Interaction of human recombinant alphaA- and alphaB-crystallins with early and late unfolding intermediates of citrate synthase on its thermal denaturation

We have investigated the role of recombinant human alphaA- and alphaB-crystallins in the heat-induced inactivation and aggregation of citrate synthase. Homo-multimers of both alphaA- and alphaB-crystallins confer protection against heat-induced inactivation in a concentration-dependent manner and also prevent aggregation. Interaction of crystallins with early unfolding intermediates of citrate synthase reduces their partitioning into aggregation-prone intermediates. This appears to result in enhanced population of early unfolding intermediates that can be reactivated by its substrate, oxaloacetate. Both these homo-multimers do not form a stable complex with the early unfolding intermediates. However, they can form a soluble, stable complex with aggregation-prone late unfolding intermediates. This soluble complex formation prevents aggregation. Thus, it appears that the chaperone activity of alpha-crystallin involves both transient and stable interactions depending on the nature of intermediates on the unfolding pathway; one leads to reactivation of the enzyme activity while the other prevents aggregation.     

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.     

Insights into hydrophobicity and the chaperone-like function of alphaA- and alphaB-crystallins: an isothermal titration calorimetric study

Alpha-crystallin, composed of two subunits, alphaA and alphaB, has been shown to function as a molecular chaperone that prevents aggregation of other proteins under stress conditions. The exposed hydrophobic surfaces of alpha-crystallins have been implicated in this process, but their exact role has not been elucidated. In this study, we quantify the hydrophobic surfaces of alphaA- and alphaB-crystallins by isothermal titration calorimetry using 8-anilino-1-napthalenesulfonic acid (ANS) as a hydrophobic probe and analyze its correlation to the chaperone potential of alphaA- and alphaB-crystallins under various conditions. Two ANS binding sites, one with low and another with high affinity, were clearly detected, with alphaB showing a higher number of sites than alphaA at 30 degrees C. In agreement with the higher number of hydrophobic sites, alphaB-crystallin demonstrated higher chaperone activity than alphaA at this temperature. Thermodynamic analysis of ANS binding to alphaA- and alphaB-crystallins indicates that high affinity binding is driven by both enthalpy and entropy changes, with entropy dominating the low affinity binding. Interestingly, although the number of ANS binding sites was similar for alphaA and alphaB at 15 degrees C, alphaA was more potent than alphaB in preventing aggregation of the insulin B-chain. Although there was no change in the number of high affinity binding sites of alphaA and alphaB for ANS upon preheating, there was an increase in the number of low affinity sites of alphaA and alphaB. Preheated alphaA, in contrast to alphaB, exhibited remarkably enhanced chaperone activity. Our results indicate that although hydrophobicity appears to be a factor in determining the chaperone-like activity of alpha-crystallins, it does not quantitatively correlate with the chaperone function of alpha-crystallins.     

Packing-induced conformational and functional changes in the subunits of alpha -crystallin

The heteroaggregate alpha-crystallin and homoaggregates of its subunits, alphaA- and alphaB-crystallins, function like molecular chaperones and prevent the aggregation of several proteins. Although modulation of the chaperone-like activity of alpha-crystallin by both temperature and chaotropic agents has been demonstrated in vitro, the mechanism(s) of its regulation in vivo have not been elucidated. The subunits of alpha-crystallin exchange freely, resulting in its dynamic and variable quaternary structure. Mixed aggregates of the alpha-crystallins and other mammalian small heat shock proteins (sHSPs) have also been observed in vivo. We have investigated the time-dependent structural and functional changes during the course of heteroaggregate formation by the exchange of subunits between homoaggregates of alphaA- and alphaB-crystallins. Native isoelectric focusing was used to follow the time course of subunit exchange. Circular dichroism revealed large tertiary structural alterations in the subunits upon subunit exchange and packing into heteroaggregates, indicating specific homologous and heterologous interactions between the subunits. Subunit exchange also resulted in quaternary structural changes as demonstrated by gel filtration chromatography. Interestingly, we found time-dependent changes in chaperone-like activity against the dithiothreitol-induced aggregation of insulin, which correlated with subunit exchange and the resulting tertiary and quaternary structural changes. Heteroaggregates of varying subunit composition, as observed during eye lens epithelial cell differentiation, generated by subunit exchange displayed differential chaperone-like activity. It was possible to alter chaperone-like activity of preexisting oligomeric sHSPs by alteration of subunit composition by subunit exchange. Our results demonstrate that subunit exchange and the resulting structural and functional changes observed could constitute a mechanism of regulation of chaperone-like activity of alpha-crystallin (and possibly other mammalian sHSPs) in vivo.     

