13C-NOESY-HSQC with split carbon evolution for increased resolution with uniformly labeled proteins

Two new pulse sequences are presented for the recording of 2D C-13-HSQC and 3D C-13-NOESY-HSQC experiments, containing two consecutive carbon evolution periods. The two periods are separated by a z-filter which creates a clean CxHz-quantum state for evolution in the second period. Each period is incremented (in a non-constant-time fashion) only to the extent that the defocusing of carbon inphase magnetization through J-coupling with neighboring carbons remains insignificant. Therefore,C-13 homonuclear J-couplings are rendered ineffective, reducing the loss of signal and peak splitting commonly associated with long C-13 evolution times. The two periods are incremented according to a special acquisition protocol employing a C-13-C-13 gradient echo to yield a data set analogous to one obtained by evolution over the added duration of both periods. The spectra recorded with the new technique on uniformly C-13-labeled proteins at twice the evolution time of the standard C-13-HSQC experiment display a nearly twofold enhancement of resolution in the carbon domain, while maintaining a good sensitivity even in the case of large proteins. Applied to the IIA(Man) protein of E. coli (31 kDa), the C-13-HSQC experiment recorded with a carbon evolution time of 2 x 8 ms showed a 36% decrease in linewidths compared to the standard C-13-HSQC experiment, and the SIN ratio of representative cross-peaks was reduced to 40%. This reduction reflects mostly the typical loss of intensity observed when recording with an increased resolution. The C-13-NOESY-HSQC experiment derived from the C-13-HSQC experiment yielded additional NOE restraints between resonances which previously had been unresolved. (C) 1998 Academic Press.