Antibody-detected folding: kinetics of surface epitope formation are distinct from other folding phases.
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 k(ad) = 0.2 s(-1) and is approximately 40-fold slower than the fastest detectable event in the folding of yeast iso-2 cytochrome c (k2f approximately 8 s(-1)), but occurs prior to the absorbance- and fluorescence-detected slow folding steps (k1a approximately 0.06 s(-1); k1b approximately 0.09 s(-1)). N5I 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.
Raman
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Department of Biochemistry, University of Texas Health Science Center, San Antonio 78229-3900, USA.
Jemmerson
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Nall
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D000818:Animals; D000911:Antibodies, Monoclonal; D003574:Cytochrome c Group / Q000737:chemistry* / Q000276:immunology; D045304:Cytochromes c; D000939:Epitopes; D006736:Horses; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D015003:Yeasts / Q000737:chemistry
D000911:Antibodies, Monoclonal; D003574:Cytochrome c Group; D000939:Epitopes; C032139:iso-2-cytochrome C; D045304:Cytochromes c