Quantitative analysis of conformational exchange contributions to H-1-N-15 multiple-quantum relaxation using field-dependent measurements. Time scale and structural characterization of exchange in a calmodulin C-terminal domain mutant
2004 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, ISSN 0002-7863, Vol. 126, no 3, 928-935 p.Article in journal (Refereed) Published
Multiple-quantum spin relaxation is a sensitive probe for correlated conformational exchange dynamics on microsecond to millisecond time scales in biomolecules. We measured differential H-1-N-15 multiple-quantum relaxation rates for the backbone amide groups of the E140Q mutant of the C-terminal domain of calmodulin at three static magnetic field strengths. The differential multiple-quantum relaxation rates range between -88.7 and 92.7 s(-1), and the mean and standard deviation are 7.0 24 s(-1), at a static magnetic field strength of 14.1 T. Together with values of the H-1 and N-15 chemical shift anisotropies (CSA) determined separately, the field-dependent data enable separation of the different contributions from dipolar-dipolar, CSA-CSA, and conformational exchange cross-correlated relaxation mechanisms to the differential multiple-quantum relaxation rates. The procedure yields precise quantitative information on the dominant conformational exchange contributions observed in this protein. The field-dependent differences between double- and zero-quantum relaxation rates directly benchmark the rates of conformational exchange, showing that these are fast on the chemical shift time scale for the large majority of residues in the protein. Further analysis of the differential H-1-N-15 multiple-quantum relaxation rates using previously determined exchange rate constants and populations, obtained from N-15 off-resonance rotating-frame relaxation data, enables extraction of the product of the chemical shift differences between the resonance frequencies of the H-1 and N-15 spins in the exchanging conformations, deltasigma(H)deltasigma(N). Thus, information on the H-1 chemical shift differences is obtained, while circumventing complications associated with direct measurements of conformational exchange effects on H-1 single-quantum coherences in nondeuterated proteins. The method significantly increases the information content available for structural interpretation of the conformational exchange process, partly because deltasigma(H)deltasigma(N) is a signed quantity, and partly because two chemical shifts are probed simultaneously. The present results support the hypothesis that the exchange in the calcium-loaded state of the E140Q mutant involves conformations similar to those of the wild-type apo (closed) and calcium-loaded (open) states.
Place, publisher, year, edition, pages
2004. Vol. 126, no 3, 928-935 p.
chemical shift anisotropy, cross-correlated relaxation, Model-free, biological macromolecules, protein, secondary structure, backbone dynamics, NMR spectroscopy
Physical Chemistry Structural Biology Physical Chemistry
IdentifiersURN: urn:nbn:se:liu:diva-51903DOI: 10.1021/ja037529rOAI: oai:DiVA.org:liu-51903DiVA: diva2:278087