Determination of (13)C (α) relaxation times in uniformly (13)C/ (15)N-enriched proteins.

Relaxation times of (13)C(α) carbons of uniformly (13)C/(15)N-enriched probes have been investigated. The relaxation behaviour was analyzed in terms of a multispin system. Pulse sequences for the determination of T(1), T(2) and the heteronuclear NOE of (13)C(α) in uniformly (13)C/(15)N-enriched ribonuclease T1 are presented. The experiments performed in order to obtain T(1) and the heteronuclear NOE were similar to those of the corresponding (15)N experiments published previously. The determination of T(2) for the C(α)-carbon in a completely labeled protein is more complicated, since the magnetization transfer during the T(2) evolution period owing to the scalar coupling of C(α)-C(β) must be suppressed. Various different pulse sequences for the T(2) evolution period were simulated in order to optimize the bandwidth for which reliable T(2) relaxation times can be obtained. A proof for the quality of these pulse sequences is given by fitting the intensity decay of individual (1)H-(13)C(α) cross peaks, in a series of ((1)H, (13)C)-ct-HSQC spectra with a modified CPMG sequence as well as a T(1p) sequence for the transverse relaxation time, to a single exponential using a simplex algorithm.