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

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Kaare Teilum, Kosuke Maki, Birthe B Kragelund, Flemming M Poulsen, Heinrich Roder

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.
Original languageEnglish
JournalProceedings of the National Academy of Science of the United States of America
Volume99
Issue number15
Pages (from-to)9807-12
Number of pages5
ISSN0027-8424
DOIs
Publication statusPublished - 2002

Bibliographical note

Keywords: Amino Acid Substitution; Cloning, Molecular; Dansyl Compounds; Diazepam Binding Inhibitor; Escherichia coli; Fluorescent Dyes; Kinetics; Models, Molecular; Mutagenesis, Site-Directed; Protein Folding; Protein Structure, Secondary; Recombinant Proteins; Spectrometry, Fluorescence; Tryptophan

ID: 21833130