Measurement of Methyl 2H Quadrupolar Couplings in Oriented Proteins. How Uniform Is the Quadrupolar Coupling Constant?

2 H quadrupolar and one-bond 13 C− 13 C dipolar couplings have been measured at methyl sites in a uniformly 13 C and fractionally 2 H labeled sample of the N-terminal drk SH3 domain, weakly aligned in a dilute solution of Pf1 phage. An average ratio between the 2 H quadrupolar and 13 C− 13 C dipolar couplings of 19.2 ± 0.1 is measured. Assuming rapid rotation about the one-bond 13 C methyl − 13 C axis and an angle of 109.5° between the unique principal axis of the electric field gradient tensor and the methyl averaging axis ( 13 C methyl − 13 C bond), an average value of 167 ± 1 kHz is obtained for the quadrupolar coupling constant, e 2 qQ / h . The profile of 2 H quadrupolar vs 13 C− 13 C dipolar couplings suggests that the use of a uniform value for the quadrupolar coupling constant is a good approximation in the analysis of 2 H relaxation data measured at methyl sites in proteins.