Resolution enhancement by homonuclear J-decoupling: application to three-dimensional solid-state magic angle spinning NMR spectroscopy
- 179 Downloads
We describe a simple protocol to achieve homonuclear J-decoupling in the indirect dimensions of multidimensional experiments, and to enhance spectral resolution of the backbone Cα carbons in the 3D NCACX experiment. In the proposed protocol, the refocusing of the Cα–CO homonuclear J-couplings is achieved by applying an off-resonance selective π pulse to the CO spectral region in the middle of Cα chemical shift evolution. As is commonly used in solution NMR, a compensatory echo period is used to refocus the unwanted chemical shift evolution of Cα spins, which takes place during the off-resonance selective pulse. The experiments were carried out on the β1 immunoglobulin binding domain of protein G (GB1). In GB1, such implementation results in significantly reduced line widths, and leads to an overall sensitivity enhancement.
KeywordsSolid-state NMR Magic angle spinning Uniformly 13C,15N labeled proteins Assignments Homonuclear decoupling Resolution enhancement
Magic angle spinning
β1 immunoglobulin binding domain of protein G
Time proportional phase increment
Small phase incremental alternation with 64 steps
This research was supported by the University of Guelph (start-up funds to V.L.), the Natural Sciences and Engineering Research Council of Canada (DG250202 to L.S.B. and Grants RG298480-04 to V.L.), the Canada Foundation for Innovation, and the Ontario Innovation Trust. V.L. holds Canada Research Chair in Biophysics, and is a recipient of an Early Researcher Award from the Ontario Ministry of Research and Innovation. L.S. is a recipient of the Ontario Graduate Scholarship. M.A. is a recipient of a Doctoral Studentship from the Ministry of Higher Education and Scientific Research of Egypt.
- Bruschweiler R, Griesinger C, Sorensen OW, Ernst RR (1988) Combined use of hard and soft pulses for omega-1 decoupling in two-dimensional NMR-spectroscopy. J Magn Reson 78:178–185Google Scholar
- Franks WT, Zhou DH, Wylie BJ, Money BG, Graesser DT, Frericks HL, Sahota G, Rienstra CM (2005) Magic-angle spinning solid-state NMR spectroscopy of the beta 1 immunoglobulin binding domain of protein G (GB1): N-15 and C-13 chemical shift assignments and conformational analysis. J Am Chem Soc 127:12291–12305CrossRefGoogle Scholar
- Keller R (2004) The computer aided resonance assignment tutorial, first ed. CANTINA Verlag GoldauGoogle Scholar
- Markley JL, Bax A, Arata Y, Hilbers CW, Kaptein R, Sykes BD, Wright PE, Wuthrich K (1998) Recommendations for the presentation of NMR structures of proteins and nucleic acids - IUPAC-IUBMB-IUPAB inter-union task group on the standardization of data bases of protein and nucleic acid structures determined by NMR spectroscopy. Eur J Biochem 256:1–15CrossRefGoogle Scholar
- Zhong L, Bamm VV, Ahmed MA, Harauz G, Ladizhansky V (2007) Solid-state NMR spectroscopy of 18.5 kDa myelin basic protein reconstituted with lipid vesicles: spectroscopic characterisation and spectral assignments of solvent-exposed protein fragments. Biochim Biophys Acta 1768:3193–3205CrossRefGoogle Scholar