Unfolding and refolding of a quinone oxidoreductase: α-crystallin, a molecular chaperone, assists its reactivation

α-Crystallin, a member of the small heat-shock protein family and present in vertebrate eye lens, is known to prevent the aggregation of other proteins under conditions of stress. However, its role in the reactivation of enzymes from their non-native inactive states has not been clearly demonstrated. We have studied the effect of α-crystallin on the refolding of ζ-crystallin, a quinone oxidoreductase, from its different urea-denatured states. Co-refolding ζ-crystallin from its denatured state in 2.5 M urea with either calf eye lens α-crystallin or recombinant human αB-crystallin could significantly enhance its reactivation yield. αB-crystallin was found to be more efficient than αA-crystallin in chaperoning the refolding of ζ-crystallin. In order to understand the nature of the denatured state(s) of ζ-crystallin that can interact with α-crystallin, we have investigated the unfolding pathway of ζ-crystallin. We find that it unfolds through three distinct intermediates: an altered tetramer, a partially unfolded dimer, which is competent to fold back to its active state, and a partially unfolded monomer. The partially unfolded monomer is inactive, exhibits highly exposed hydrophobic surfaces and has significant secondary structural elements with little or no tertiary structure. This intermediate does not refold into the active state without assistance. α-Crystallin provides the required assistance and improves the reactivation yield several-fold

Oligomeric Hsp33 with enhanced chaperone activity

Hsp33, an Escherichia coli cytosolic chaperone, is inactive under normal conditions but becomes active upon oxidative stress. It was previously shown to dimerize upon activation in a concentration- and temperature-dependent manner. This dimer was thought to bind to aggregation-prone target proteins, preventing their aggregation. In the present study, we report small angle x-ray scattering (SAXS), steady state and time-resolved fluorescence, gel filtration, and glutaraldehyde cross-linking analysis of full-length Hsp33. Our circular dichroism and fluorescence results show that there are significant structural changes in oxidized Hsp33 at different temperatures. SAXS, gel filtration, and glutaraldehyde cross-linking results indicate, in addition to the dimers, the presence of oligomeric species. Oxidation in the presence of physiological salt concentration leads to significant increases in the oligomer population. Our results further show that under conditions that mimic the crowded milieu of the cytosol, oxidized Hsp33 exists predominantly as an oligomeric species. Interestingly, chaperone activity studies show that the oligomeric species is much more efficient compared with the dimers in preventing aggregation of target proteins. Taken together, these results indicate that in the cell, Hsp33 undergoes conformational and quaternary structural changes leading to the formation of oligomeric species in response to oxidative stress. Oligomeric Hsp33 thus might be physiologically relevant under oxidative stress

Rapid refolding studies on the chaperone-like α-crystallin effect of α-crystallin on refolding of β- and γ-crystallins

α-Crystallin, a multimeric protein present in the eye lens, is shown to have chaperone-like activity in preventing thermally induced aggregation of enzymes and other crystallins. We have studied the rapid refolding of α-crystallin, and compared it with other calf eye lens proteins, namely β- and γ-crystallins. α-Crystallin forms a clear solution upon rapid refolding from 8 M urea. The refolded α-crystallin has native-like secondary, tertiary, and quaternary structures as revealed by circular dichroism and fluorescence characteristics as well as gel filtration and sedimentation velocity measurements. On rapid refolding, β- and γ-crystallins aggregate and form turbid solutions. The presence of α-crystallin in the refolding buffer marginally increases the recovery of β- and γ-crystallins in the soluble form. However, unfolding of these crystallins together with α-crystallin using 8 M urea and subsequent refolding significantly increases the recovery of these proteins in the soluble form. These results indicate that an intermediate of α-crystallin formed during refolding is more effective in preventing the aggregation of β- and γ-crystallins. This supports our earlier hypothesis (Raman, B., and Rao, C. M.(1994) J. Biol. Chem. 269, 27264-27268) that the chaperone-like activity of α-crystallin is more pronounced in its structurally perturbed state

Refolding of denatured and denatured/reduced lysozyme at high concentrations

Refolding of proteins at high concentrations often results in aggregation. To gain insight into the molecular aspects of refolding and to improve the yield of active protein, we have studied the refolding of lysozyme either from its denatured state or from its denatured/reduced state. Refolding of denatured lysozyme, even at 1 mg/ml, yields fully active enzyme without aggregation. However, refolding of denatured/reduced lysozyme into buffer that lacks thiol/disulfide reagents leads to aggregation. Thiol/disulfide redox reagents such as cysteine/cystine and reduced/oxidized glutathione facilitate the renaturation, with the yield depending on their absolute concentrations. We have obtained an ~70% renaturation yield upon refolding of lysozyme at 150 μg/ml. The cysteine/cystine redox system is more efficient compared with the glutathione redox system. When lysozyme is refolded in the absence of redox reagents, a transient intermediate that has regained a significant amount of secondary structure is formed. The tryptophans in this intermediate are as exposed to water as in the fully unfolded protein. It shows increased exposure of hydrophobic surfaces compared with the native or completely unfolded enzyme. This aggregation-prone intermediate folds to active enzyme upon addition of oxidized glutathione before the aggregation process starts. These properties of the intermediate in the refolding pathway of lysozyme are similar to those proposed for the molten globule

HspB2/Myotonic Dystrophy Protein Kinase Binding Protein (MKBP) as a Novel Molecular Chaperone: Structural and Functional Aspects

