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Early kinetic intermediate in the folding of acyl-CoA binding protein detected by fluorescence labeling and ultrarapid mixing.

Abstract：

:Early conformational events during folding of acyl-CoA binding protein (ACBP), an 86-residue alpha-helical protein, were explored by using a continuous-flow mixing apparatus with a dead time of 70 micros to measure changes in intrinsic tryptophan fluorescence and tryptophan-dansyl fluorescence energy transfer. Although the folding of ACBP was initially described as a concerted two-state process, the tryptophan fluorescence measurements revealed a previously unresolved phase with a time constant tau = 80 micros, indicating formation of an intermediate with only slightly enhanced fluorescence of Trp-55 and Trp-58 relative to the unfolded state. To amplify this phase, a dansyl fluorophore was introduced at the C terminus by labeling an I86C mutant of ACBP with 5-IAEDANS [5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid]. Continuous-flow refolding of guanidine HCl-denatured ACBP showed a major increase in tryptophan-dansyl fluorescence energy transfer, indicating formation of a partially collapsed ensemble of states on the 100-micros time scale. A subsequent decrease in dansyl fluorescence is attributed to intramolecular quenching of donor fluorescence on formation of the native state. The kinetic data are fully accounted for by three-state mechanisms with either on- or off-pathway intermediates. The intermediate accumulates to a maximum population of 40%, and its stability depends only weakly on denaturant concentration, which is consistent with a marginally stable ensemble of partially collapsed states with approximately 1/3 of the solvent-accessible surface buried. The findings indicate that ultrafast mixing methods combined with sensitive conformational probes can reveal transient accumulation of intermediate states in proteins with apparent two-state folding mechanisms.

journal_name

Proc Natl Acad Sci U S A

journal_title

Proceedings of the National Academy of Sciences of the United States of America

authors

Teilum K,Maki K,Kragelund BB,Poulsen FM,Roder H

doi

10.1073/pnas.152321499

keywords:

subject

Has Abstract

pub_date

2002-07-23 00:00:00

pages

9807-12

issue

eissn

0027-8424

issn

1091-6490

pii

152321499

journal_volume

pub_type

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Early kinetic intermediate in the folding of acyl-CoA binding protein detected by fluorescence labeling and ultrarapid mixing.