Alpha-crystallin-type heat shock proteins: socializing minichaperones in the context of a multichaperone network.

Alpha-crystallins were originally recognized as proteins contributing to the transparency of the mammalian eye lens. Subsequently, they have been found in many, but not all, members of the Archaea, Bacteria, and Eucarya. Most members of the diverse alpha-crystallin family have four common structural and functional features: (i) a small monomeric molecular mass between 12 and 43 kDa; (ii) the formation of large oligomeric complexes; (iii) the presence of a moderately conserved central region, the so-called alpha-crystallin domain; and (iv) molecular chaperone activity. Since alpha-crystallins are induced by a temperature upshift in many organisms, they are often referred to as small heat shock proteins (sHsps) or, more accurately, alpha-Hsps. Alpha-crystallins are integrated into a highly flexible and synergistic multichaperone network evolved to secure protein quality control in the cell. Their chaperone activity is limited to the binding of unfolding intermediates in order to protect them from irreversible aggregation. Productive release and refolding of captured proteins into the native state requires close cooperation with other cellular chaperones. In addition, alpha-Hsps seem to play an important role in membrane stabilization. The review compiles information on the abundance, sequence conservation, regulation, structure, and function of alpha-Hsps with an emphasis on the microbial members of this chaperone family.     

A critical motif for oligomerization and chaperone activity of bacterial alpha-heat shock proteins.

Oligomerization into multimeric complexes is a prerequisite for the chaperone function of almost all alpha-crystallin type heat shock proteins (alpha-Hsp), but the molecular details of complex assembly are poorly understood. The alpha-Hsp proteins from Bradyrhizobium japonicum are suitable bacterial models for structure-function studies of these ubiquitous stress proteins. They fall into two distinct classes, A and B, display chaperone activity in vitro and form oligomers of approximately 24 subunits. We constructed 19 derivatives containing truncations or point mutations within the N- and C-terminal regions and analyzed them by gel filtration, citrate synthase assay and coaffinity purification. Truncation of more than the initial few amino acids of the N-terminal region led to the formation of distinct dimeric to octameric structures devoid of chaperone activity. In the C-terminal extension, integrity of an isoleucine-X-isoleucine (I-X-I) motif was imperative for alpha-Hsp functionality. This I-X-I motif is one of the characteristic consensus motifs of the alpha-Hsp family, and here we provide experimental evidence of its structural and functional importance. alpha-Hsp proteins lacking the C-terminal extension were inactive, but still able to form dimers. Here, we demonstrate that the central alpha-crystallin domain alone is not sufficient for dimerization. Additional residues at the end of the N-terminal region were required for the assembly of two subunits.     

Analysis of the alphaB-crystallin domain responsible for inhibiting tubulin aggregation

The cytoskeleton has a unique property such that changes of conformation result in polymerization into a filamentous form. alphaB-Crystallin, a small heat shock protein (sHsp), has chaperone activities for various substrates, including proteins constituting the cytoskeleton, such as actin; intermediate filament; and tubulin. However, it is not clear whether the "alpha-crystallin domain" common to sHsps also has chaperone activity for the protein cytoskeleton. To investigate the possibility that the C-terminal alpha-crystallin domain of alpha-crystallin has the aggregation-preventing ability for tubulin, we constructed an N-terminal domain deletion mutant of alphaB-crystallin. We characterized its structural properties and chaperone activities. Far-ultraviolet (UV) circular dichroism measurements showed that secondary structure in the alpha-crystallin domain of the deletion mutant is maintained. Ultracentrifuge analysis of molecular masses indicated that the deletion mutant formed smaller oligomers than did the full-length protein. Chaperone activity assays demonstrated that the N-terminal domain deletion mutant suppressed heat-induced aggregation of tubulin well. Comparison of chaperone activities for 2 other substrates (citrate synthase and alcohol dehydrogenase) showed that it was less effective in the suppression of their aggregation. These results show that alphaB-crystallin recognizes a variety of substrates and especially that alpha-crystallin domain binds free cytoskeletal proteins. We suggest that this feature would be advantageous in its functional role of holding or folding multiple proteins denatured simultaneously under stress conditions.     

