Off-resonance R(1rho) NMR studies of exchange dynamics in proteins with low spin-lock fields: an application to a Fyn SH3 domain.
An (15)N NMR R(1rho) relaxation experiment is presented for the measurement of millisecond time scale exchange processes in proteins. On- and off-resonance R(1rho) relaxation profiles are recorded one residue at a time using a series of one-dimensional experiments in concert with selective Hartmann-Hahn polarization transfers. The experiment can be performed using low spin-lock field strengths (values as low as 25 Hz have been tested), with excellent alignment of magnetization along the effective field achieved. Additionally, suppression of the effects of cross-correlated relaxation between dipolar and chemical shift anisotropy interactions and (1)H-(15)N scalar coupled evolution is straightforward to implement, independent of the strength of the (15)N spin-locking field. The methodology is applied to study the folding of a G48M mutant of the Fyn SH3 domain that has been characterized previously by CPMG dispersion experiments. It is demonstrated through experiment that off-resonance R(1rho) data measured at a single magnetic field and one or more spin-lock field strengths, with amplitudes on the order of the rate of exchange, allow a complete characterization of a two-site exchange process. This is possible even in the case of slow exchange on the NMR time scale, where complementary approaches involving CPMG-based experiments fail. Advantages of this methodology in relation to other approaches are described.
Korzhnev
Dmitry M
DM
Protein Engineering Network Centers of Excellence and Department of Medical Genetics, The University of Toronto, Toronto, Ontario, Canada M5S 1A8.
Orekhov
Vladislav Yu
VY
Kay
Lewis E
LE
D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D017510:Protein Folding; D011518:Proto-Oncogene Proteins / Q000737:chemistry*; D051076:Proto-Oncogene Proteins c-fyn; D018909:src Homology Domains; D019061:src-Family Kinases / Q000737:chemistry*
D009587:Nitrogen Isotopes; D011518:Proto-Oncogene Proteins; D051076:Proto-Oncogene Proteins c-fyn; D019061:src-Family Kinases