N2 - We present a highly sensitive pulse sequence, carbonyl carbon label selective 1H-15N HSQC (CCLS-HSQC) for the detection of signals from 1H-15N units involved in 13C′-15N linkages. This work was supported by NIH Grants RR02301 and GM66326 (Markley, J.L.), GM64742, GM072701 (Veglia, G.) and American Heart Association support from 0615546Z (Masterson, L.R.).Ĭopyright 2009 Elsevier B.V., All rights reserved. Vickery for graciously lending the HscB sample. Alessandro Mascioni for assistance with NMR sample preparation. Taylor for generously supplying the plasmid for PKA-C and Dr. T1 - Carbonyl carbon label selective (CCLS) 1H-15N HSQC experiment for improved detection of backbone 13 C-15N cross peaks in larger proteinsĪcknowledgements We thank Professor Susan S. This approach will be applicable to families of proteins that have been resistant to NMR structural and dynamic analysis, such as large enzymes, and partially folded or unfolded proteins.', The method also is tolerant to the partial loss of signal from isotopic dilution (scrambling). The method is suitable for larger proteins and is less affected by conformational exchange than HNCO experiments, which require a longer period of transverse 15N magnetization. Although the approach requires that the 1H-15N HSQC cross peaks be observable, it does not require deuteration of the protein. We then show that the CCLS-HSQC experiment yields assignments in the case of a 41 kDa protein incorporating pairs of 15N- and 13C′- labeled amino acids, where a TROSY 2D-HN(CO) had failed. By collecting CCLS-HSQC and HNCO data for two proteins (8 kDa ubiquitin and 20 kDa HscB) at various temperatures (5-40☌) in order to vary correlation times, we demonstrate the superiority of the CCLS-HSQC pulse sequence for proteins with long correlation times (i.e. The CCLS-HSQC pulse sequence utilizes a modified 15N CT evolution period equal to 1/(21JNC′) (∼33 ms) to select for 13C′-15N pairs. This approach will be applicable to families of proteins that have been resistant to NMR structural and dynamic analysis, such as large enzymes, and partially folded or unfolded proteins.Ībstract = 'We present a highly sensitive pulse sequence, carbonyl carbon label selective 1H-15N HSQC (CCLS-HSQC) for the detection of signals from 1H-15N units involved in 13C′-15N linkages. Although the approach requires that the 1H- 15N HSQC cross peaks be observable, it does not require deuteration of the protein.

The CCLS-HSQC pulse sequence utilizes a modified 15N CT evolution period equal to 1/(2 1J NC′) (∼33 ms) to select for 13C′- 15N pairs. We present a highly sensitive pulse sequence, carbonyl carbon label selective 1H- 15N HSQC (CCLS-HSQC) for the detection of signals from 1H- 15N units involved in 13C′- 15N linkages.

Labeling carbon backbone amino acid