Determination of 13C? relaxation times in uniformly 13C/15N-enriched proteins

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Abstract
Relaxation times of 13Cα carbons of uniformly 13C/15N-enriched probes have been investigated. The relaxation behaviour was analyzed in terms of a multispin system. Pulse sequences for the determination of T1, T2 and the heteronuclear NOE of 13Cα in uniformly 13C/15N-enriched ribonuclease T1 are presented. The experiments performed in order to obtain T1 and the heteronuclear NOE were similar to those of the corresponding 15N experiments published previously. The determination of T2 for the Cα-carbon in a completely labeled protein is more complicated, since the magnetization transfer during the T2 evolution period owing to the scalar coupling of Cα−Cβ must be suppressed. Various different pulse sequences for the T2 evolution period were simulated in order to optimize the bandwidth for which reliable T2 relaxation times can be obtained. A proof for the quality of these pulse sequences is given by fitting the intensity decay of individual 1H−13Cα cross peaks, in a series of (1H, 13C)-ct-HSQC spectra with a modified CPMG sequence as well as a T1p sequence for the transverse relaxation time, to a single exponential using a simplex algorithm.