Structural perturbation of alphaB-crystallin by zinc and temperature related to its chaperone-like activity

alphaB-crystallin is a small heat shock protein that shows chaperone-like activity, as it protects the aggregation of denatured proteins. In this work, the possible relationships between structural characteristics and the biological activity of alphaB-crystallin were investigated on the native protein and on the protein undergoing the separate effects of metal ligation and temperature. The chaperone-like activity of alphaB-crystallin increased in the presence of zinc and when temperature was increased. By using fluorescent probes to monitor hydrophobic surfaces on alphaB-crystallin, it was found that exposed hydrophobic patches on the protein surface increased significantly both in the presence of zinc and when the temperature was raised from 25 to 37 degrees C. The zinc-induced increased exposure of lipophilic residues is in agreement with theoretical calculations performed on 3D-models of monomeric alphaB-crystallin, and may be significant to its increased biological activity.     

Effects of divalent metal ions on the alphaB-crystallin chaperone-like activity: spectroscopic evidence for a complex between copper(II) and protein

AlphaB-crystallin is a small heat shock protein, showing chaperone-like activity, that is expressed in the lens and in several other tissues. The role of some metal ions in the alphaB-crystallin biology starts to be well documented. In some neuro-degenerative pathologies, like Parkinson and Alzheimer's diseases, alphaB-crystallin is expressed at high levels. In the same pathologies an accumulation of divalent metal cations is observed. In order to investigate the interactions between human alphaB-crystallin and divalent metal ions, the effect of copper, zinc and calcium on the chaperone-like activity of the protein has been studied. Copper and zinc at concentrations 0.1 and 1 mM significantly increase the chaperone-like activity, whereas calcium 1 mM completely inhibits activity. Electron paramagnetic resonance (EPR) and circular dichroism (CD) spectra indicate the possible complex formation between Cu(II) and protein at physiological pH. Molecular modeling calculations, carried out for the probable Cu(II) binding site, suggest that a complex with three histidine residues is possible.     

Differential temperature-dependent chaperone-like activity of alphaA- and alphaB-crystallin homoaggregates

alpha-Crystallin, a heteromultimeric protein made up of alphaA- and alphaB-crystallins, functions as a molecular chaperone in preventing the aggregation of proteins. We have shown earlier that structural perturbation of alpha-crystallin can enhance its chaperone-like activity severalfold. The two subunits of alpha-crystallin have extensive sequence homology and individually display chaperone-like activity. We have investigated the chaperone-like activity of alphaA- and alphaB-crystallin homoaggregates against thermal and nonthermal modes of aggregation. We find that, against a nonthermal mode of aggregation, alphaB-crystallin shows significant protective ability even at subphysiological temperatures, at which alphaA-crystallin or heteromultimeric alpha-crystallin exhibit very little chaperone-like activity. Interestingly, differences in the protective ability of these homoaggregates against the thermal aggregation of beta(L)-crystallin is negligible. To investigate this differential behavior, we have monitored the temperature-dependent structural changes in both the proteins using fluorescence and circular dichroism spectroscopy. Intrinsic tryptophan fluorescence quench-ing by acrylamide shows that the tryptophans in alphaB-crystallin are more accessible than the lone tryptophan in alphaA-crystallin even at 25 degrees C. Protein-bound 8-anilinonaphthalene-1-sulfonate fluorescence demonstrates the higher solvent accessibility of hydrophobic surfaces on alphaB-crystallin. Circular dichroism studies show some tertiary structural changes in alphaA-crystallin above 50 degrees C. alphaB-crystallin, on the other hand, shows significant alteration of tertiary structure by 45 degrees C. Our study demonstrates that despite a high degree of sequence homology and their generally accepted structural similarity, alphaB-crystallin is much more sensitive to temperature-dependent structural perturbation than alphaA- or alpha-crystallin and shows differences in its chaperone-like properties. These differences appear to be relevant to temperature-dependent enhancement of chaperone-like activity of alpha-crystallin and indicate different roles for the two proteins both in alpha-crystallin heteroaggregate and as separate proteins under stress conditions.     

