Interactions of Chaperone α-Crystallin with the Molten Globule State of Xylose Reductase

Abstract

␣-Crystallin is a multimeric protein that has been shown to function as a molecular chaperone. Present investigations were undertaken to understand its mechanism of chaperoning. For this functional in vitro analysis of ␣-crystallin we used xylose reductase (XR) from Neurospora crassa as the model system. Denaturation studies using the structure-perturbing agent guanidinium chloride indicated that XR folds through a partially folded state that resembles the molten globule. Fluorescence and delay experiments revealed that ␣-crystallin interacts with the molten globule state of XR (XR-m) and prevents its aggregation. Cold lability of ␣-crystallin⅐XR-m interaction was revealed by temperature shift experiments implicating the involvement of hydrophobic interactions in the formation of the complex. Reconstitution of active XR was observed on cooling the ␣-crystallin⅐XR-m complex to 4°C or on addition of ATP at 37°C. ATP hydrolysis is not a prerequisite for XR release since the nonhydrolyzable analogue 5-adenylyl imidodiphosphate (AMP-PNP) was capable of reconstitution of active XR. Experimental evidence has been provided for temperature-and ATP-mediated structural changes in the ␣-crystallin⅐XR-m complex that shed some light on the mechanism of reconstitution of active XR by this chaperone. The relevance of our finding to the role of ␣-crystallin in vivo is discussed.

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

“…But, when a nonnatural substrate carbonic anhydrase remained bound to the chaperone, further loss in subunit exchange rate was Differential Recognition of Natural and Nonnatural Substrate by Molecular Chaperone -Crystallin-A Subunit Exchange Study -Crystallin prevents the aggregation of a large variety of substrates, e.g., insulin, -lactalbumin, lysozyme, conalbumin, alcohol dehydrogenase, citrate synthase, xylose reductase, etc. [9][10][11][12][13][14][15][16][17][18][19] These substrates include proteins, which are of low molecular weight [e.g., insulin (6 kDa)], comparable to -crystallin subunit molecular weight [e.g., carbonic anhydrase (29 kDa)] and high molecular weight [e.g., alcohol dehydragenase (150 kDa)]. Besides, it recognizes substrates under various stress conditions, such as heat, disulphide cleavage, UV light exposure, oxidative stress, etc.…”

“…Besides, it recognizes substrates under various stress conditions, such as heat, disulphide cleavage, UV light exposure, oxidative stress, etc. [9][10][11][12][13][14][15][16][18][19][20][21] These in vitro substrates belong to no particular category in terms of sequence or threedimensional structure. In addition to these ''nonnatural'' substrates, -crystallin prevents the aggregation of a number of its own substrates, which are present in the lens.…”

Role of ATP on the Interaction of α-Crystallin with Its Substrates and Its Implications for the Molecular Chaperone Function

“…Thus, ␣-crystallin has been reported to have autophosphorylation activity by some workers (29,30), whereas others failed detect significant ATPase activity (23). Some spectroscopic (16,24) and proteolytic (31) studies suggested interaction between ATP and ␣-crystallin with concomitant conformational changes, but the nature of the changes and its functional implications are far from clear. According to a totally different viewpoint existing in the literature, most sHSPs having the conserved ␣-crystallin domain have no dependence on ATP for their functions (22).…”

“…It has already been established that the yield of several substrates refolded in vitro in the presence of ␣-crystallin somewhat increased when ATP was present in the system (16,17,25,26), but mechanistic details are not known. ATP enhanced the yield of ␣-crystallin-mediated refolding of citrate synthase (17,25,26), xylose reductase (16), and luciferase (25).…”

“…␣-Crystallin, like other sHSPs, acts like a molecular chaperone in preventing the aggregation of other proteins (8 -10). An intense research activity on the structure and function of ␣-crystallin has grown since then (11)(12)(13)(14)(15)(16)(17)(18)(19)(20), and the subject matter has been extensively reviewed in recent years (21)(22)(23).…”