The lack of chaperonelike activity of Caenorhabditis elegans Hsp12.2 cannot be restored by domain swapping with human αB-crystallin

The small heat shock proteins Hsp12.2 and alphaB-crystallin differ in that the former occurs as tetramers, without chaperonelike activity, whereas the latter forms multimers and is a good chaperone. To investigate whether the lack of chaperone activity of Hsp12.2 is primarily due to its tetrameric structure or rather to intrinsic sequence features, we engineered chimeric proteins by swapping the N-terminal, C-terminal, and tail regions of Hsp12.2 and alphaB-crystallin, designated as n-c-t and N-C-T, respectively. Three of the chimeric sHsps, namely N-c-T, n-c-T, and N-C-t, showed nativelike secondary and quaternary structures as measured by circular dichroism and gel permeation chromatography. Combining the conserved alpha-crystallin domain of Hsp12.2 with the N-terminal and tail regions of alphaB-crystallin (N-c-T) resulted in multimeric complexes, but did not restore chaperonelike activity. Replacing the tail region of Hsp12.2 with that of alphaB-crystallin (n-c-T) did not alter the tetrameric structure and lack of chaperone activity. Similarly, providing alphaB-crystallin with the tail of Hsp12.2 (N-C-t) did not substantially influence the multimeric complex size, but it reduced the chaperoning ability, especially for small substrates. These results suggest that the conserved alpha-crystallin domain of Hsp12.2 is intrinsically unsuitable to confer chaperonelike activity and confirms that the tail region in alphaB-crystallin modulates chaperonelike capacity in a substrate-dependent manner.     

Wrapping the alpha-crystallin domain fold in a chaperone assembly

Small heat shock proteins (sHsps) are oligomers that perform a protective function by binding denatured proteins. Although ubiquitous, they are of variable sequence except for a C-terminal approximately 90-residue "alpha-crystallin domain". Unlike larger stress response chaperones, sHsps are ATP-independent and generally form polydisperse assemblies. One proposed mechanism of action involves these assemblies breaking into smaller subunits in response to stress, before binding unfolding substrate and reforming into larger complexes. Two previously solved non-metazoan sHsp multimers are built from dimers formed by domain swapping between the alpha-crystallin domains, adding to evidence that the smaller subunits are dimers. Here, the 2.5A resolution structure of an sHsp from the parasitic flatworm Taenia saginata Tsp36, the first metazoan crystal structure, shows a new mode of dimerization involving N-terminal regions, which differs from that seen for non-metazoan sHsps. Sequence differences in the alpha-crystallin domains between metazoans and non-metazoans are critical to the different mechanism of dimerization, suggesting that some structural features seen for Tsp36 may be generalized to other metazoan sHsps. The structure also indicates scope for flexible assembly of subunits, supporting the proposed process of oligomer breakdown, substrate binding and reassembly as the chaperone mechanism. It further shows how sHsps can bind coil and secondary structural elements by wrapping them around the alpha-crystallin domain. The structure also illustrates possible roles for conserved residues associated with disease, and suggests a mechanism for the sHsp-related pathogenicity of some flatworm infections. Tsp36, like other flatworm sHsps, possesses two divergent sHsp repeats per monomer. Together with the two previously solved structures, a total of four alpha-crystallin domain structures are now available, giving a better definition of domain boundaries for sHsps.     

Site-directed mutations within the core "alpha-crystallin" domain of the small heat-shock protein, human alphaB-crystallin, decrease molecular chaperone functions.

