Asymmetric effect of domain interactions on the kinetics of folding in yeast phosphoglycerate kinase

Szabolcs Osváth, Gottfried Köhler, Péter Závodszky, Judit Fidy

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

The aim of this work is to shed more light on the effect of domain-domain interactions on the kinetics and the pathway of protein folding. A model protein system consisting of several single-tryptophan variants of the two-domain yeast phosphoglycerate kinase (PGK) and its individual domains was studied. Refolding was initiated from the guanidine-unfolded state by stopped-flow or manual mixing and monitored by tryptophan fluorescence from 1 msec to 1000 sec. Denaturant titrations of both individual domains showed apparent two-state unfolding transitions. Refolding kinetics of the individual domains from different denaturant concentrations, however, revealed the presence of intermediate structures during titration for both domains. Refolding of the same domains within the complete protein showed that domain-domain interactions direct the folding of both domains, but in an asymmetric way. Folding of the N domain was already altered within 1 msec, while detectable changes in the folding of the C domain occurred only 60-100 msec after initiating refolding. All mutants showed a hyperfluorescent kinetic intermediate. Both the disappearance of this intermediate and the completion of the folding were significantly faster in the individual N domain than in the complete protein. On the contrary, folding of the individual C domain was slower than in the complete protein. The presence of the C domain directs the refolding of the N domain along a completely different pathway than that of the individual N domain, while folding of the individual C domain follows the same path as within the complete protein.

Original languageEnglish
Pages (from-to)1609-1616
Number of pages8
JournalProtein Science
Volume14
Issue number6
DOIs
Publication statusPublished - Jun 1 2005

Keywords

  • Domain interactions
  • Phosphoglycerate kinase
  • Protein folding
  • Tryptophan fluorescence
  • Two-state folding

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Fingerprint Dive into the research topics of 'Asymmetric effect of domain interactions on the kinetics of folding in yeast phosphoglycerate kinase'. Together they form a unique fingerprint.

  • Cite this