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1 Department of Biophysics and Radiation Biology and 2 Research Group for Biophysics, Hungarian Academy of Sciences–Semmelweis University, H-1088 Budapest, Hungary
3 Institute for Theoretical Chemistry and Structural Biology, University of Vienna, A-1090 Wien, Austria
4 Institute of Enzymology, Biological Research Center of Szeged, H-1113 Budapest, Hungary
(RECEIVED January 14, 2005; FINAL REVISION March 13, 2005; ACCEPTED March 13, 2005)
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 Ndomain 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.
Keywords: protein folding; domain interactions; phosphoglycerate kinase; two-state folding; tryptophan fluorescence
Abbreviations: GuHCl, Guanidine hydrochloride • DTT, 1,4-Dithio-Lthreitol • EDTA, Ethylenediamine-tetraacetic acid disodium salt • PGK, phosphoglycerate kinase • hisPGK, histidine-tagged variant of yeast PGK • hisN, histidine-tagged variant of the N-terminal domain (1–186) of yeast PGK • hisC, histidine-tagged variant of the C-terminal domain (187–412) of yeast PGK • hisPGK W333, W308F mutant of hisPGK • hisPGK W122, W308F, W333F, Y122W triple mutant of hisPGK • hisN W122, Y122W mutant of hisN • hisCW333,W308Fmutant of hisC
Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051359905.
Reprint requests to: Szabolcs Osváth, H-1444, Budapest Pf: 263, Hungary; e-mail: osvath{at}puskin.sote.hu; fax: +36-1-266-6656.
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