Interactions of chaperone alpha-crystallin with the molten globule state of xylose reductase. Implications for reconstitution of the active enzyme.
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.
Rawat
U
U
Division of Biochemical Sciences, National Chemical Laboratory, Pune 411008, India.
Rao
M
M
D000449:Aldehyde Reductase / Q000737:chemistry* / Q000378:metabolism*; D002942:Circular Dichroism; D003459:Crystallins / Q000737:chemistry* / Q000378:metabolism*; D019791:Guanidine / Q000494:pharmacology; D007700:Kinetics; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism*; D009492:Neurospora crassa / Q000201:enzymology; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence
D003459:Crystallins; D018832:Molecular Chaperones; D000449:Aldehyde Reductase; D019791:Guanidine