Interactions of chaperone alpha-crystallin with the molten globule state of xylose reductase. Implications for reconstitution of the active enzyme

J Biol Chem. 1998 Apr 17;273(16):9415-23. doi: 10.1074/jbc.273.16.9415.

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.

MeSH terms

  • Aldehyde Reductase / chemistry*
  • Aldehyde Reductase / metabolism*
  • Circular Dichroism
  • Crystallins / chemistry*
  • Crystallins / metabolism*
  • Guanidine / pharmacology
  • Kinetics
  • Molecular Chaperones / chemistry
  • Molecular Chaperones / metabolism*
  • Neurospora crassa / enzymology
  • Protein Conformation*
  • Protein Denaturation
  • Protein Folding*
  • Spectrometry, Fluorescence

Substances

  • Crystallins
  • Molecular Chaperones
  • Aldehyde Reductase
  • Guanidine