Temperature dependence of chaperone-like activity and oligomeric state of alphaB-crystallin

The chaperone-like activity and the oligomeric state of alphaB-crystallin were studied at different temperatures and in the presence of urea and thiocyanate. The activity, assessed measuring the ability of alphaB-crystallin to prevent the aggregation of denatured insulin, strongly depends on temperature. While a significant activity increase was detected at 42 degrees C, the presence of urea and thiocyanate does not affect the protein activity in an irreversible way. In-solution SAXS measurements performed in the same experimental conditions showed that alphaB-crystallin forms near-spherical, hollowed, polydisperse oligomers, whose dimensions change above 42 degrees C. Moreover, in the presence of urea and thiocyanate, a global fit analysis confirms the high stability of alphaB-crystallin assemblies in relationship with their variable quaternary structure. In particular, the changes in the inner radius as well as the thickness and dispersion of the protein shell, account for the preservation of the chaperone-like activity.     

C-terminal lysine truncation increases thermostability and enhances chaperone-like function of porcine alphaB-crystallin

The carboxyl-terminal segment of alpha-crystallin, a major lens protein of all vertebrates, has a short and flexible peptide extension of about 20 amino acid residues that are very susceptible to proteolytic truncation and modifications under physiological conditions. To investigate its role in crystallin aggregation and chaperone-like activity, we constructed a mutant of porcine alphaB-crystallin with C-terminal lysine truncated end, which unexpectedly showed better chaperone-like function than wild-type alphaB-crystallin. From circular dichroism (CD) spectra, we show that the mutant possesses similar secondary and tertiary structures to those of native purified and recombinant alphaB-crystallins. Analytical ultracentrifugation revealed that the truncated mutant was smaller than wild-type alphaB-crystallin in aggregation size and mass. The observed higher thermostability and anti-thermal aggregation propensity of the truncated alphaB-crystallin mutant than wild-type alphaB-crystallin are in contrast to the prevailing notion that mutations at the C-terminal lysines of alphaB-crystallin result in substantial loss of chaperone-like activity, despite the overall preservation of secondary structure. The detailed characterization of the C-terminal deletion mutants may provide some deeper insight into the chaperoning mechanism of the structurally related small heat-shock protein family.     

Mini-alphaB-crystallin: a functional element of alphaB-crystallin with chaperone-like activity

Alpha-crystallin is a member of the family of small heat-shock proteins (sHSP) and is composed of two subunits, alphaA-crystallin and alphaB-crystallin, which exhibit molecular chaperone-like properties. In a previous study, we found that residues 70-88 in alphaA-crystallin can function like a molecular chaperone by preventing the aggregation and precipitation of denaturing substrate proteins [Sharma, K. K., et al. (2000) J. Biol. Chem. 275, 3767-3771]. In this study, we show that the complementary sequence in alphaB-crystallin, residues 73-92 (DRFSVNLDVKHFSPEELKVK), is the functional chaperone site of alphaB-crystallin. Like the mini-alphaA-crystallin chaperone, the mini-alphaB-crystallin chaperone interacts with 1,1'-bi(4-anilino) naphthalene-5,5'-disulphonic acid (bis-ANS) and also possesses significant beta-sheet and random coil structure. Deletion of four residues (DRFS) from the N-terminus or deletion of C-terminus LKVK residues from the 73-92 peptide abolishes the chaperone-like activity against denaturing alcohol dehydrogenase. However, removal of DRFS or HFSPEELKVK is necessary to completely abolish the antiaggregation property of the peptide in insulin reduction assay. Substitution of Asp at a site corresponding to D80 in alphaB-crystallin with d-Asp or beta-Asp results in a significant loss of chaperone-like activity. Kynurenine modification of His in the peptide abolishes the antiaggregation property of the mini-chaperone. These data suggest that the 73-92 region in alphaB-crystallin is one of the substrate binding sites during chaperone activity.     

