Differential recognition of natural and nonnatural substrate by molecular chaperone α‐crystallin—A subunit exchange study

Abstract

Abstractα‐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 α‐crystallin has any preference for its natural substrates compared to the nonnatural one. Our results clearly show that α‐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 α‐crystallin. Using suitable fluorescent‐labeled α‐crystallins and performing fluorescence resonance energy transfer experiments, we were able to determine the subunit exchange kinetics between the α‐crystallin oligomers. It was found that while α‐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 β‐sheet structure of both α‐crystallin and its natural substrates may be the reason for the preference in molecular recognition in comparison with the nonnatural substrate. © 2006 Wiley Periodicals, Inc. Biopolymers 85:189–197, 2007.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

Structure, stability, and chaperone function of αA‐crystallin: Role of N‐terminal region

“…Recently we demonstrated that subunit exchange rate can be a valid mechanism by which a-crystallin can distinguish between the native and non-native substrate. 75 Results presented here ( Figure 8 and unfavorable for keeping the chaperone-substrate interaction soluble folding competent state. It appears to us that the subunit exchange rate is in someway related to the major b-sheet content of the wild type protein.…”

“…These two probes have been previously characterized and used to monitor exchange of subunits. 50,51,54,55,75 FðU The subunit exchange reaction was initiated 50 by mixing an equimolar concentration of the donor (AIAS) with the acceptor (LYI) labeled mutant at 378C. AIAS emission intensity at 415 nm is decreased in a time dependent manner (Figures 8A and 8B) with a concomitant increase in intensity at 525 nm indicating FRET because of the close proximity of LYI and AIAS.…”

“…It appears to us that the subunit exchange rate is in someway related to the major b-sheet content of the wild type protein. 75 As the b-sheet structure of the chaperone gets collapsed with deletion of the N-terminal residues, the exchange rate is decreased. Therefore subunit exchange is a valid indicator of chaperone activity.…”

Stabilization of oligomeric structure of α‐crystallin by Zn+2 through intersubunit bridging

“…32,33 We earlier reported that subunit exchange rate of aA-crystallin decreased significantly when non-natural substrate was bound to it 33 or when its N-terminal region was deleted. 26 Since intersubunit metal ion bridge formation is expected to influence the subunit exchange rate, we decided to measure the same in aA-crystallin in presence and absence of Zn 12 using FRET.…”

“…In order to carry out the FRET study recombinant aA-crystallin was separately labeled with AIAS and LYI through available cystein residues. 32,33 Recombinant human aA-crystallin contains two cystein residues at positions 131 and 142 among which the first one is exposed while the later is buried. We did not use human aB-crystallin because it did not contain any cystein residue.…”