Precision measurements of relaxation rates of degenerate 1H transitions in methyl groups of small proteins

Abstract

An NMR experiment is designed for accurate and robust measurement of transverse relaxation rates of degenerate ¹H transitions in selectively ¹³CH₃-labeled, deuterated small proteins. The measurement is based on the use of acute (<90°) angle ¹H radio-frequency pulses and relies on selection of the slow- and fast-relaxing components of methyl magnetization following the relaxation period in separate experiments. The R₂ decay series recorded with selection of the fast-relaxing components serves as a useful complement to the R₂ series acquired with selection of the slow-relaxing part, and permits the extension of the range of relative contributions of the fast- and slow-relaxing parts to apparent signal decay. The approach is experimentally verified on ¹³CH₃ methyl groups of the ILV-{¹³CH₃}-labeled protein ubiquitin at 10 °C and 25 °C. The obtained methyl ¹H relaxation rates are in remarkably good agreement with the values obtained from well-established NMR techniques.

Keywords: (1)H relaxation in magnetically equivalent spin-systems; (13)CH(3) spin-system; Acute angle RF pulses; Methyl NMR.