Conserved F84 and P86 residues in alphaB-crystallin are essential to effectively prevent the aggregation of substrate proteins

Previously, we have shown that residues 73-92 (sequence DRFSVNLDVKHFSPEELKVK) in alphaB-crystallin are involved in preventing the formation of light scattering aggregates by substrate proteins. In this study, we made single substitutions of three conserved amino acid residues (H83 --> A, F84 --> G, and P86 --> A) and a nonconserved amino acid residue (K90 --> C) in the functional region of alphaB-crystallin and evaluated their role in anti-aggregation activity. Mutation of conserved residues led to changes in intrinsic tryptophan intensity, bis-ANS binding, and in the secondary and tertiary structures. The H83A mutation led to a twofold increase in molar mass, while the other mutants did not produce significant changes in the molar mass when compared to that of wild-type protein. The chaperone-like activity of the H83A mutant was enhanced by 15%-20%, and the chaperone-like activity of F84G and P86A mutants was reduced by 50%-65% when compared to the chaperone-like activity of wild-type alphaB-crystallin. The substitution of the nonconserved residue (K90 --> C) did not induce an appreciable change in the structure and function of the mutant protein. Fluorescence resonance energy transfer (FRET) assay demonstrated that destabilized ADH interacted near the K90 region in alphaB-crystallin. The data show that F84 and P86 residues are essential for alphaB-crystallin to effectively prevent the aggregation of substrate proteins. This study further supports the involvement of the residues in the 73-92 region of alphaB-crystallin in substrate protein binding and chaperone-like action.     

AlphaA-crystallin interacting regions in the small heat shock protein, alphaB-crystallin

Amino acid sequences of alphaB-crystallin, involved in interaction with alphaA-crystallin, were determined by using peptide scans. Positionally addressable 20-mer overlapping peptides, representing the entire sequence of alphaB-crystallin, were synthesized on a PVDF membrane. The membrane was blocked with albumin and incubated with purified alphaA-crystallin. Probing the membrane with alphaA-crystallin-specific antibodies revealed residues 42-57, 60-71, and 88-123 in alphaB-crystallin to interact with alphaA-crystallin. Residues 42-57 and 60-71 interacted more strongly with alphaA-crystallin than the 88-123 sequence of alphaB-crystallin. Binding of one of the alphaB peptides (42-57) to alphaA-crystallin was also confirmed by gel filtration studies and HPLC analysis. The alphaB-crystallin sequences involved in interaction with alphaA-crystallin were distinct from the chaperone sites reported earlier as binding of the alphaB sequence from residues 42-57 does not alter the chaperone-like function of alphaA-crystallin. To identify the critical residues involved in interaction with alphaA-crystallin, R50G and P51A mutants of alphaB-crystallin were made and tested for their ability to interact with alphaA-crystallin. The oligomeric size and hydrophobicity of the mutants were similar. Circular dichroism studies showed that the P51A mutation increased the alpha-helical content of the protein. While the alphaBR50G mutant showed chaperone-like activity similar to wild-type alphaB, alphaBP51A showed reduced chaperone function. Fluorescence resonance energy transfer studies showed that the P51A mutation decreased the rate of subunit exchange with alphaA by 63%, whereas the R50G mutation reduced the exchange rate by 23%. Similar to wild-type alphaB, alphaB-crystallin peptide (42-57) effectively competed with alphaBP51A and alphaBR50G for interaction with alphaA. Thus, our studies showed that the alphaB-crystallin sequence (42-57) is one of the interacting regions in alphaB and alphaA oligomer formation.     

