| Autor: |
Philipp, Sabine, Kim, Young-Mi, Dürr, Ingolf, Wenzl, Gabriele, Vogt, Michael, Flecker, Peter |
| Předmět: |
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| Zdroj: |
European Journal of Biochemistry; Feb98 Part1, Vol. 251 Issue 3, p854-862, 9p, 6 Diagrams, 1 Chart, 3 Graphs |
| Abstrakt: |
It is widely believed that protein folding is a hierarchical process proceeding from secondary structure via subdomains and domains towards the complete tertiary structure. Accordingly, protein subdomains should behave as independent folding units. However, this prediction would underestimate the well-established structural significance of tertiary context and domain interfaces in proteins. The principal objective of this work was to distinguish between autonomous and cooperative refolding of protein subdomains by means of mutational analysis. The double-headed Bowman-Birk inhibitor of trypsin and chymotrypsin of known crystal structure was selected for study. The relative orientation of the two subdomains is stabilized by intramolecular and water-mediated hydrogen bonds and close ion pairs across a polar domain interface. The binary arrangement of a trypsin-reactive and a chymotrypsin-reactive subdomain facilitates the distinction of local and global irregularities in the mutants of this protein by means of functional assays. The functional consequences of five replacements in the S-S bond framework of the trypsin-reactive subdomain are analyzed in the present report. The mutants were subjected to refolding experiments in a refolding buffer and on trypsin-Sepharose as a template with complementary structure leading into a fully active state. The stability of the variants was assessed by means of subsequent equilibration experiments in solution. The mutants may be grouped into the following two classes : the class-I mutations located within β-strand A are characterized by a breakdown of the trypsin- and the chymotrypsin-reactive subdomain upon refolding in solution and a complicated behavior in the equilibration experiments; by contrast, the Class-II mutations (β-strand B) display rather local perturbations and a reversible return to the initial ratio of the two subdomains. This points to a significance of polar interactions connecting the... [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
| Externí odkaz: |
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