Differential protective activity of alpha A- and alphaB-crystallin in lens epithelial cells

alphaA- and alphaB-crystallins are molecular chaperones expressed at low levels in lens epithelial cells, and their expression increases dramatically during differentiation to lens fibers. However, the functions of alphaA- and alphaB-crystallins in lens epithelial cells have not been studied in detail. In this study, the relative ability of alphaA- and alphaB-crystallin, in protecting lens epithelial cells from apoptotic cell death was determined. The introduction of alphaA-crystallin in the transformed human lens epithelial (HLE) B-3 lens epithelial cell line (which expresses low endogenous levels of alphaB-crystallin) led to a nearly complete protection of cell death induced by staurosporine, Fas monoclonal antibody, or the cytokine tumor necrosis factor alpha. To further study the relative protective activities of alphaA- and alphaB-crystallins, we created a cell line derived from alphaA-/-alphaB-/- double knockout mouse lens epithelia by infecting primary cells with Ad12-SV40 hybrid virus. The transformed cell line alphaAalphaBKO1 derived from alphaA/alphaB double knockout cells was transfected with alphaA- or alphaB-crystallin cDNA contained in pCIneo mammalian expression vector. Cells expressing different amounts of either alphaA-crystallin or alphaB-crystallin were isolated. The ability of alphaA- or alphaB-crystallin to confer protection from apoptotic cell death was determined by annexin labeling and flow cytometry of staurosporine- or UVA- treated cells. The results indicate that the anti-apoptotic activity of alphaA-crystallin was two to three-fold higher than that of alphaB-crystallin. Our work suggests that comparing the in vitro annexin labeling of lens epithelial cells is an effective way to measure the protective activity of alphaA- and alphaB-crystallin. Since the expression of alphaA-crystallin is largely restricted to the lens, its greater protective effect against apoptosis suggests that it may play a significant role in protecting lens epithelial cells from stress.     

alphaA- and alphaB-crystallins protect glucose-6-phosphate dehydrogenase against UVB irradiation-induced inactivation

alpha-Crystallin, a major eye lens protein, has been shown to function like a molecular chaperone by suppressing the aggregation of other proteins induced by various stress conditions. Ultraviolet (UV) radiation is known to cause structural and functional alterations in the lens macromolecules. Earlier we observed that exposure of rat lens to in vitro UV radiation led to inactivation of many lens enzymes including glucose-6-phosphate dehydrogenase (G6PD). In the present paper, we show that alpha-crystallin (alphaA and alphaB) protects G6PD from UVB irradiation induced inactivation. While, at 25 degrees C, there was a time-dependent decrease in G6PD activity upon irradiation at 300 nm, at 40 degrees C there was a complete loss of activity within 30 min even without irradiation. The loss of activity of G6PD was prevented significantly, if alphaA- or alphaB-crystallin was present during irradiation. At 25 degrees C, alphaB-crystallin was slightly a better chaperone in protecting G6PD against UVB inactivation. Interestingly, at 40 degrees C, alphaA- and alphaB-crystallins not only prevent the loss of G6PD activity but also protect against UVB inactivation. However, alphaA- and alphaB-crystallins were equally efficient at 40 degrees C in protecting G6PD.     

