chimera. Interestingly, swapping domains results in complete loss of chaperone-like activity of alphaANBC, whereas alphaBNAC shows severalfold increase in its protective ability. Our findings show the importance of the N- and C-terminal domains of alphaA and alphaB crystallins in subunit oligomerization and chaperone-like activity. Domain swapping results in an engineered protein with significantly enhanced chaperone-like activity.     

Inhibition of α-synuclein aggregation by small heat shock proteins

The fibrillization of α-synuclein (α-syn) is a key event in the pathogenesis of α-synucleinopathies. Mutant α-syn (A53T, A30P, or E46K), each linked to familial Parkinson's disease, has altered aggregation properties, fibril morphologies, and fibrillization kinetics. Besides α-syn, Lewy bodies also contain several associated proteins including small heat shock proteins (sHsps). Since α-syn accumulates intracellularly, molecular chaperones like sHsps may regulate α-syn folding and aggregation. Therefore, we investigated if the sHsps αB-crystallin, Hsp27, Hsp20, HspB8, and HspB2B3 bind to α-syn and affect α-syn aggregation. We demonstrate that all sHsps bind to the various α-syns, although the binding kinetics suggests a weak and transient interaction only. Despite this transient interaction, the various sHsps inhibited mature α-syn fibril formation as shown by a Thioflavin T assay and atomic force microscopy. Interestingly, HspB8 was the most potent sHsp in inhibiting mature fibril formation of both wild-type and mutant α-syn. In conclusion, sHsps may regulate α-syn aggregation and, therefore, optimization of the interaction between sHsps and α-syn may be an interesting target for therapeutic intervention in the pathogenesis of α-synucleinopathies.     

Structural and functional specificity of small heat shock protein HspB1 and HspB4, two cellular partners of HspB5: role of the in vitro hetero-complex formation in chaperone activity

The ubiquitous small heat shock proteins are essential elements in cellular protection, through a molecular chaperone activity. Among them, human small heat shock protein HspB1, HspB4 and HspB5 are involved in oncogenesis, anti-apoptotic activity and lens transparency. Therefore, these proteins are potential therapeutic targets in many diseases. Their general chaperone activity is related to their dynamic and multiple oligomeric structures, which are still poorly understood. The tissue selective distribution of HspB1 and HspB4, two cellular partners of HspB5, suggests that these two proteins might have evolved to play distinct physiological functions. Moreover, hetero-complex formation seems to be favoured in?vivo, yet the functional specificity of the HspB1-HspB5 and HspB4-HspB5 hetero-complexes compared to the homo-oligomers remains unclear in the stress response pathway. A powerful approach combining biochemistry, biophysics and bioinformatics, allowed us to compare the different assemblies, with a special emphasis on the structural data, subunit exchange properties, activity and sequence evolution. We showed that they all exhibit different properties, from structural organization in physiological versus stress conditions, to chaperone-like activity, whatever the level of sequence conservation. Subunit exchange kinetics leading to HspB1-HspB5 or HspB4-HspB5 hetero-complex formation is also different between these two complexes: HspB5 exchanges more rapidly subunits with HspB1 than with HspB4. The relative sequence conservation in the sHSP superfamily does hide important structural heterogeneity and flexibility, which confer an enlarged range of different surface necessary to efficiently form complexes with various stress-induced cellular targets. Our data suggest that the formation of hetero-complexes could be an original evolutionary strategy to gain new cellular functions.     

Phosphorylation of human small heat shock protein HspB8 (Hsp22) by ERK1 protein kinase

