Abstract
| Originalsprog | Engelsk |
|---|---|
| Tidsskrift | Proceedings of the National Academy of Science of the United States of America |
| Vol/bind | 99 |
| Udgave nummer | 15 |
| Sider (fra-til) | 9807-12 |
| Antal sider | 5 |
| ISSN | 0027-8424 |
| DOI | |
| Status | Udgivet - 2002 |
Bibliografisk 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; TryptophanAdgang til dokumentet
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Early kinetic intermediate in the folding of acyl-CoA binding protein detected by fluorescence labeling and ultrarapid mixing. / Teilum, Kaare; Maki, Kosuke; Kragelund, Birthe B; Poulsen, Flemming M; Roder, Heinrich.
I: Proceedings of the National Academy of Science of the United States of America, Bind 99, Nr. 15, 2002, s. 9807-12.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review
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TY - JOUR
T1 - Early kinetic intermediate in the folding of acyl-CoA binding protein detected by fluorescence labeling and ultrarapid mixing
AU - Teilum, Kaare
AU - Maki, Kosuke
AU - Kragelund, Birthe B
AU - Poulsen, Flemming M
AU - Roder, Heinrich
N1 - 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
PY - 2002
Y1 - 2002
N2 - 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.
AB - 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.
U2 - 10.1073/pnas.152321499
DO - 10.1073/pnas.152321499
M3 - Journal article
C2 - 12096190
VL - 99
SP - 9807
EP - 9812
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
SN - 0027-8424
IS - 15
ER -