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

Kaare Teilum; Flemming M Poulsen; Mikael Akke

doi:10.1073/pnas.0509100103

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

Kaare Teilum, Flemming M Poulsen, Mikael Akke

21 Citations (Scopus)

Abstract

The folding kinetics of bovine acyl-CoA binding protein was studied by 15N 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 k(f) 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 k(f) 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.

Original language	English
Journal	Proceedings of the National Academy of Science of the United States of America
Volume	103
Issue number	18
Pages (from-to)	6877-82
Number of pages	5
ISSN	0027-8424
DOIs	https://doi.org/10.1073/pnas.0509100103
Publication status	Published - 2006
Externally published	Yes

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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; Poulsen, Flemming M; Akke, Mikael.
In: Proceedings of the National Academy of Science of the United States of America, Vol. 103, No. 18, 2006, p. 6877-82.

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

abstract = "The folding kinetics of bovine acyl-CoA binding protein was studied by 15N 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 k(f) 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 k(f) 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 = "Kaare Teilum and Poulsen, {Flemming M} and Mikael Akke",

note = "Keywords: Anilino Naphthalenesulfonates; Animals; Cattle; Diazepam Binding Inhibitor; Fluorescent Dyes; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Protein Denaturation; Protein Folding; Protein Structure, Secondary; Temperature",

year = "2006",

doi = "10.1073/pnas.0509100103",

language = "English",

volume = "103",

pages = "6877--82",

journal = "Proceedings of the National Academy of Science of the United States of America",

issn = "0027-8424",

publisher = "The National Academy of Sciences of the United States of America",

number = "18",

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T1 - The inverted chevron plot measured by NMR relaxation reveals a native-like unfolding intermediate in acyl-CoA binding protein

AU - Teilum, Kaare

AU - Poulsen, Flemming M

AU - Akke, Mikael

N1 - Keywords: Anilino Naphthalenesulfonates; Animals; Cattle; Diazepam Binding Inhibitor; Fluorescent Dyes; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Protein Denaturation; Protein Folding; Protein Structure, Secondary; Temperature

PY - 2006

Y1 - 2006

N2 - The folding kinetics of bovine acyl-CoA binding protein was studied by 15N 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 k(f) 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 k(f) 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.

AB - The folding kinetics of bovine acyl-CoA binding protein was studied by 15N 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 k(f) 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 k(f) 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.

U2 - 10.1073/pnas.0509100103

DO - 10.1073/pnas.0509100103

M3 - Journal article

C2 - 16641108

SN - 0027-8424

VL - 103

SP - 6877

EP - 6882

JO - Proceedings of the National Academy of Science of the United States of America

JF - Proceedings of the National Academy of Science of the United States of America

IS - 18

ER -

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

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