Structural perturbation and enhancement of the chaperone-like activity of alpha-crystallin by arginine hydrochloride

Structural perturbation of alpha-crystallin is shown to enhance its molecular chaperone-like activity in preventing aggregation of target proteins. We demonstrate that arginine, a biologically compatible molecule that is known to bind to the peptide backbone and negatively charged side-chains, increases the chaperone-like activity of calf eye lens alpha-crystallin as well as recombinant human alphaA- and alphaB-crystallins. Arginine-induced increase in the chaperone activity is more pronounced for alphaB-crystallin than for alphaA-crystallin. Other guanidinium compounds such as aminoguanidine hydrochloride and guanidine hydrochloride also show a similar effect, but to different extents. A point mutation, R120G, in alphaB-crystallin that is associated with desmin-related myopathy, results in a significant loss of chaperone-like activity. Arginine restores the activity of mutant protein to a considerable extent. We have investigated the effect of arginine on the structural changes of alpha-crystallin by circular dichroism, fluorescence, and glycerol gradient sedimentation. Far-UV CD spectra show no significant changes in secondary structure, whereas near-UV CD spectra show subtle changes in the presence of arginine. Glycerol gradient sedimentation shows a significant decrease in the size of alpha-crystallin oligomer in the presence of arginine. Increased exposure of hydrophobic surfaces of alpha-crystallin, as monitored by pyrene-solubilization and ANS-fluorescence, is observed in the presence of arginine. These results show that arginine brings about subtle changes in the tertiary structure and significant changes in the quaternary structure of alpha-crystallin and enhances its chaperone-like activity significantly. This study should prove useful in designing strategies to improve chaperone function for therapeutic applications.     

An atypical form of alphaB-crystallin is present in high concentration in some human cataractous lenses. Identification and characterization of aberrant N- and C-terminal processing.

Two unique polypeptides, 22.4 and 16.4 kDa, were prominent in some human cataracts. Both proteins were identified as modified forms of the small heat shock protein, alphaB-crystallin. The concentration of total alphaB-crystallin in most of these cataracts was significantly increased. The 22.4-kDa protein was subsequently designated as alphaB(g). Mass spectrometric analyses of tryptic and Asp-N digests showed alphaB(g) is alphaB-crystallin minus the C-terminal lysine. alphaB(g) constituted 10-90% of the total alphaB-crystallin in these cataracts and was preferentially phosphorylated over the typical form of alphaB-crystallin. Human alphaB(g) and alphaB-crystallin were cloned and expressed in Escherichia coli. The differences in electrophoretic mobility and the large difference in native pI values suggest some structural differences exist. The chaperone-like activity of recombinant human alphaB(g) was comparable to that of recombinant human alphaB-crystallin in preventing the aggregation of lactalbumin induced by dithiothreitol. The mechanism involved in generating alphaB(g) is not known, but a premature termination of the alphaB-crystallin gene was ruled out by sequencing the polymerase chain reaction products of the last exon for the alphaB-crystallin gene from lenses containing alphaB(g). The 16.4-kDa protein was an N-terminally truncated fragment of alphaB(g). The high concentration of alphaB-crystallin in these cataracts is the first observation of this kind in human lenses.     

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.     

Small heat shock protein alphaB-crystallin is part of cell cycle-dependent Golgi reorganization

AlphaB-crystallin is a developmentally regulated small heat shock protein known for its binding to a variety of denatured polypeptides and suppression of protein aggregation in vitro. Elevated levels of alphaB-crystallin are known to be associated with a number of neurodegenerative pathologies such as Alzheimer disease and multiple sclerosis. Mutations in alphaB-crystallin gene have been linked to desmin related cardiomyopathy and cataractogenesis. The physiological function of this protein, however, is unknown. Using discontinuous sucrose density gradient fractionation of post-nuclear supernatants, prepared from rat tissues and human glioblastoma cell line U373MG, we have identified discrete membrane-bound fractions of alphaB-crystallin, which co-sediment with the Golgi matrix protein, GM130. Confocal microscopy reveals co-localization of alphaB-crystallin with BODIPY TR ceramide and the Golgi matrix protein, GM130, in the perinuclear Golgi in human glioblastoma U373MG cells. Examination of synchronized cultures indicated that alphaB-crystallin follows disassembly of the Golgi at prometaphase and its reassembly at the completion of cytokinesis, suggesting that this small heat shock protein, with its chaperone-like activity, may have an important role in the Golgi reorganization during cell division.     

