Characterization of the denaturation and renaturation of human plasma vitronectin. I. Biophysical characterization of protein unfolding and multimerization.
Upon treatment with denaturing agents, vitronectin has been observed to exhibit conformational alterations which are similar to the structural changes detected when vitronectin binds the thrombin-antithrombin complex or associates with the terminal attack complex of complement. Denaturation and renaturation of vitronectin isolated from human plasma were characterized by changes in intrinsic fluorescence. Unfolding by chemical denaturants was irreversible and accompanied by self-association of the protein to form vitronectin multimers. Self-association was evaluated by equilibrium analytical ultracentrifugation which demonstrated that multimers form only during the refolding process after removal of denaturant, that multimeric vitronectin dissociates to constituent subunits readily upon treatment with chemical denaturant, and that intermolecular disulfide cross-linking occurs primarily at the dimer level among a subset of constituent vitronectin subunits within the multimer. The monomeric form of vitronectin isolated from human plasma partially unfolds at intermediate concentrations of denaturant to an altered conformation with a high propensity to associate into multimers. Folding of vitronectin in vivo appears to be regulated by partitioning of folding intermediates toward either of two conformations, one that exists as a stable monomer and another that associates into a multimeric form.
Zhuang
P
P
Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996, USA.
Blackburn
M N
MN
Peterson
C B
CB
D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008962:Models, Theoretical; D008970:Molecular Weight; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D014461:Ultracentrifugation; D014508:Urea / Q000494:pharmacology; D019096:Vitronectin / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification
D006146:Guanidines; D046911:Macromolecular Substances; D019096:Vitronectin; D014508:Urea; D019791:Guanidine