Snapshots of a protein folding intermediate.

Abstract:

:We have investigated the folding dynamics of Thermus thermophilus cytochrome c(552) by time-resolved fluorescence energy transfer between the heme and each of seven site-specific fluorescent probes. We have found both an equilibrium unfolding intermediate and a distinct refolding intermediate from kinetics studies. Depending on the protein region monitored, we observed either two-state or three-state denaturation transitions. The unfolding intermediate associated with three-state folding exhibited native contacts in β-sheet and C-terminal helix regions. We probed the formation of a refolding intermediate by time-resolved fluorescence energy transfer between residue 110 and the heme using a continuous flow mixer. The intermediate ensemble, a heterogeneous mixture of compact and extended polypeptides, forms in a millisecond, substantially slower than the ∼100-μs formation of a burst-phase intermediate in cytochrome c. The surprising finding is that, unlike for cytochrome c, there is an observable folding intermediate, but no microsecond burst phase in the folding kinetics of the structurally related thermostable protein.

authors

Yamada S,Bouley Ford ND,Keller GE,Ford WC,Gray HB,Winkler JR

doi

10.1073/pnas.1221832110

subject

Has Abstract

pub_date

2013-01-29 00:00:00

pages

1606-10

issue

5

eissn

0027-8424

issn

1091-6490

pii

1221832110

journal_volume

110

pub_type

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