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J Biol Chem, Vol. 274, Issue 47, 33209-33212, November 19, 1999

COMMUNICATION
Insight into the Secondary Structure of Non-native Proteins Bound to a Molecular Chaperone -Crystallin
AN ISOTOPE-EDITED INFRARED SPECTROSCOPIC STUDY

Kali P. Das, Lin-P'ing Choo-Smith, J. Mark Petrash^¶, and Witold K. Surewicz

From the  Department of Pathology, Case Western Reserve University, Cleveland, Ohio 44106 and the ^¶ Department of Ophthalmology and Visual Sciences, Washington University, St. Louis, Missouri 63110

-Crystallin, the major lens protein, acts as a molecular chaperone by preventing the aggregation of proteins damaged by heat and other stress conditions. To characterize the backbone conformation of protein folding intermediates that are recognized by the chaperone, we prepared the uniformly ¹³C-labeled A-crystallin. The labeling greatly reduced the overlapping between the conformation-sensitive amide I bands of -crystallin and unlabeled substrate proteins. This procedure has allowed us to gain insight into the secondary structure of -crystallin-bound species, an understanding which has previously been unattainable. Analysis of the infrared spectra of two substrate proteins (- and _L-crystallins) indicates that heat-destabilized conformers captured by -crystallin are characterized by a high proportion of native-like secondary structure. In contrast to the chaperone-bound species, the same proteins subjected to heat treatment in the absence of -crystallin preserve very little native secondary structure. These data show that -crystallin specifically recognizes very early intermediates on the denaturation pathway of proteins. These aggregation-prone species are characterized by native-like secondary structure but compromised tertiary interactions. The experimental approach described in this study can be further applied to probe the backbone conformation of proteins bound to chaperones other than -crystallin.

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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.

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