Subunit Exchange of αA-Crystallin

Abstract

␣-Crystallin, the major protein in the mammalian lens, is a molecular chaperone that can bind denaturing proteins and prevent their aggregation. Like other structurally related small heat shock proteins, each ␣-crystallin molecule is composed of an average of 40 subunits that can undergo extensive reorganization. In this study we used fluorescence resonance energy transfer to monitor the rapid exchange of recombinant ␣-crystallin subunits. We labeled ␣A-crystallin with stilbene iodoacetamide (4-acetamido-4-((iodoacetyl)amino)stilbene-2,2-disulfonic acid), which serves as an energy donor and with lucifer yellow iodoacetamide, which serves as an energy acceptor. Upon mixing the two populations of labeled ␣A-crystallin, we observed a reversible, time-dependent decrease in stilbene iodoacetamide emission intensity and a concomitant increase in lucifer yellow iodoacetamide fluorescence. This result is indicative of an exchange reaction that brings the fluorescent ␣A-crystallin subunits close to each other. We further showed that the exchange reaction is strongly dependent on temperature, with a rate constant of 0.075 min ؊1 at 37°C and an activation energy of 60 kcal/mol. The subunit exchange is independent of pH and calcium concentration but decreases at low and high ionic strength, suggesting the involvement of both ionic and hydrophobic interactions. It is also markedly reduced by the binding of large denatured proteins. The degree of inhibition is directly proportional to the molecular mass and the amount of bound polypeptide, suggesting an interaction of several ␣A-crystallin subunits with multiple binding sites of the denaturing protein. Our findings reveal a dynamic organization of ␣A-crystallin subunits, which may be a key factor in preventing protein aggregation during denaturation.␣-Crystallin, the major lens protein of the mammalian eye, is a member of the small heat shock protein family (1, 2). Like other small heat shock proteins, ␣-crystallin is a high molecular mass complex consisting of a large number of subunits. The two polypeptides of ␣-crystallin found in the lens of the mammalian eye, ␣A and ␣B, are encoded by evolutionarily related genes and share more than 50% identity in amino acid sequence (3, 4). For many years, ␣-crystallin was thought to be lens-specific. However, recent advances in detection methods have revealed much wider non-lenticular tissue distributions in heart, thymus, skin, lung, retina, and brain (5-8).

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

“…Substrate binding to -crystallin slows down the exchange reaction. 27 We have chosen natural and nonnatural substrates of comparable molecular weight to that of the subunit of -crystallin. Our data show that the binding of CA substantially slows down the subunit exchange process compared to the binding of -crystallin and the rate constant for the subunit exchange process for the previous situation becomes nearly half of the later (Table II).…”

“…The concentrations of LYI and AIAS were determined from their absorption spectra using molar extinction coefficients of 13,000 and 35,000 cm À1 M À1 at 435 and 335 nm, respectively. 27 …”

“…The significant overlap of the emission spectrum of the AIAS fluorophore with the absorption spectrum of LYI fluorophore indicates that they are excellent donor-acceptor pair for fluorescence resonance energy transfer (FRET). 27 This energy transfer efficiency is dependent upon the distance between the two fluorophores. Subunit exchange brings the fluorophores tagged to Acrystallin close enough for the energy transfer to take place.…”

“…The intensity at 415 nm was determined. The subunit exchange rate was calculated from the equation 27 :…”

Small heat-shock proteins: important players in regulating cellular proteostasis

“…adding green fluorescent protein or one of its derivatives for fluorescence energy transfer studies) as these can directly interfere with the dynamism of the sHsps, their ability to form oligomers and thus their chaperone function [87,88]. The formation of hetero-oligomers between sHsps is temperature dependent [8,81,82,89] and, upon heat shock, the hetero-oligomeric assemblies of Hsp27 and αBc in cells dissociate and reform once the cell has recovered [79]. This raises questions as to whether homo-and heterooligomeric forms of sHsps play different functional roles in the cells.…”