A number of phosphomimicking mutants (replacement of Ser/Thr residues by Asp) of human small heat shock protein HspB8 were obtained and phosphorylation of the wild type HspB8 and its mutants by ERK1 kinase was analyzed in vitro. Mutation S159D does not affect phosphorylation, whereas mutations S24D and S27D equally moderately inhibited and mutation T87D strongly inhibited phosphorylation of HspB8. The double mutations S24D/T87D and S27D/T87D induced very strong inhibitory effect and the triple mutations S24D/S27D/T87D completely prevented phosphorylation catalyzed by ERK1. Thus, Ser24 and Thr87, found to be phosphorylated in vivo, are among the sites phosphorylated by ERK1 in HspB8 in vitro. Mutations S24D and T87D affect intrinsic tryptophan fluorescence and susceptibility to chymotrypsinolysis of HspB8. Phosphomimicking mutations and phosphorylation promote concentration-dependent association of HspB8 subunits. Mutations S24D and S27D decrease, whereas mutation T87D increases the chaperone-like activity of HspB8. It is concluded that phosphorylation catalyzed by ERK1 might affect the structure and chaperone-like activity of HspB8 and therefore can be important for regulation of interaction of HspB8 with different target proteins.     

The problem of protein kinase activity of small heat shock protein Hsp22 (H11 or HspB8)

The recently described protein denoted H11, Hsp22 or HspB8 seems to participate in regulation of proliferation, apoptosis, and cardiac hypertrophy. Mutation of Hsp22 causes distal motor neuropathy. Multitude action of Hsp22 is supposed to be due to its protein kinase and/or chaperone-like activities. There are many indirect evidences indicating that Hsp22 possesses intrinsic protein kinase activity. However, low homology to protein kinases, low extent of autophosphorylation, lack of significant protein kinase activity with commonly used substrates, and lack of information on stoichiometry, kinetics, and substrate specificity make the existence of intrinsic protein kinase activity of Hsp22 questionable. It is supposed that protein kinase activity ascribed to Hsp22 is due to contaminating protein kinases. Hsp22 is highly homologous to small heat shock proteins and effectively prevents aggregation of denatured protein both in vitro and in vivo. Therefore, it is supposed that chaperone-like activity is of great importance for Hsp22 functioning.     

Heterooligomeric complexes of human small heat shock proteins

Oligomeric association of human small heat shock proteins HspB1, HspB5, HspB6 and HspB8 was analyzed by means of size-exclusion chromatography, analytical ultracentrifugation and chemical cross-linking. Wild-type HspB1 and Cys mutants of HspB5, HspB6 and HspB8 containing a single Cys residue in position homologous to that of Cys137 of human HspB1 were able to generate heterodimers cross-linked by disulfide bond. Cross-linked heterodimers between HspB1/HspB5, HspB1/HspB6 and HspB5/HspB6 were easily produced upon mixing, whereas formation of any heterodimers with participation of HspB8 was significantly less efficient. The size of heterooligomers formed by HspB1/HspB6 and HspB5/HspB6 was different from the size of the corresponding homooligomers. Disulfide cross-linked homodimers of small heat shock proteins were unable to participate in heterooligomer formation. Thus, monomers can be involved in subunit exchange leading to heterooligomer formation and restriction of flexibility induced by disulfide cross-linking prevents subunit exchange.     

Temperature and concentration-controlled dynamics of rhizobial small heat shock proteins.

A hallmark of alpha-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 nanoelectrospray 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 glycine residue at the C-terminal end of the alpha-crystallin domain, formed labile complexes that disassembled more readily than the corresponding wild-type proteins. Reduced complex stability coincided with reduced chaperone activity.     

Chaperone Activity of Small Heat Shock Proteins Underlies Therapeutic Efficacy in Experimental Autoimmune Encephalomyelitis

To determine whether the therapeutic activity of αB crystallin, small heat shock protein B5 (HspB5), was shared with other human sHsps, a set of seven human family members, a mutant of HspB5 G120 known to exhibit reduced chaperone activity, and a mycobacterial sHsp were expressed and purified from bacteria. Each of the recombinant proteins was shown to be a functional chaperone, capable of inhibiting aggregation of denatured insulin with varying efficiency. When injected into mice at the peak of disease, they were all effective in reducing the paralysis in experimental autoimmune encephalomyelitis. Additional structure activity correlations between chaperone activity and therapeutic function were established when linear regions within HspB5 were examined. A single region, corresponding to residues 73–92 of HspB5, forms amyloid fibrils, exhibited chaperone activity, and was an effective therapeutic for encephalomyelitis. The linkage of the three activities was further established by demonstrating individual substitutions of critical hydrophobic amino acids in the peptide resulted in the loss of all of the functions.