Early kinetic intermediate in the folding of acyl-CoA binding protein detected by fluorescence labeling and ultrarapid mixing.
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.
Teilum
Kaare
K
Department of Protein Chemistry, Institute of Molecular Biology, University of Copenhagen, DK-1353 Copenhagen K, Denmark.
Maki
Kosuke
K
Kragelund
Birthe B
BB
Poulsen
Flemming M
FM
Roder
Heinrich
H
D019943:Amino Acid Substitution; D003001:Cloning, Molecular; D003619:Dansyl Compounds; D026261:Diazepam Binding Inhibitor / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005456:Fluorescent Dyes; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
D003619:Dansyl Compounds; D026261:Diazepam Binding Inhibitor; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D014364:Tryptophan