Biomedical subjects

Ashis Biswas

Publications and source records attributed to Ashis Biswas.

4 recordsLinked to original sources

PubMed2007

Differential recognition of natural and nonnatural substrate by molecular chaperone alpha-crystallin-A subunit exchange study.

alpha-Crystallin is a molecular chaperone that recognizes proteins substrates in stress. It binds to the unstable conformer of a large variety of related or unrelated substrates and thus prevents them aggregating and holds them in a folding competent state. In this article, we have tried to critically analyze, from experimental point of view, whether alpha-crystallin has any preference for its natural substrates compared to the nonnatural one. Our results clearly show that alpha-crystallin is exceptionally active and sensitive in preventing aggregation of its natural substrates and can fully prevent such an aggregation in a substoichiometric ratio, but nonnatural substrates require a considerably higher amount of alpha-crystallin. Using suitable fluorescent-labeled alpha-crystallins and performing fluorescence resonance energy transfer experiments, we were able to determine the subunit exchange kinetics between the alpha-crystallin oligomers. It was found that while alpha-crystallin was bound to its natural substrate, the rate of subunit exchange was slightly decreased. But, when a nonnatural substrate carbonic anhydrase remained bound to the chaperone, further loss in subunit exchange rate was observed. Nonnatural substrate was found to create higher activation energy barrier for the subunit exchange reaction compared to the native substrates. Similarities in major beta-sheet structure of both alpha-crystallin and its natural substrates may be the reason for the preference in molecular recognition in comparison with the nonnatural substrate.

Animals↗

PubMed2006

Effect of site-directed mutagenesis of methylglyoxal-modifiable arginine residues on the structure and chaperone function of human alphaA-crystallin.

We reported previously that chemical modification of human alphaA-crystallin by a metabolic dicarbonyl compound, methylglyoxal (MGO), enhances its chaperone-like function, a phenomenon which we attributed to formation of argpyrimidine at arginine residues (R) 21, 49, and 103. This structural change removes the positive charge on the arginine residues. To explore this mechanism further, we replaced these three R residues with a neutral alanine (A) residue one at a time or in combination and examined the impact on the structure and chaperone function. Measurement of intrinsic tryptophan fluorescence and near-UV CD spectra revealed alteration of the microenvironment of aromatic amino acid residues in mutant proteins. When compared to wild-type (wt) alphaA-crystallin, the chaperone function of R21A and R103A mutants increased 20% and 18% as measured by the insulin aggregation assay and increased it as much as 39% and 28% when measured by the citrate synthase (CS) aggregation assay. While the R49A mutant lost most of its chaperone function, R21A/R103A and R21A/R49A/R103A mutants had slightly better function (6-14% and 10-14%) than the wt protein in these assays. R21A and R103A mutants had higher surface hydrophobicity than wt alphaA-crystallin, but the R49A mutant had lower hydrophobicity. R21A and R103A mutants, but not the R49A mutant, were more efficient than wt protein in refolding guanidine hydrochloride-treated malate dehydrogenase to its native state. Our findings indicate that the positive charges on R21, R49, and R103 are important determinants of the chaperone function of alphaA-crystallin and suggest that chemical modification of arginine residues may play a role in protein aggregation during lens aging and cataract formation.

Arginine↗

PubMed2004

Role of ATP on the interaction of alpha-crystallin with its substrates and its implications for the molecular chaperone function.

ATP plays a significant role in the function of molecular chaperones of the large heat shock protein families. However, its role in the functions of chaperones of the small heat shock protein families is not understood very well. We report here a study on the role of ATP on the structure and function of the major eye lens chaperone alpha-crystallin. Our in vitro study shows that at physiological temperature, ATP induces the association of alpha-crystallin with substrate proteins. The association process is reversible and low affinity in nature with unit binding stoichiometry. 4,4'-Dianilino-1,1'-binaphthyl-5,5-disulfonic acid, dipotassium salt, binding studies show that ATP induces the exposure of additional hydrophobic sites on alpha-crystallin, but no appreciable enhancement of the same was observed for the substrate protein gamma-crystallin or carbonic anhydrase. An equilibrium unfolding study reveals that ATP at 3 mgm concentration stabilizes the alpha-crystallin structure by 4.5 kJ/mol. The compactness induced by ATP makes it more resistant to tryptic cleavage. ATP-induced association of chaperone alpha-crystallin with substrate enhanced its aggregation prevention ability and also enhanced the refolding yield of lactate dehydrogenase from the unfolded state. Our results suggest that the binding of ATP to alpha-crystallin and not its hydrolysis is required for all these effects, as replacement of ATP by its nonhydrolyzable analogue adenosine-5'-O-(3-thiotriphosphate), tetralithium salt, reproduced all the results faithfully. The implication of the ATP-induced reversible protein-protein association at physiological temperatures on the functional role of alpha-crystallin in vivo is discussed.

Adenosine Monophosphate↗

PubMed2004

SDS induced structural changes in alpha-crystallin and it's effect on refolding.

Alpha-crystallin, a major eye lens protein and a key member of the small heat shock protein family, acts like a chaperone by preventing aggregation of substrate proteins. One of the hallmarks of most small heat shock proteins is their existence as a large oligomer, the role of which in its function is not understood at present. We have studied the role of the oligomer in the stability of its structure against SDS induced destabilization by CD measurements. Alpha-crystallin from bovine source as well as recombinant preparation was used for this purpose. As SDS concentration was gradually increased, the beta-sheet structure was diminished followed by concomitant increase in the alpha-helical structure. The quaternary structural changes in presence of SDS were also monitored by light scattering, polarization and anisotropy measurements. It was found that the breakdown of the oligomeric structure was nearly complete above 1 mM SDS concentration. The results were compared with that of a monomeric gamma-crystallin, which is also a major beta-sheet protein like alpha-crystallin. When alpha-crystallin was first converted into monomeric random coil structure in presence of 6 M urea and allowed to refold in SDS solution, amount of alpha-helix was more than that incubated directly in the same concentration of SDS. The results show that alpha-crystallin attains extra structural stability against external stress due to its oligomeric structure. The implication for the extra stability is discussed in reference to its function as molecular chaperone.

Animals↗