Phi-value analysis of a three-state protein folding pathway by NMR relaxation dispersion spectroscopy.
Experimental studies of protein folding frequently are consistent with two-state folding kinetics. However, recent NMR relaxation dispersion studies of several fast-folding mutants of the Fyn Src homology 3 (SH3) domain have established that folding proceeds through a low-populated on-pathway intermediate, which could not be detected with stopped-flow experiments. The dispersion experiments provide precise kinetic and thermodynamic parameters that describe the folding pathway, along with a detailed site-specific structural characterization of both the intermediate and unfolded states from the NMR chemical shifts that are extracted. Here we describe NMR relaxation dispersion Phi-value analysis of the A39V/N53P/V55L Fyn SH3 domain, where the effects of suitable point mutations on the energy landscape are quantified, providing additional insight into the structure of the folding intermediate along with per-residue structural information of both rate-limiting transition states that was not available from previous studies. In addition to the advantage of delineating the full three-state folding pathway, the use of NMR relaxation dispersion as opposed to stopped-flow kinetics to quantify Phi values facilitates their interpretation because the obtained chemical shifts monitor any potential structural changes along the folding pathway that might be introduced by mutation, a significant concern in their analysis. Phi-Value analysis of several point mutations of A39V/N53P/V55L Fyn SH3 establishes that the beta(3)-beta(4)-hairpin already is formed in the first transition state, whereas strand beta(1), which forms nonnative interactions in the intermediate, does not fully adopt its native conformation until after the final transition state. The results further support the notion that on-pathway intermediates can be stabilized by nonnative contacts.
Neudecker
Philipp
P
Department of Medical Genetics, University of Toronto, Toronto, ON, Canada M5S 1A8.
Zarrine-Afsar
Arash
A
Davidson
Alan R
AR
Kay
Lewis E
LE
D019943:Amino Acid Substitution; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D009154:Mutation; D017354:Point Mutation; D011487:Protein Conformation; D017510:Protein Folding; D051076:Proto-Oncogene Proteins c-fyn / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D018909:src Homology Domains
D011994:Recombinant Proteins; D051076:Proto-Oncogene Proteins c-fyn