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 ¹³C-HSQC and 3D ¹³C-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, ¹³C homonuclear J-couplings are rendered ineffective, reducing the loss of signal and peak splitting commonly associated with long ¹³C evolution times. The two periods are incremented according to a special acquisition protocol employing a ¹³C-¹³C 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 ¹³C-labeled proteins at twice the evolution time of the standard ¹³C-HSQC experiment display a nearly two-fold 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 ¹³C-HSQC experiment recorded with a carbon evolution time of 2 × 8 ms showed a 36% decrease in linewidths compared to the standard ¹³C-HSQC experiment, and the S/N 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 ¹³C-NOESY-HSQC experiment derived from the ¹³C-HSQC experiment yielded additional NOE restraints between resonances which previously had been unresolved.