Site-directed mutagenesis was used to evaluate the effects on structure and function of selected substitutions within and N-terminal to the core "alpha-crystallin" domain of the small heat-shock protein (sHsp) and molecular chaperone, human alphaB-crystallin. Five alphaB-crystallin mutants containing single amino acid substitutions within the core alpha-crystallin domain displayed a modest decrease in chaperone activity in aggregation assays in vitro and in protecting cell viability of E. coli at 50 degrees C in vivo. In contrast, seven alphaB-crystallin mutants containing substitutions N-terminal to the core alpha-crystallin domain generally resembled wild-type alphaB-crystallin in chaperone activity in vitro and in vivo. Size-exclusion chromatography, ultraviolet circular dichroism spectroscopy and limited proteolysis were used to evaluate potential structural changes in the 12 alphaB-crystallin mutants. The secondary, tertiary and quaternary structures of mutants within and N-terminal to the core alpha-crystallin domain were similar to wild-type alphaB-crystallin. SDS-PAGE patterns of chymotryptic digestion were also similar in the mutant and wild-type proteins, indicating that the mutations did not introduce structural modifications that altered the exposure of proteolytic cleavage sites in alphaB-crystallin. On the basis of the similarities between the sequences of human alphaB-crystallin and the sHsp Mj HSP16.5, the only sHsp for which there exists high resolution structural information, a three-dimensional model for alphaB-crystallin was constructed. The mutations at sites within the core alpha-crystallin domain of alphaB-crystallin identify regions that may be important for the molecular chaperone functions of sHsps.     

Structural and functional roles for beta-strand 7 in the alpha-crystallin domain of p26, a polydisperse small heat shock protein from Artemia franciscana

Oviparous development in the extremophile crustacean, Artemia franciscana, generates encysted embryos which enter a profound state of dormancy, termed diapause. Encystment is marked by the synthesis of p26, a polydisperse small heat shock protein thought to protect embryos from stress. In order to elucidate structural/functional relationships within p26 and other polydisperse small heat shock proteins, and to better define the protein's role during diapause, amino acid substitutions R110G, F112R, R114A and Y116D were generated within the p26 alpha-crystallin domain by site-directed mutagenesis. These residues were chosen because they are highly conserved across species boundaries, and molecular modelling indicates that they are part of a key structural interface between dimers. The F112R mutation, which had the greatest impact on oligomerization, placed two charged residues at the p26 dimer-dimer interface, demonstrating the importance of beta-strand 7 in tetramer formation. All mutated versions of p26 were less able than wild-type p26 to confer thermotolerance on transformed bacteria and they exhibited diminished chaperone action in three in vitro assays; however, all variants retained protective activity. This apparent stability of p26 may, by prolonging effective chaperone life in vivo, enhance embryo stress resistance. All substitutions modified p26 intrinsic fluorescence, surface hydrophobicity and secondary structure, and the pronounced changes in variant R114A, as indicated by these physical measurements, correlated with the greatest loss of function. Although mutation R114A had the greatest effect on p26 chaperoning, it had the least on oligomerization. These results demonstrate that in contrast to many other small heat shock proteins, p26 effectiveness as a chaperone is independent of oligomerization. The results also reinforce the idea, occasioned by modelling, that R114 is removed slightly from dimer-dimer interfaces. Moreover, beta-strand 7 is shown to have an important role in oligomerization of p26, a function first proposed for this structural element upon crystallization of wheat Hsp16.9, a small heat shock protein with different quaternary structure.     

Alpha-crystallin and ATP facilitate the in vitro renaturation of xylanase: enhancement of refolding by metal ions

Alpha-crystallin is a multimeric protein that functions as a molecular chaperone and shares extensive structural homology to small heat shock proteins. For the functional in vitro analysis of alpha-crystallin, the xylanase Xyl II from alkalophilic thermophilic Bacillus was used as a model system. The mechanism of chaperone action of alpha-crystallin is less investigated. Here we studied the refolding of Gdn HCl-denatured Xyl II in the presence and absence of alpha-crystallin to elucidate the molecular mechanism of chaperone-mediated in vitro folding. Our results, based on intrinsic tryptophan fluorescence and hydrophobic fluorophore 8-anilino-1-naphthalene sulfonate binding studies, suggest that alpha-crystallin formed a complex with a putative molten globule-like intermediate in the refolding pathway of Xyl II. The alpha-crystallin.Xyl II complex exhibited no functional activity. Addition of ATP to the complex initiated the renaturation of Xyl II with 30%-35% recovery of activity. The nonhydrolyzable analog 5'-adenylyl imidodiphosphate (AMP-PNP) was capable of reconstitution of active Xyl II to a lesser extent than ATP. Although the presence of Ca(2+) was not required for the in vitro refolding of Xyl II, the renaturation yield was enhanced in its presence. Experimental evidence indicated that the binding of ATP to the alpha-crystallin.Xyl II complex brought about conformational changes in alpha-crystallin facilitating the dissociation of xylanase molecules. This is the first report of the enhancement of alpha-crystallin chaperone functions by metal ions.     

