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.
Biswas
Ashis
A
Protein Chemistry Laboratory, Department of Chemistry, Bose Institute, 93/1 A. P. C. Road, Kolkata 700 009, India.
Das
K P
KP
D000818:Animals; D001688:Biological Products / Q000378:metabolism*; D002417:Cattle; D021122:Protein Subunits / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D038201:alpha-Crystallins / Q000378:metabolism*
D001688:Biological Products; D021122:Protein Subunits; D038201:alpha-Crystallins