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

The heteroaggregate α-crystallin and homoaggregates of its subunits, αA- and αB-crystallins, function like molecular chaperones and prevent the aggregation of several proteins. Although modulation of the chaperone-like activity of α-crystallin by both temperature and chaotropic agents has been demonstrated in vitro, the mechanism(s) of its regulationin vivo have not been elucidated. The subunits of α-crystallin exchange freely, resulting in its dynamic and variable quaternary structure. Mixed aggregates of the α-crystallins and other mammalian small heat shock proteins (sHSPs) have also been observedin 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 αA- and αB-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 α-crystallin (and possibly other mammalian sHSPs) in vivo

UV-Light Exposed Prion Protein Fails to Form Amyloid Fibrils

Amyloid fibril formation involves three steps; structural perturbation, nucleation and elongation. We have investigated amyloidogenesis using prion protein as a model system and UV-light as a structural perturbant. We find that UV-exposed prion protein fails to form amyloid fibrils. Interestingly, if provided with pre-formed fibrils as seeds, UV-exposed prion protein formed amyloid fibrils albeit with slightly different morphology. Atomic force microscopy and electron microscopic studies clearly show the formation of fibrils under these conditions. Circular dichroism study shows loss in helicity in UV-exposed protein. UV-exposed prion protein fails to form amyloid fibrils. However, it remains competent for fibril extension, suggesting that UV-exposure results in loss of nucleating capability. This work opens up possibility of segregating nucleation and elongation step of amyloidogenesis, facilitating screening of new drug candidates for specifically inhibiting either of these processes. In addition, the work also highlights the importance of light-induced structural and functional alterations which are important in protein based therapeutics

Molten-globule state of carbonic anhydrase binds to the chaperone-like α-crystallin

α-Crystallin, a multimeric protein, exhibits chaperone-like activity in preventing aggregation of several proteins. We have studied the chaperone-like activity of α-crystallin toward heat-induced aggregation of bovine and human carbonic anhydrase. Human carbonic anhydrase aggregates at 60°C, while bovine carbonic anhydrase does not aggregate significantly at this temperature. Removal of the enzyme-bound metal ion, Zn2+, by EDTA modulates the aggregation behavior of bovine carbonic anhydrase. Fluorescence and circular dichroism studies show that removal of the metal ion from the bovine carbonic anhydrase by a chelator such as EDTA enhances the propensity of the enzyme to adopt the molten-globule state. α-Crystallin binds to this state of the enzyme and prevents aggregation. Fluorescence and circular dichroism studies on the α-crystallin-enzyme complexes show that the enzymes in the complex are in the molten-globule state. These results are of relevance to the interaction of chaperones with the partially unfolded states of target proteins

Domain swapping in human αA and αB crystallins affects oligomerization and enhances chaperone-like activity

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

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

Co-refolding denatured-reduced hen egg white lysozyme with acidic and basic proteins

Refolding of denatured-reduced lysozyme and the effect of co-refolding it with other proteins such as RNase A, bovine serum albumin, histone, myelin basic protein, alcohol dehydrogenase and DNase I on the renaturation yield and the aggregation of lysozyme have been studied. Basic proteins consistently increase the renaturation yield of the basic protein lysozyme (10–20% more than in their absence) with little or no aggregation. On the other hand, co-refolding of lysozyme with acidic proteins leads to aggregation and a significant decrease in renaturation yields. Our results show that hetero-interchain interactions (non-specific interactions) occur when the basic protein lysozyme is refolded together with acidic proteins such as bovine serum albumin, alcohol dehydrogenase or DNase I. Our results also suggest that the net charge on proteins plays a significant role in such non-specific aggregation. These results should prove useful in understanding the hetero-interchain interactions between folding polypeptide chains

Oxidative refolding of lysozyme in trifluoroethanol (TFE) and ethylene glycol: interfering role of preexisting α-helical structure and intermolecular hydrophobic interactions

The oxidative refolding of equilibrium intermediates of lysozyme stabilized in trifluoroethanol (TFE) and ethylene glycol was monitored. Equilibrium intermediates of disulfide reduced lysozyme in TFE are known to contain considerable amounts of α-helical structure and resemble the early intermediate in the oxidative refolding of lysozyme. We find that the intermediates in TFE do not proceed to folding; they form aggregates. However, interestingly, intermediates in ethylene glycol refold to the native state with improved folding yield. Secondary structure of these intermediates was monitored by far-UV circular dichroism. Our results indicate that formation of α-helical structure prior to oxidative refolding does not help the process in the case of lysozyme. Interfering with intermolecular hydrophobic interactions in the unfolded state is more productive

Redox-regulated chaperone function and conformational changes of Escherichia coli Hsp33

We have studied the chaperone activity and conformation of Escherichia coli heat shock protein (Hsp)33, whose activity is known to be switched on by oxidative conditions. While oxidized Hsp33 completely prevents the heat-induced aggregation of ζ-crystallin at 42°C at a ratio of 1:1 (w/w), the reduced form exhibits only a marginal effect on the aggregation. Far UV–circular dichroism (CD) spectra show that reduced Hsp33 contains a significant α-helical component. Oxidation results in significant changes in the far UV–CD spectrum. Near UV–CD spectra show changes in tertiary structural packing upon oxidation. Polarity-sensitive fluorescent probes report enhanced hydrophobic surfaces in the oxidized Hsp33. Our studies show that the oxidative activation of the chaperone function of Hsp33 involves observable conformational changes accompanying increased exposure of hydrophobic pockets

Role of Fluorescein angiography in evaluation of posterior segment disorders

Objective: To study the role of fluorescein angiography in the evaluation of posterior segment diseases. Materials & Methods: A hospital based prospective randomized study was done which included 80 patients. Detailed patient history was taken and a thorough ocular and systemic examination was done. All patients were examined by ophthalmoscopy (direct, indirect and slit lamp examination with +90 D lens), followed by fluorescein angiography. Ophthalmoscopic and fluorescein angiography findings were analyzed and categorized. Patients were advised necessary ocular and systemic treatment. Results: 80 cases with posterior segment diseases were analyzed and sub-divided into categories of Diabetic retinopathy, vascular occlusive disorders, age related macular degenerations, Central serous chorioretinopathy, inflammatory disorders and miscellaneous conditions. Fundus Fluorescein Angiography (FFA) altered the diagnosis in 37.5% of cases and categorized the lesions in all cases. 11% of patients experienced adverse reactions like nausea and vomiting. On statistical analysis, FFA proved to be a far superior diagnostic modality than clinical examination (ophthalmoscopy) in diagnosing fundus pathology. Conclusion: FFA is a superior diagnostic tool and is a necessity for evaluating, localizing and categorization of lesions in Retinal, Macular and Choroidal pathologies