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.     

Distinct roles of alphaA- and alphaB-crystallins under thermal and UV stresses

alpha-Crystallin, a major protein of all vertebrate lenses, consists of two subunits, alphaA and alphaB, which form polymeric aggregates with an average molecular mass of about 800kDa. In this study, we have employed various biophysical methods to study aggregate sizes and conformational properties of purified alphaA, alphaB subunits, and cloned recombinant alphaB subunit. From far- and near-UV CD spectra, native alpha-, alphaA-, alphaB-, and recombinant alphaB-crystallins from porcine lenses all show similar beta-sheet conformation to that from bovine and human lenses as reported previously. By means of gel-filtration chromatography and dynamic light scattering, we have found that the molecular sizes of all four crystallin aggregates are polydispersedly distributed in the following order of aggregate sizes, i.e., native alpha>alphaA>alphaB approximately recombinant alphaB. To investigate the structural and functional relationships, we have also compared the chaperone activities of all four alpha-crystallin aggregates at different temperatures. From the results of chaperone-activity assays, ANS (8-anilinonaphthalene-1-sulfonic acid) binding and thermal stability studies, there appeared to be at least two factors playing major roles in the chaperone-like activity of these lens proteins: one is the hydrophobicity of the exposed protein surface and the other is the structural stability associated with each protein. We showed that alphaA-crystallin is a better chaperone to protect gamma-crystallin against UV irradiation than alphaB-crystallin, in contrast to the observation that alphaB is generally a better chaperoning protein than alphaA for enzyme protective assays at physiological temperatures.     

Chaperone-like activity and hydrophobicity of alpha-crystallin

alpha-Crystallin, a prominent member of small heat shock protein (sHsp) family and a major structural protein of the eye lens is a large polydisperse oligomer of two isoforms, alphaA- and alphaB-crystallins. Numerous studies have demonstrated that alpha-crystallin functions like a molecular chaperone in preventing the aggregation of various proteins under a wide range of stress conditions. The molecular chaperone function of alpha-crystallin is thus considered to be vital in the maintenance of lens transparency and in cataract prevention. alpha-Crystallin selectively interacts with non-native proteins thereby preventing them from aggregation and helps maintain them in a folding competent state. It has been proposed and generally accepted that alpha-crystallin suppresses the aggregation of other proteins through the interaction between hydrophobic patches on its surface and exposed hydrophobic sites of partially unfolded substrate protein. However, a quantifiable relationship between hydrophobicity and chaperone-like activity remains a matter to be concerned about. On an attentive review of studies on alpha-crystallin chaperone-like activity, particularly the studies that have direct or indirect implications to hydrophobicity and chaperone-like activity, we found several instances wherein the correlation between hydrophobicity and its chaperone-like activity is paradoxical. We thus attempted to provide an overview on the role of hydrophobicity in chaperone-like activity of alpha-crystallin, the kind of evaluation done for the first time.     

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.     

In vitro dephosphorylation of alpha-crystallin is dependent on the state of oligomerization

alphaA- and alphaB-crystallin, members of the small heat shock protein family, are present in lens cell extracts as large aggregates. Both alpha-crystallins are found partially phosphorylated. This study tests the ability of five phosphatases (protein phosphatase PP1, PP2A, PP2B, alkaline and acid phosphatases) to dephosphorylate alphaA- and alphaB-crystallin in vitro. Activity of a phosphatase was dependent on the size of the aggregate. Each of the phosphatases tested showed different specificity and efficiency towards alphaA- and alphaB-crystallins, which depended on the oligomeric state of the alpha-crystallin aggregate. Alkaline phosphatase dephosphorylated both alphaA- and alphaB-crystallin. The reaction was faster when alpha-crystallin was in a tetrameric form. PP2A dephosphorylated primarily alphaA-crystallin but only after the conversion of alpha-crystallin to tetramers. PP1 and PP2B did not dephosphorylate either alphaA- or alphaB-crystallins present as large aggregates but could not be tested on the lower molecular weight form of alphaA-crystallin. Acid phosphatase dephosphorylated both alphaA- and alphaB-crystallin. The results suggest that an important relationship exists between the structure of alpha-crystallin and its level of phosphorylation in the cell.     