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.     

Phosphorylation of alphaB-crystallin alters chaperone function through loss of dimeric substructure

Phosphorylation is the most common posttranslational modification of the alpha-crystallins in the human lens. These phosphorylated forms are not only important because of their abundance in aging lenses and the implications for cataract but also because they have been identified in patients with degenerative brain disease. By using mimics corresponding to the reported in vivo phosphorylation sites in the human lens, we have examined the effects of phosphorylation upon the chaperone-like properties and structure of alphaB-crystallin. Here we show that phosphorylation of alphaB-crystallin at Ser-45 results in uncontrolled aggregation. By using an innovative tandem mass spectrometry approach, we demonstrate how this alteration in behavior stems from disruption of dimeric substructure within the polydisperse alphaB-crystallin assembly. This structural perturbation appears to disturb the housekeeping role of alphaB-crystallin and consequently has important implications for the disease states caused by protein aggregation in the lens and deposition in non-lenticular tissue.     

Chaperone-like activity revealed in the matricellular protein SPARC

SPARC (Secreted Protein, Acidic and Rich in Cysteine) is a matricellular glycoprotein that modulates cell proliferation, adhesion, migration, and extracellular matrix (ECM) production. In this report chaperone-like activity of SPARC was identified in a thermal aggregation assay in vitro. Ultraviolet circular dichroism (UVCD) spectroscopy determined that SPARC was stable at temperatures up to 50 degrees C. Unfolding and aggregation of the chaperone target protein, alcohol dehydrogenase (ADH), were initiated at 50 degrees C. SPARC inhibited the thermal aggregation of ADH in a concentration-dependent manner, with maximal inhibition at a 1:4 molar ratio of SPARC:ADH. Synergy between the chaperone-like activities of SPARC and alphaB-crystallin, a small heat shock protein and molecular chaperone in the lens, was observed in SPARC-alphaB-crystallin double -/- mice.     

Thermodynamic stability of human lens recombinant alphaA- and alphaB-crystallins

Lens alpha-crystallin is a 600-800-kDa heterogeneous oligomer protein consisting of two subunits, alphaA and alphaB. The homogeneous oligomers (alphaA- and alphaB-crystallins) have been prepared by recombinant DNA technology and shown to differ in the following biophysical/biochemical properties: hydrophobicity, chaperone-like activity, subunit exchange rate, and thermal stability. In this study, we studied their thermodynamic stability by unfolding in guanidine hydrochloride. The unfolding was probed by three spectroscopic parameters: absorbance at 235 nm, Trp fluorescence intensity at 320 nm, and far-UV circular dichroism at 223 nm. Global analysis indicated that a three-state model better describes the unfolding behavior than a two-state model, an indication that there are stable intermediates for both alphaA- and alphaB-crystallins. In terms of standard free energy (DeltaG(NU)(H(2)(O))), alphaA-crystallin is slightly more stable than alphaB-crystallin. The significance of the intermediates may be related to the functioning of alpha-crystallins as chaperone-like molecules.     

Phosphorylation-induced change of the oligomerization state of alpha B-crystallin

