Off-resonance rotating-frame amide proton spin relaxation experiments measuring microsecond chemical exchange in proteins
2005 (English)In: Journal of Biomolecular NMR, ISSN 0925-2738, Vol. 32, no 2, 163-173 p.Article in journal (Refereed) Published
NMR spin relaxation in the rotating frame (R-1 rho) is a unique method for atomic-resolution characterization of conformational ( chemical) exchange processes occurring on the microsecond time scale. Here, we use amide H-1 off-resonance R-1 rho relaxation experiments to determine exchange parameters for processes that are significantly faster than those that can be probed using N-15 or C-13 relaxation. The new pulse sequence is validated using the E140Q mutant of the C-terminal domain of calmodulin, which exhibits significant conformational exchange contributions to the transverse relaxation rates. The H-1 off-resonance R-1 rho data sample the entire relaxation dispersion profiles for the large majority of residues in this protein, which exchanges between conformations with a time constant of approximately 20 mu s. This is in contrast to the case for N-15, where additional laboratory-frame relaxation data are required to determine the exchange parameters reliably. Experiments were performed on uniformly N-15-enriched samples that were either highly enriched in H-2 or fully protonated. In the latter case, dipolar cross-relaxation with aliphatic protons were effectively decoupled to first order using a selective inversion pulse. Deuterated and protonated samples gave the same results, within experimental errors. The use of deuterated samples increases the sensitivity towards exchange contributions to the H-1 transverse relaxation rates, since dipolar relaxation is greatly reduced. The exchange correlation times determined from the present H-1 off-resonance R-1 rho experiments are in excellent agreement with those determined previously using a combination of N-15 laboratory-frame and off-resonance R-1 rho relaxation data, with average values of <tau(ex)> = 19 +/- 7 and 21 +/- 3 mu s, respectively.
Place, publisher, year, edition, pages
2005. Vol. 32, no 2, 163-173 p.
CONFORMATIONAL EXCHANGE; TRANSVERSE RELAXATION; R-1-RHO RELAXATION; CROSS-CORRELATION; NMR-SPECTRA; TIME-SCALE; CALMODULIN
IdentifiersURN: urn:nbn:se:liu:diva-51906DOI: 10.1007/s10858-005-5027-3OAI: oai:DiVA.org:liu-51906DiVA: diva2:278088