Detection of oligomerisation and substrate recognition sites of small heat shock proteins by peptide arrays

Small heat shock proteins (sHsps) form large oligomers that are characterised by their dynamic behaviour, e.g., complex disassembly/reassembly and extensive subunit exchange. These processes are interrelated with sHsp/substrate interaction. sHsps bind a broad spectrum of unrelated substrate proteins under denaturing conditions. Detailed knowledge about the binding process and regions critical for sHsp/substrate interaction is missing. In this study, we screened cellulose-bound peptide spot libraries derived from a bacterial sHsp and the model-substrate citrate synthase to detect oligomerisation and substrate interaction sites, respectively. In line with previous results, it was demonstrated that multiple contacts involving the N- and C-terminal extensions and the central alpha-crystallin domain are required for oligomerisation. Incubation of the citrate synthase membrane with sHsps revealed a putative substrate interaction site. A soluble peptide with the sequence RTKYWELIYEDCMDL (CS(191-205)) corresponding to that site inhibited chaperone activity of sHsps, presumably by blocking their substrate-binding sites.     

p23 and HSP20/alpha-crystallin proteins define a conserved sequence domain present in other eukaryotic protein families

We identified families of proteins characterized by the presence of a domain similar to human p23 protein, which include proteins such as Sgt1, involved in the yeast kinetochore assembly; melusin, involved in specific interactions with the cytoplasmic integrin beta1 domain; Rar1, related to pathogenic resistance in plants, and to development in animals; B5+B5R flavo-hemo cytochrome NAD(P)H oxidoreductase type B in humans and mice; and NudC, involved in nucleus migration during mitosis. We also found that p23 and the HSP20/alpha-crystallin family of heat shock proteins, which share the same three-dimensional folding, show a pattern of conserved residues that points to a common origin in the evolution of both protein domains. The p23 and HSP20/alpha-crystallin phylogenetic relationship and their similar role in chaperone activity suggest a common function, probably involving protein-protein interaction, for those proteins containing p23-like domains.     

Role of the C-terminal extensions of alpha-crystallins. Swapping the C-terminal extension of alpha-crystallin to alphaB-crystallin results in enhanced chaperone activity

Several small heat shock proteins contain a well conserved alpha-crystallin domain, flanked by an N-terminal domain and a C-terminal extension, both of which vary in length and sequence. The structural and functional role of the C-terminal extension of small heat shock proteins, particularly of alphaA- and alphaB-crystallins, is not well understood. We have swapped the C-terminal extensions between alphaA- and alphaB-crystallins and generated two novel chimeric proteins, alphaABc and alphaBAc. We have investigated the domain-swapped chimeras for structural and functional alterations. We have used thermal and non-thermal models of protein aggregation and found that the chimeric alphaB with the C-terminal extension of alphaA-crystallin, alphaBAc, exhibits dramatically enhanced chaperone-like activity. Interestingly, however, the chimeric alphaA with the C-terminal extension of alphaB-crystallin, alphaABc, has almost lost its activity. Pyrene solubilization and bis-1-anilino-8-naphthalenesulfonate binding studies show that alphaBAc exhibits more solvent-exposed hydrophobic pockets than alphaA, alphaB, or alphaABc. Significant tertiary structural changes are revealed by tryptophan fluorescence and near-UV CD studies upon swapping the C-terminal extensions. The far-UV CD spectrum of alphaBAc differs from that of alphaB-crystallin whereas that of alphaABc overlaps with that of alphaA-crystallin. Gel filtration chromatography shows alteration in the size of the proteins upon swapping the C-terminal extensions. Our study demonstrates that the unstructured C-terminal extensions play a crucial role in the structure and chaperone activity, in addition to generally believed electrostatic "solubilizer" function.     

