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The inverted chevron plot measured by NMR relaxation reveals a native-like unfolding intermediate in acyl-CoA binding protein

Teilum, Kaare LU ; Poulsen, Flemming M. and Akke, Mikael LU (2006) In Proceedings of the National Academy of Sciences 103(18). p.6877-6882
Abstract
The folding kinetics of bovine acyl-CoA binding protein was studied by N-15 relaxation dispersion measurements under equilibrium conditions. Relaxation dispersion profiles were measured at several concentrations of guanidine hydrochloride (GuHCl). The unfolding rate constant (k(u)) was determined under conditions favoring folding, for which the folding rate constant (k(f)) dominates the relaxation in stopped-flow kinetic measurements. Conversely, k(f) was determined under conditions favoring unfolding, for which k(u) dominates stopped-flow data. The rates determined by NMR therefore complement those from stopped-flow kinetics and define an "inverted chevron" plot. The combination of NMR relaxation and stopped-flow kinetic measurements... (More)
The folding kinetics of bovine acyl-CoA binding protein was studied by N-15 relaxation dispersion measurements under equilibrium conditions. Relaxation dispersion profiles were measured at several concentrations of guanidine hydrochloride (GuHCl). The unfolding rate constant (k(u)) was determined under conditions favoring folding, for which the folding rate constant (k(f)) dominates the relaxation in stopped-flow kinetic measurements. Conversely, k(f) was determined under conditions favoring unfolding, for which k(u) dominates stopped-flow data. The rates determined by NMR therefore complement those from stopped-flow kinetics and define an "inverted chevron" plot. The combination of NMR relaxation and stopped-flow kinetic measurements allowed determination of kf and k(u) in the range from 0.48 M GuHCl to 1.28 M GuHCl. Individually, the stopped-flow and NMR data fit two-state models for folding. However, although the values of kf determined by the two methods agree, the values of k(u) do not. As a result, a combined analysis of all data does not comply with a two-state model but indicates that an unfolding intermediate exists on the native side of the dominant energy barrier. The denaturant and temperature dependencies of the chemical shifts and k(u) indicate that the intermediate state is structurally similar to the native state. Equilibrium unfolding monitored by optical spectroscopy corroborate these conclusions. The temperature dependence of the chemical shifts identifies regions of the protein that are selectively destabilized in the intermediate. These results illustrate the power of combining stopped-flow kinetics and NMR spectroscopy to analyze protein folding. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
flow, protein folding, kinetic analysis, NMR relaxation dispersion, stopped
in
Proceedings of the National Academy of Sciences
volume
103
issue
18
pages
6877 - 6882
publisher
National Acad Sciences
external identifiers
  • pmid:16641108
  • wos:000237399900019
  • scopus:33646474847
ISSN
1091-6490
DOI
10.1073/pnas.0509100103
language
English
LU publication?
yes
id
f3fbae3c-0762-4af2-940d-781d4869ddf6 (old id 572475)
date added to LUP
2007-10-20 10:30:00
date last changed
2019-02-20 04:14:28
@article{f3fbae3c-0762-4af2-940d-781d4869ddf6,
  abstract     = {The folding kinetics of bovine acyl-CoA binding protein was studied by N-15 relaxation dispersion measurements under equilibrium conditions. Relaxation dispersion profiles were measured at several concentrations of guanidine hydrochloride (GuHCl). The unfolding rate constant (k(u)) was determined under conditions favoring folding, for which the folding rate constant (k(f)) dominates the relaxation in stopped-flow kinetic measurements. Conversely, k(f) was determined under conditions favoring unfolding, for which k(u) dominates stopped-flow data. The rates determined by NMR therefore complement those from stopped-flow kinetics and define an "inverted chevron" plot. The combination of NMR relaxation and stopped-flow kinetic measurements allowed determination of kf and k(u) in the range from 0.48 M GuHCl to 1.28 M GuHCl. Individually, the stopped-flow and NMR data fit two-state models for folding. However, although the values of kf determined by the two methods agree, the values of k(u) do not. As a result, a combined analysis of all data does not comply with a two-state model but indicates that an unfolding intermediate exists on the native side of the dominant energy barrier. The denaturant and temperature dependencies of the chemical shifts and k(u) indicate that the intermediate state is structurally similar to the native state. Equilibrium unfolding monitored by optical spectroscopy corroborate these conclusions. The temperature dependence of the chemical shifts identifies regions of the protein that are selectively destabilized in the intermediate. These results illustrate the power of combining stopped-flow kinetics and NMR spectroscopy to analyze protein folding.},
  author       = {Teilum, Kaare and Poulsen, Flemming M. and Akke, Mikael},
  issn         = {1091-6490},
  keyword      = {flow,protein folding,kinetic analysis,NMR relaxation dispersion,stopped},
  language     = {eng},
  number       = {18},
  pages        = {6877--6882},
  publisher    = {National Acad Sciences},
  series       = {Proceedings of the National Academy of Sciences},
  title        = {The inverted chevron plot measured by NMR relaxation reveals a native-like unfolding intermediate in acyl-CoA binding protein},
  url          = {http://dx.doi.org/10.1073/pnas.0509100103},
  volume       = {103},
  year         = {2006},
}