alphaB-crystallin in cells can be phosphorylated at three serine residues in response to stress or during mitosis (Ito, H., Okamoto, K., Nakayama, H., Isobe, T., and Kato, K. (1997) J. Biol. Chem. 272, 29934-29941 and Kato, K., Ito, H., Kamei, K., Inaguma, Y., Iwamoto, I., and Saga, S. (1998) J. Biol. Chem. 273, 28346-28354). In the present study, we determined effects of phosphorylation of alphaB-crystallin on its oligomerization state, mainly by using site-directed mutagenesis, in which all three phosphorylation sites were substituted with aspartate to mimic the phosphorylation state (3D-alphaB). From results of sucrose density gradient centrifugation, we found that wild type alphaB-crystallin (wt-alphaB) and 3D-alphaB sedimented in fractions corresponding to apparent molecular masses of about 500 and 300 kDa, respectively. Chaperone-like activity of 3D-alphaB was significantly weaker than that of wt-alphaB. When wt-alphaB and 3D-alphaB were expressed in COS-m6 cells, they sedimented at positions corresponding to apparent molecular masses of about 500 and 100 kDa, respectively. In U373 MG human glioma cells, alphaB-crystallin was observed as large oligomers with apparent molecular masses about 500 kDa and the oligomerization size was reduced after phosphorylation induced by phorbol 12-myristate 13-acetate and okadaic acid. Coexpression of luciferase and wt-alphaB or 3D-alphaB in Chinese hamster ovary cells caused protection of the enzyme from heat inactivation although the degree of protection with 3D-alphaB was less than that with wt-alphaB. From these observations, it is suggested that phosphorylation of alphaB-crystallin causes dissociation of large oligomers to smaller sizes molecules and reduction of chaperone-like activity, like in the case of HSP27.     

Recognition sequence 2 (residues 60-71) plays a role in oligomerization and exchange dynamics of alphaB-crystallin

Previously, using the peptide scan method, we have determined that residues 42-57 and 60-71 in alphaB-crystallin (TSLSPFYLRPPSFLRA, named recognition sequence 1 or RS-1, and WFDTGLSEMRLE, named recognition sequence 2 or RS-2) are involved in interaction with alphaA-crystallin. To understand the significance of the RS-2 region in interactions between alphaA- and alphaB-crystallins, W60R, F61N, and S66G mutants of alphaB-crystallin were made and tested for their ability to interact with alphaA-crystallin. W60R and S66G mutations increased the oligomeric size of alphaB-crystallin by 1.6- and 2.7-fold respectively, whereas the F61N mutation had no effect. The tryptophan fluorescence intensity of alphaBS66G was 1.5-fold higher than that for the wild type. The intrinsic fluorescence of alphaBF61N was marginally lower than that of alphaB, whereas the fluorescence intensity of alphaBW60R decreased by 40% compared with that of alphaB. The relative availability of hydrophobic sites in the mutants was in the following order: alphaBS66G > alphaB = alphaBF61N = alphaBW60R. The far-UV CD profiles for the wild type and alphaB-crystallin mutants indicated no significant changes in their secondary structures, except for alphaBS66G, which showed an increase in alpha-helical content. The near-UV CD profiles of alphaBW60R and alphaBF61N were nearly similar to that of wild type alphaB. On the other hand, alphaBS66G beyond 270 nm exhibited a signature completely different from that of wild type alphaB. Mutations did not alter the chaperone-like activity of these proteins. The W60R mutation did not affect the rate of subunit exchange between alphaB- and alphaA-crystallins. On the other hand, the S66G mutation increased the subunit exchange rate by 100%, whereas the F61N mutation decreased the rate of subunit exchange between alphaB- and alphaA-crystallins by 36%. Our results establish the importance of residues 60-71 in oligomerization of alphaB-crystallin and subunit interaction between alphaB- and alphaA-crystallins.     

Electrostatic interactions play a critical role in Mycobacterium tuberculosis Hsp16.3 binding of substrate proteins

Mycobacterium tuberculosis Hsp16.3, a member of a small heat shock protein family, has chaperone-like activity in vitro and suppresses thermally or chemically induced aggregation of proteins. The nature of the interactions between Hsp16.3 and the denatured substrate proteins was investigated. A dramatic enhancement of chaperone-like activity of Hsp16.3 upon increasing temperature was accompanied by decreased ANS-detectable surface hydrophobicity. Hsp16.3 exhibited significantly enhanced chaperone-like activity after preincubation at 100°C with almost unchanged surface hydrophobicity. The interaction between Hsp16.3 and dithiothreitol-treated insulin B chains was markedly weakened in the presence of NaCl but greatly enhanced by the addition of a low-polarity alcohol, accompanied by significantly increased and decreased surface hydrophobicity, respectively. A working model for Hsp16.3 binding to its substrate proteins is proposed.