The inverted chevron plot measured by NMR relaxation reveals a native-like unfolding intermediate in acyl-CoA binding protein.
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.
Teilum
Kaare
K
Department of Biophysical Chemistry, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden.
Poulsen
Flemming M
FM
Akke
Mikael
M
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D000818:Animals; D002417:Cattle; D026261:Diazepam Binding Inhibitor / Q000737:chemistry* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013696:Temperature
D000817:Anilino Naphthalenesulfonates; D026261:Diazepam Binding Inhibitor; D005456:Fluorescent Dyes; C027132:1-anilino-8-naphthalenesulfonate