Oligomerization, chaperone activity, and nuclear localization of p26, a small heat shock protein from Artemia franciscana

Artemia franciscana embryos undergo encystment, developmental arrest and diapause, the last characterized by profound metabolic dormancy and extreme stress resistance. Encysted embryos contain an abundant small heat shock protein termed p26, a molecular chaperone that undoubtedly has an important role in development. To understand better the role of p26 in Artemia embryos, the structural and functional characteristics of full-length and truncated p26 expressed in Escherichia coli and COS-1 cells were determined. p26 chaperone activity declined with increasing truncation of the protein, and those deletions with the greatest adverse effect on protection of citrate synthase during thermal stress had the most influence on oligomerization. When produced in either prokaryotic or eukaryotic cells the p26 alpha-crystallin domain consisting of amino acid residues 61-152 existed predominantly as monomers, and p26 variants lacking the amino-terminal domain but with intact carboxyl-terminal extensions were mainly monomers and dimers. The amino terminus was, therefore, required for efficient dimer formation. Assembly of higher order oligomers was enhanced by the carboxyl-terminal extension, although removing the 10 carboxyl-terminal residues had relatively little effect on oligomerization and chaperoning. Full-length and carboxyl-terminal truncated p26 resided in the cytoplasm of transfected COS-1 cells; however, variants missing the complete amino-terminal domain and existing predominantly as monomers/dimers entered the nuclei. A mechanism whereby oligomer disassembly assisted entry of p26 into nuclei was suggested, this of importance because p26 translocates into Artemia embryo nuclei during development and stress. However, when examined in Artemia, the p26 oligomer size was unchanged under conditions that allowed movement into nuclei, suggesting a process more complex than just oligomer dissociation.     

The excised heat-shock domain of alphaB crystallin is a folded, proteolytically susceptible trimer with significant surface hydrophobicity and a tendency to self-aggregate upon heating

The lens protein, alpha-crystallin, is a molecular chaperone that prevents the thermal aggregation of other proteins. The C-terminal domain of this protein (homologous to domains present in small heat-shock proteins) is implicated in chaperone function, although the domain itself has been reported to show no chaperone activity. Here, we show that the domain can be excised out of the intact alphaB polypeptide and recovered directly in pure form through the transfer of CNBr digests of whole lens homogenates into urea-containing buffer, followed by dialysis-based refolding of digests under acidic conditions and a single gel-filtration purification step. The folded (beta sheet) domain thus obtained is found to be (a) predominantly trimeric, and to display (b) significant surface hydrophobicity, (c) a marked tendency to undergo degradation, and (d) a tendency to aggregate upon heating, and on exposure to UV light. Thus, the twin 'chaperone' features of multimericity and surface hydrophobicity are clearly seen to be insufficient for this domain to function as a chaperone. Since alpha-crystallin interacts with its substrates through hydrophobic interactions, the hydrophobicity of the excised domain indicates that separation of domains may regulate function; at the same time, the fact is also highlighted that surface hydrophobicity is a liability in a chaperone since heating strengthens hydrophobic interactions and can potentially promote self-aggregation. Thus, it would appear that the role of the N-terminal domain in alpha-crystallin is to facilitate the creation of a porous, hollow structural framework of >/=24 subunits in which solubility is effected through increase in the ratio of exposed surface area to buried volume. Trimers of interacting C-terminal domains anchored to this superstructure, and positioned within its interior, might allow hydrophobic surfaces to remain accessible to substrates without compromising solubility.     