The small heat shock protein, human HspB2, also known as Myotonic Dystrophy Kinase Binding Protein (MKBP), specifically associates with and activates Myotonic Dystrophy Protein Kinase (DMPK), a serine/threonine protein kinase that plays an important role in maintaining muscle structure and function. The structure and function of HspB2 are not well understood. We have cloned and expressed the protein in E.coli and purified it to homogeneity. Far-UV circular dichroic spectrum of the recombinant HspB2 shows a b-sheet structure. Fluorescence spectroscopic studies show that the sole tryptophan residue at the 130 th position is almost completely solvent-exposed. Bis-ANS binding shows that though HspB2 exhibits accessible hydrophobic surfaces, it is significantly less than that exhibited by another well characterized small HSP, aB-crystallin. Sedimentation velocity measurements show that the protein exhibits concentration-dependent oligomerization. Fluorescence resonance energy transfer study shows that HspB2 oligomers exchange subunits. Interestingly, HspB2 exhibits target protein-dependent chaperone-like activity: it exhibits significant chaperone-like activity towards dithiothreitol (DTT)-induced aggregation of insulin and heat-induced aggregation of alcohol dehydrogenase, but only partially prevents the heat-induced aggregation of citrate synthase, co-precipitating with the target protein. It also significantly prevents the ordered amyloid fibril formation of a-synuclein. Thus, our study, for the first time, provides biophysical characterization on the structural aspects of HspB2, and shows that it exhibits target protein-dependen

Fibrillogenic and non-fibrillogenic ensembles of SDS-bound human α-synuclein

Fibril formation of α-synuclein is associated with several neurodegenerative diseases, including Parkinson's disease in humans. The anionic detergent sodium dodecyl sulfate (SDS) can accelerate the fibril formation in vitro. However, the molecular basis of this acceleration is not clear. Our study shows that native α-synuclein exhibits relatively less fibril growth despite providing fibril seeds for nucleation. The presence of SDS promotes the seeded fibril growth in a concentration-dependent manner, with an optimal concentration of 0.5–0.75 mM. We used isothermal calorimetry, hydrophobic dye binding and circular dichroism spectroscopy to characterize the protein–detergent interactions as a function of the concentration of SDS. Interaction of SDS with α-synuclein when studied by isothermal titration calorimetry and hydrophobic dye-binding reveals a similar characteristic optimal behavior between 0.5 mM and 0.75 mM SDS. The study shows two types of ensembles of α-synuclein and SDS: the fibrillogenic ensembles formed with optimal concentration of SDS around 0.5–0.75 mM are characterized by enhanced accessible hydrophobic surfaces and extended to partially helical conformation, while the less or non-fibrillogenic ensembles formed above 2 mM SDS are characterized by less accessible hydrophobic surfaces and maximal helical content. Little or no fibrillogenicity of the ensembles observed above 2 mM SDS could be partly because of the observed intrinsic instability of the fibrils under the condition

Detection and assay of proteases using calf lens β-crystallin aggregate as substrate

The eye lens protein, βL-crystallin, aggregates and yields a turbid solution upon refolding from its denatured state. We have observed that the addition of trace amounts of protease results in clearing of this turbidity. Based on this observation, we have developed a simple and rapid method for the detection and assay of proteases. This assay can be performed in the pH range of 6.0–9.0. We could assay the activity of trypsin at a concentration as low as 5 μg/ml

Structural aspects and chaperone activity of human HspB3: role of the “C-terminal extension”

HspB3, an as yet uncharacterized sHsp, is present in muscle, brain, heart, and in fetal tissues. A point mutation correlates with the development of axonal motor neuropathy. We purified recombinant human HspB3. Circular dichroism studies indicate that it exhibits β-sheet structure. Gel filtration and sedimentation velocity experiments show that HspB3 exhibits polydisperse populations with predominantly trimeric species. HspB3 exhibits molecular chaperone-like activity in preventing the heat-induced aggregation of alcohol dehydrogenase (ADH). It exhibits moderate chaperone-like activity towards heat-induced aggregation of citrate synthase. However, it does not prevent the DTT-induced aggregation of insulin, indicating that it exhibits target protein-dependent molecular chaperone-like activity. Unlike other sHsps, it has a very short C-terminal extension. Fusion of the C-terminal extension of αB-crystallin results in altered tertiary and quaternary structure, and increase in polydispersity of the chimeric protein, HspB3αB-CT. The chimeric protein shows comparable chaperone-like activity towards heat-induced aggregation of ADH and citrate synthase. However, it shows enhanced activity towards DTT-induced aggregation of insulin. Our study, for the first time, provides the structural and chaperone functional characterization of HspB3 and also sheds light on the role of the C-terminal extension of sHsps

The cataract-causing mutation G98R in human αA-crystallin leads to folding defects and loss of chaperone activity

Purpose: The objective of this study is to understand the molecular basis of cataract that develops due to the mutation of the glycine-98 residue to arginine in αA-crystallin. Methods: The glycine-98 residue was mutated to arginine by site-directed mutagenesis. The expression, structural and chaperone properties and thermal stability of the mutant, G98RαA-crystallin have been studied. The secondary and tertiary structure of the wild type and the mutant protein was studied using circular dichroism and fluorescence spectroscopy. The quaternary structure was studied by gel filtration chromatography and dynamic light scattering. Chaperone activity studies were carried out using DTT-induced aggregation of insulin. Results: Unlike the wild type protein, the heterologous expression of G98R αA-crystallin in E.coli results in the formation of inclusion bodies. Upon dissolving the inclusion bodies in 3 M urea and subjecting to refolding, it yielded a clear solution. The refolded mutant protein exhibits altered secondary, tertiary and quaternary structure, which lacks chaperone function, and is susceptible to heat-induced aggregation. Conclusions: The G98R mutation in αA-crystallin results in altered folding and becomes aggregation-prone leading to formation of large oligomers lacking chaperone function. Tendency to aggregate and loss of chaperone activity could be contributing to turbidity and loss of transparency of the lens