Temperature-dependent chaperone activity and structural properties of human alphaA- and alphaB-crystallins

The chaperone activity and biophysical properties of recombinant human alphaA- and alphaB-crystallins were studied by light scattering and spectroscopic methods. While the chaperone function of alphaA-crystallin markedly improves with an increase in temperature, the activity of alphaB homopolymer appears to change very little upon heating. Compared with alphaB-crystallin, the alphaA-homopolymer is markedly less active at low temperatures, but becomes a more active species at high temperatures. At physiologically relevant temperatures, the alphaB homopolymer appears to be modestly (two times or less) more potent chaperone than alphaA homopolymer. In contrast to very similar thermotropic changes in the secondary structure of both homopolymers, alphaA- and alphaB-crystallins markedly differ with respect to the temperature-dependent surface hydrophobicity profiles. Upon heating, alphaA-crystallin undergoes a conformational transition resulting in the exposure of additional hydrophobic sites, whereas no such transition occurs for alphaB-crystallin. The correlation between temperature-dependent changes in the chaperone activity and hydrophobicity properties of the individual homopolymers supports the view that the chaperone activity of alpha-crystallin is dependent on the presence of surface-exposed hydrophobic patches. However, the present data also show that the surface hydrophobicity is not the sole determinant of the chaperone function of alpha-crystallin.     

Unfolding of human lens recombinant betaB2- and gammaC-crystallins

betaB2- and gammaC-crystallins belong to the betagamma-crystallin superfamily and have very similar structures. Molecular spectroscopic techniques such as UV-visible absorption, circular dichroism, and fluorescence indicate they have similar biophysical properties. Their structures are characterized by the presence of two domains consisting of four Greek key motifs. The only difference is the connecting peptide of the two domains, which is flexible in gamma-crystallin but extended in beta-crystallin; thus, an intradomain association and a monomer are formed in gamma-crystallin and an interdomain association and a dimer are formed in beta-crystallin. The difference may be reflected in the thermodynamic stability. In the present study, we calculated the standard free-energy by equilibrium unfolding transition in guanidine hydrochloride using three spectroscopic parameters: absorbance at 235nm, Trp fluorescence intensity at 320nm, and far-UV circular dichroism at 223nm. Global analyses indicate that both dimeric betaB2- and monomeric gammaC-crystallins are a better fit to a three-state model than to a two-state model. In terms of standard free-energy, deltaG(0)(H(2)O,i) both betaB2-crystallin and gammaC-crystallin are stable proteins and dimeric betaB2-crystallin is more stable than the monomeric gammaC-crystallin. The significance of the thermodynamic stability for betaB2- and gammaC-crystallins may be related to their functions in the lens.     