Transthyretin is up-regulated by sex hormones in mice liver

The chaperone function of alpha-crystallin is significantly affected in diabetes. Increased formation of advanced glycation end products (AGEs) is the likely cause. This study was aimed to investigate the effect of AGE crosslinks on the chaperone activity of alpha-crystallin and to show the effect of an AGEs crosslink breaker, phenacyl-4,5-dimethylthiazolium bromide (DMPTB). Recombinant alphaA-crystallin was prepared by expressing it in Escherichia coli and purified by size exclusion chromatography. Glycation of alphaA-crystallin was performed with 1-100 mM glucose-6-phosphate (G6P) as the glycating agent for a period of 1-15 days. To break AGE crosslinks, pre-glycated alphaA-crystallin was treated with 0.1-20 mM DMPTB for 3 days. Excess G6P and DMPTB were removed by gel filtration before performing additional experiments. AGEs and crosslinked proteins were estimated by measuring non-tryptophan fluorescence and by SDS-PAGE. Chaperone activity was determined with alcohol dehydrogenase as the target protein. With increasing duration of glycation and G6P concentration, chaperone activity of alpha-crystallin decreased. When pre-glycated alphaA-crystallin was treated with 5-20 mM DMPTB, a DMPTB concentration-dependent recovery of chaperone activity was seen. Lower concentrations, 0.1, 0.5, and 1.0 mM, of DMPTB also showed significant recovery of the chaperone activity. SDS-PAGE analysis after DMPTB treatment showed 40% decrease in crosslinked proteins and fluorescence scan indicated 30% decrease in AGEs. DMPTB is expected to regain alpha-crystallin chaperone activity and provide structural stability to other eye lens proteins that are in aggregation mode which emphasizes the clinical importance of the present finding.     

The N-terminal domain of alphaB-crystallin is protected from proteolysis by bound substrate

Alpha-crystallin, a major structural protein of the lens can also function as a molecular chaperone by binding to unfolding substrate proteins. We have used a combination of limited proteolysis at low temperature, and mass spectrometry to identify the regions of alpha-crystallin directly involved in binding to the structurally compromised substrate, reduced alpha-lactalbumin. In the presence of trypsin, alpha-crystallin which had been pre-incubated with substrate showed markedly reduced proteolysis at the C-terminus compared with a control, indicating that the bound substrate restricted access of trypsin to R157, the main cleavage site. Chymotrypsin was able to cleave at residues in both the N- and C-terminal domains. In the presence of substrate, alpha-crystallin showed markedly reduced proteolysis at four sites in the N-terminal domain when compared with the control. Minor differences in cleavage were observed within the C-terminal domain suggesting that the N-terminal region of alpha-crystallin contains the major substrate interaction sites.     

A novel quaternary structure of the dimeric alpha-crystallin domain with chaperone-like activity

alphaB-crystallin, a member of the small heat-shock protein family and a major eye lens protein, is a high molecular mass assembly and can act as a molecular chaperone. We report a synchrotron radiation x-ray solution scattering study of a truncation mutant from the human alphaB-crystallin (alphaB57-157), a dimeric protein that comprises the alpha-crystallin domain of the alphaB-crystallin and retains a significant chaperone-like activity. According to the sequence analysis (more than 23% identity), the monomeric fold of the alpha-crystallin domain should be close to that of the small heat-shock protein from Methanococcus jannaschii (MjHSP16.5). The theoretical scattering pattern computed from the crystallographic model of the dimeric MjHSP16.5 deviates significantly from the experimental scattering by the alpha-crystallin domain, pointing to different quaternary structures of the two proteins. A rigid body modeling against the solution scattering data yields a model of the alpha-crystallin domain revealing a new dimerization interface. The latter consists of a strand-turn-strand motif contributed by each of the monomers, which form a four-stranded, antiparallel, intersubunit composite beta-sheet. This model agrees with the recent spin labeling results and suggests that the alphaB-crystallin is composed by flexible building units with an extended surface area. This flexibility may be important for biological activity and for the formation of alphaB-crystallin complexes of variable sizes and compositions.     

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.     

