HRMAS: 1H-13C HSQC of fractionally deuterated (mostly at Calpha) KcsA

Citation

A.G. Palmer III, J. Cavanagh, P.E. Wright & M. Rance, J. Magn. Reson. 93, 151-170 (1991) https://www.sciencedirect.com/science/article/pii/002223649190036S L.E. Kay, P. Keifer & T. Saarinen, J. Am. Chem. Soc. 114, 10663-5 (1992) (https://pubs.acs.org/doi/abs/10.1021/ja00052a088](https://pubs.acs.org/doi/abs/10.1021/ja00052a088) J. Schleucher, M. Schwendinger, M. Sattler, P. Schmidt, O. Schedletzky, S.J. Glaser, O.W. Sorensen & C. Griesinger, J. Biomol. NMR 4, 301-306 (1994) [https://www.ncbi.nlm.nih.gov/pubmed/8019138]https://www.ncbi.nlm.nih.gov/pubmed/8019138) S. Grzesiek & A. Bax, J. Am. Chem. Soc. 115, 12593-12594 (1993) https://pubs.acs.org/doi/abs/10.1021/ja00079a052

Protocol

a. Increasing spinning frequency above 8kHz doesn't bring any improvements in most cases. b. It's better to optimize 1H-13C coupling parameter. The optimal value is often 1.2-1.5 times longer than the literature number of 145Hz c. There are two water populations due to the liposome sample. They can be both efficiently suppressed by the pulse sequence. d. Use EA only e. SMSQ10.100 have much better performance than SINE.100 f. You can adjust p28 trim pulse length between 500us and 1000us to optimize water suppression vs protein signal loss. g. Make sure you use -DLABEL_CN flag on with hard pulse on 15N for 13C-15N labeled sample. The pulse refocuses 15N and improves 13C linewidth somewhat.

Samples

The experiments were performed with a precipitated 2F-2H, U-13C/15N KcsA, LPR =1, 9:1 DOPE-DOPS D2O citrate buffer pH 3.5

Processing and analysis

Pulse Sequence and Parameter Set