Amyloidogenic unfolding intermediates differentiate sheep prion protein variants

Sheep is a unique example among mammalian species to present a strong correlation between genotype and prion disease susceptibility phenotype. Indeed a well-defined set of PrP polymorphisms at positions 136, 154 and 171 (sheep numbering) govern scrapie susceptibility, ranging from very high susceptibility for V136-R154-Q171 variant (VRQ) to resistance for A136-R154-R171 variant (ARR).To get better insight into the molecular mechanisms of scrapie susceptibility/resistance, the unfolding pathways of the different full-length recombinant sheep prion protein variants were analysed by differential scanning calorimetry in a wide range of pH. In the pH range 4.5-6.0, thermal unfolding occurs through a reversible one-step process while at pH <4.5 and >6.0 unfolding intermediates are formed, which are stable in the temperature range 65-80 degrees C. While these general behaviours are shared by all variants, VRQ and ARQ (susceptibility variants) show higher thermal stability than AHQ and ARR (resistance variants) and the formation of their unfolding intermediates requires higher activation energy than in the case of AHQ and ARR. Furthermore, secondary structures of the unfolding intermediates differentiate variants: ARR unfolding intermediate exhibits random coil structure, contrasting with the beta-sheet structure of VRQ and ARQ unfolding intermediates. The rate of the unfolding intermediate formation allows us to classify genetic variants along increasing scrapie susceptibility at pH 4.0, VRQ and ARQ rates being the highest. Rather poor correlation is observed at pH 7.2. Upon cooling, these intermediates refold into stable species, which are rich in beta-type secondary structures and, as revealed by thioflavin T fluorescence and electron microscopy, share amyloid characteristics. These results highlight the prion protein plasticity genetically modulated in sheep, and might provide a molecular basis for sheep predisposition to scrapie taking into account both thermodynamic stability and transconformation rate of prion protein.     

Human alphaA- and alphaB-crystallins bind to Bax and Bcl-X(S) to sequester their translocation during staurosporine-induced apoptosis

AlphaA- and alphaB-crystallins are distinct antiapoptotic regulators. Regarding the antiapoptotic mechanisms, we have recently demonstrated that alphaB-crystallin interacts with the procaspase-3 and partially processed procaspase-3 to repress caspase-3 activation. Here, we demonstrate that human alphaA- and alphaB-crystallins prevent staurosporine-induced apoptosis through interactions with members of the Bcl-2 family. Using GST pulldown assays and coimmunoprecipitations, we demonstrated that alpha-crystallins bind to Bax and Bcl-X(S) both in vitro and in vivo. Human alphaA- and alphaB-crystallins display similar affinity to both proapoptotic regulators, and so are true with their antiapoptotic ability tested in human lens epithelial cells, human retina pigment epithelial cells (ARPE-19) and rat embryonic myocardium cells (H9c2) under treatment of staurosporine, etoposide or sorbitol. Two prominent mutants, R116C in alphaA-crystallin and R120G, in alphaB-crystallin display much weaker affinity to Bax and Bcl-X(S). Through the interaction, alpha-crystallins prevent the translocation of Bax and Bcl-X(S) from cytosol into mitochondria during staurosporine-induced apoptosis. As a result, alpha-crystallins preserve the integrity of mitochondria, restrict release of cytochrome c, repress activation of caspase-3 and block degradation of PARP. Thus, our results demonstrate a novel antiapoptotic mechanism for alpha-crystallins.     

Stability and folding of dihydrofolate reductase from the hyperthermophilic bacterium Thermotoga maritima.

Dihydrofolate reductase (DHFR) has been a well-established model system for protein folding. The enzyme DHFR from the hyperthermophilic bacterium Thermotoga maritima (TmDHFR) displays distinct adaptations toward high temperatures at the level of both structure and stability. The enzyme represents an extremely stable dimer; no isolated structured monomers could be detected in equilibrium or during unfolding. The equilibrium unfolding strictly follows the two-state model for a dimer (N(2) right harpoon over left harpoon 2U), with a free energy of stabilization of DeltaG = -142 +/- 10 kJ/mol at 15 degrees C. The two-state model is applicable over the whole temperature range (5-70 degrees C), yielding a DeltaG vs T profile with maximum stability at around 35 degrees C. There is no flattening of the stability profile. Instead, the enhanced thermostability is characterized by shifts toward higher overall stability and higher temperature of maximum stability. TmDHFR unfolds in a highly cooperative manner via a nativelike transition state without intermediates. The unfolding reaction is much slower (ca. 10(8) times) compared to DHFR from Escherichia coli (EcDHFR). In contrast to EcDHFR, no evidence for heterogeneity of the native state is detectable. Refolding proceeds via at least two intermediates and a burst-phase of rather low amplitude. Reassociation of monomeric intermediates is not rate-limiting on the folding pathway due to the high association constant of the dimer.     