Thermal stability of human alpha-crystallins sensed by amide hydrogen exchange

The alpha-crystallins, alphaA and alphaB, are major lens structural proteins with chaperone-like activity and sequence homology to small heat-shock proteins. As yet, their crystal structures have not been determined because of the large size and heterogeneity of the assemblies they form in solution. Because alpha-crystallin chaperone activity increases with temperature, understanding structural changes of alpha-crystallin as it is heated may help elucidate the mechanism of chaperone activity. Although a variety of techniques have been used to probe changes in heat-stressed alpha-crystallin, the results have not yet yielded a clear understanding of chaperone activity. We report examination of native assemblies of human lens alpha-crystallin using hydrogen/deuterium exchange in conjunction with enzymatic digestion and analysis by mass spectrometry. This technique has the advantage of sensing structural changes along much of the protein backbone and being able to detect changes specific to alphaA and alphaB in the native assembly. The reactivity of the amide linkages to hydrogen/deuterium exchange was determined for 92% of the sequence of alphaA and 99% of alphaB. The behavior of alphaA and alphaB is remarkably similar. At low temperatures, there are regions at the beginning of the alpha-crystallin domains in both alphaA and alphaB that have high protection to isotope exchange, whereas the C termini offer little protection. The N terminus of alphaA also has low protection. With increasing temperatures, both proteins show gradual unfolding. The maximum percent change in exposure with increasing temperatures was found in alphaA 72-75 and alphaB 76-79, two regions considered critical for chaperone activity.     

Tsp36, a tapeworm small heat-shock protein with a duplicated alpha-crystallin domain, forms dimers and tetramers with good chaperone-like activity

Small heat shock proteins (sHSPs), which range in monomer size between 12 and 42 kDa, are characterized by a conserved C-terminal alpha-crystallin domain of 80-100 residues. They generally form large homo- or heteromeric complexes, and typically have in vitro chaperone-like activity, keeping unfolding proteins in solution. A special type of sHSP, with a duplicated alpha-crystallin domain, is present in parasitic flatworms (Platyhelminthes). Considering that an alpha-crystallin domain is essential for the oligomerization and chaperone-like properties of sHSPs, we characterized Tsp36 from the tapeworm Taenia saginata. Both wild-type Tsp36 and a mutant (Tsp36C-->R) in which the single cysteine has been replaced by arginine were expressed and purified. Far-UV CD measurements of Tsp36 were in agreement with secondary structure predictions, which indicated alpha-helical structure in the N-terminal region and the expected beta-sandwich structure for the two alpha-crystallin domains. Gel permeation chromatography and nano-ESI-MS showed that wild type Tsp36 forms dimers in a reducing environment, and tetramers in a non-reducing environment. The tetramers are stabilized by disulfide bridges involving a large proportion of the Tsp36 monomers. Tsp36C-->R exclusively occurs as dimers according to gel permeation chromatography, while the nondisulfide bonded fraction of wild type Tsp36 dissociates from tetramers into dimers under nonreducing conditions at increased temperature (43 degrees C). The tetrameric form of Tsp36 has a greater chaperone-like activity than the dimeric form.     

Structure-based analysis of the beta8 interactive sequence of human alphaB crystallin

The functional importance of the beta8 sequence ((131)LTITSSLS(138)), which is on the surface of the alpha crystallin core domain of human alphaB crystallin, was evaluated using site-directed mutagenesis. Ultraviolet circular dichroism determined that mutating the surface-exposed, nonconserved residues, Leu-131, Thr-132, Thr-134, Ser-135, Ser-136, and Ser-138 individually or in combination (alphaAbeta8 and CEbeta8), had no measurable effect on secondary and tertiary structure. Size exclusion chromatography determined the size of the complexes formed by the beta8 mutants to be 6-8 subunits larger than wt alphaB crystallin. In chaperone assays, the protective effect of the L131S, T132A, and S135C mutants of the beta8 sequence was similar to wt alphaB crystallin when beta(L) crystallin and alcohol dehydrogenase were the chaperone substrates and decreased to 66% when citrate synthase was the chaperone substrate. In contrast, the chaperone activity for all three substrates was dramatically reduced for the T134K, S138A, S136H, and CEbeta8 mutants. The prominent location of Thr-134, Ser-136, and Ser-138 on the exposed surface of the alpha crystallin core domain could account for the effect on complex assembly and chaperone activity. Modulation of chaperone activity by the exposed residues of the beta8 sequence in the alpha crystallin core domain was independent of complex size. The results established the beta3-beta8-beta9 surface of the alpha crystallin core domain as an interface for complex assembly and chaperone activity.