Abstract
alpha-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 alpha-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 alpha-crystallin interacts with the molten globule state of XR (XR-m) and prevents its aggregation. Cold lability of alpha-crystallin.XR-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 alpha-crystallin.XR-m complex to 4 degrees C or on addition of ATP at 37 degrees 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 alpha-crystallin.XR-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 alpha-crystallin in vivo is discussed.
Highlights
␣-Crystallin is a multimeric protein that has been shown to function as a molecular chaperone
Experimental evidence has been provided for temperature- and ATP-mediated structural changes in the ␣-crystallin1⁄7XR-m complex that shed some light on the mechanism of reconstitution of active xylose reductase (XR) by this chaperone
XR was incubated with increasing concentrations of the denaturant GdmCl, and the changes in the negative Circular dichroism (CD) band in the far UV region were monitored
Summary
␣-Crystallin is a multimeric protein that has been shown to function as a molecular chaperone. Experimental evidence has been provided for temperature- and ATP-mediated structural changes in the ␣-crystallin1⁄7XR-m complex that shed some light on the mechanism of reconstitution of active XR by this chaperone.
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