Multiple unfolding intermediates of human placental alkaline phosphatase in equilibrium urea denaturation.

Alkaline phosphatase is an enzyme with a typical alpha/beta hydrolase fold. The conformational stability of the human placental alkaline phosphatase was examined with the chemical denaturant urea. The red shifts of fluorescence spectra show a complex unfolding process involving multiple equilibrium intermediates indicating differential stability of the subdomains of the enzyme. None of these unfolding intermediates were observed in the presence of 83 mM NaCl, indicating the importance of ionic interactions in the stabilization of the unfolding intermediates. Guanidinium chloride, on the other hand, could stabilize one of the unfolding intermediates, which is not a salt effect. Some of the unfolding intermediates were also observed in circular dichroism spectroscopy, which clearly indicates steady loss of helical structure during unfolding, but very little change was observed for the beta strand content until the late stage of the unfolding process. The enzyme does not lose its phosphate-binding ability after substantial tertiary structure changes, suggesting that the substrate-binding region is more resistant to chemical denaturant than the other structural domains. Global analysis of the fluorescence spectral change demonstrated the following folding-unfolding process of the enzyme: N <--> I(1) <--> I(2) <--> I(3) <--> I(4) <--> I(5) <--> D. These discrete intermediates are stable at urea concentrations of 2.6, 4.1, 4.7, 5.5, 6.6, and 7.7 M, respectively. These intermediates are further characterized by acrylamide and/or potassium iodide quenching of the intrinsic fluorescence of the enzyme and by the hydrophobic probes, 1-anilinonaphthalene-8-sulfonic acid and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid. The stepwise unfolding process was interpreted by the folding energy landscape in terms of the unique structure of the enzyme. The rigid central beta-strand domain is surrounded by the peripheral alpha-helical and coil structures, which are marginally stable toward a chemical denaturant.     

Three-dimensional models corresponding to the C-terminal domain of human alphaA- and alphaB-crystallins based on the crystal structure of the small heat-shock protein HSP16.9 from wheat

We propose three-dimensional models corresponding to the C-terminal domain of human alphaA- and alphaB-crystallins by using the comparative modeling program Modeler and the more closely related crystal structure of the small heat-shock protein (sHSP) belonging to the eukaryotic species from wheat HSP16.9 as template structure. The sequence alignments differ slightly from alignments that were used previously to construct alpha-crystallin models based on homology and the crystal structure of the more distantly related small heat-shock protein from archaeal species; Methanococcus jannaschii Mj HSP16.5, the only related structure then available as a template. The alpha-crystallin models based on HSP16.9 show better 3-D profile scores and reflect the relative shifts in the beta-strands corresponding to the beta-sandwich associated with the core C-terminal domain that is common to small heat-shock proteins and the alpha-crystallins. The loop between the equivalent beta5-beta7 strands corresponds to a region of seven amino acid residues deletion in alpha-crystallins and defines the new set of amino acid residues likely to be associated with a dimer interface. The models may be useful to examine sites of mutations that are known to affect chaperone-like activity and provide the structural basis for dimerization in alpha-crystallins.     

Biophysical Characterization and Folding Studies of Plant Protease,Wrightin: Identification of Folding Intermediate Under Different Conditions

Wrightin, a serine protease from Wrightia tinctoria, has been used as model system to examine structure-function and stability. Our studies show high stability of the enzyme with major elements of secondary structure being β-sheets. Under neutral conditions the enzyme is stable in 8 M urea and high temperature. GuHCl induced unfolding of wrightin at lower pH cannot be satisfactorily fit to a two state model for unfolding. Multiple intermediates were identified during unfolding of wrightin. Further, two intermediates, early and late are identified in the urea induced unfolding pathway at pH 3.0. Spectroscopic properties of intermediate states are analyzed and interpreted.