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Antibody-detected folding: Kinetics of surface epitope formation are distinct from other folding phases

Published online by Cambridge University Press:  01 January 2000

C.S. RAMAN
Affiliation:
Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229-3900 Present address: Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697-3900.
RONALD JEMMERSON
Affiliation:
Department of Microbiology and Center for Immunology, University of Minnesota, Minneapolis, Minnesota 55455-0312
BARRY T. NALL
Affiliation:
Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229-3900
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Abstract

The rate of macromolecular surface formation in yeast iso-2 cytochrome c and its site-specific mutant, N52I iso-2, has been studied using a monoclonal antibody that recognizes a tertiary epitope including K58 and H39. The results indicate that epitope refolding occurs after fast folding but prior to slow folding, in contrast to horse cytochrome c where surface formation occurs early. The antibody-detected (ad) kinetic phase accompanying epitope formation has kad = 0.2 s−1 and is ∼40-fold slower than the fastest detectable event in the folding of yeast iso-2 cytochrome c (k2f ∼ 8 s−1), but occurs prior to the absorbance- and fluorescence-detected slow folding steps (k1a ∼ 0.06 s−1; k1b ∼ 0.09 s−1). N52I iso-2 cytochrome c exhibits similar kinetic behavior with respect to epitope formation. A detailed dissection of the mechanistic differences between the folding pathways of horse and yeast cytochromes c identifies possible reasons for the slow surface formation in the latter. Our results suggest that non-native ligation involving H33 or H39 during refolding may slow down the formation of the tertiary epitope in iso-2 cytochrome c. This study illustrates that surface formation can be coupled to early events in protein folding. Thus, the rate of macromolecular surface formation is fine tuned by the residues that make up the surface and the interactions they entertain during refolding.

Type
Research Article
Copyright
© 2000 The Protein Society

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