Off-resonance R1rho relaxation outside of the fast exchange limit: an experimental study of a cavity mutant of T4 lysozyme.
An (15)N off-resonance R(1rho) spin relaxation study of an L99A point mutant of T4 lysozyme is presented. Previous CPMG-based relaxation dispersion studies of exchange in this protein have established that the molecule interconverts between a populated ground state and an excited state (3.4%) with an exchange rate constant of 1450 s(-1) at 25 degrees C. It is shown that for the majority of residues in this protein the offset dependence of the R(1rho) relaxation rates cannot be well fit using models which are only valid in the fast exchange regime. In contrast, a recently derived expression by Trott and Palmer (J. Magn. Reson., 154, 157-160, 2002) which is valid over a wider window of exchange than other relations, is shown to fit the data well. Values of (signed) chemical shift differences between exchanging sites have been extracted and are in reasonable agreement with shift differences measured using CPMG methods. A set of simulations is presented which help establish the exchange regimes that are best suited to analysis by off-resonance R(1rho) techniques.
Korzhnev
Dmitry M
DM
Protein Engineering Network Centers of Excellence, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Orekhov
Vladislav Yu
VY
Dahlquist
Frederick W
FW
Kay
Lewis E
LE
D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008962:Models, Theoretical; D009113:Muramidase / Q000737:chemistry*; D009587:Nitrogen Isotopes; D017354:Point Mutation; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D009587:Nitrogen Isotopes; D009113:Muramidase