Spectral editing of two-dimensional magic-angle-spinning solid-state NMR spectra for protein resonance assignment and structure determination
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Several techniques for spectral editing of 2D 13C–13C correlation NMR of proteins are introduced. They greatly reduce the spectral overlap for five common amino acid types, thus simplifying spectral assignment and conformational analysis. The carboxyl (COO) signals of glutamate and aspartate are selected by suppressing the overlapping amide N–CO peaks through 13C–15N dipolar dephasing. The sidechain methine (CH) signals of valine, lecuine, and isoleucine are separated from the overlapping methylene (CH2) signals of long-chain amino acids using a multiple-quantum dipolar transfer technique. Both the COO and CH selection methods take advantage of improved dipolar dephasing by asymmetric rotational-echo double resonance (REDOR), where every other π-pulse is shifted from the center of a rotor period tr by about 0.15 tr. This asymmetry produces a deeper minimum in the REDOR dephasing curve and enables complete suppression of the undesired signals of immobile segments. Residual signals of mobile sidechains are positively identified by dynamics editing using recoupled 13C–1H dipolar dephasing. In all three experiments, the signals of carbons within a three-bond distance from the selected carbons are detected in the second spectral dimension via 13C spin exchange. The efficiencies of these spectral editing techniques range from 60 % for the COO and dynamic selection experiments to 25 % for the CH selection experiment, and are demonstrated on well-characterized model proteins GB1 and ubiquitin.
KeywordsSpectral editing REDOR CH selection Protein secondary structure
We thank Professor Chad Rienstra for providing the microcrystalline 13C, 15N-labeled GB1 and Tuo Wang for help with the experiments. This work was supported by the National Institutes of Health grant GM088204 (M. H., K.J. F and S.Y. L.) for the 600 MHz NMR experiments and by the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering under Award AL-90-360-001 (to K.S-R) for the 400 MHz NMR experiments.
- Bax A, Szeverenyi NM, Maciel GE (1983) Chemical shift anisotropy in powdered solids studied by 2D Fourier transform NMR with flipping of the spinning axis. J Magn Reson 55:494–497Google Scholar
- Franks WT, Zhou DH, Wylie BJ, Money BG, Graesser DT, Frericks HL, Sahota G, Rienstra CM (2005b) Magic-angle spinning solid-state NMR spectroscopy of the beta1 immunoglobulin binding domain of protein G (GB1): 15N and 13C chemical shift assignments and conformational analysis. J Am Chem Soc 127:12291–12305CrossRefGoogle Scholar
- Franks WT, Wylie BJ, Schmidt HL, Nieuwkoop AJ, Mayrhofer RM, Shah GJ, Graesser DT, Rienstra CM (2008) Magic-angle spinning solid-state NMR spectroscopy of the β1 immunoglobulin binding domain of protein G (GB1): 15N and 13C chemical shift assignments and conformational analysis. Proc Natl Acad Sci USA 105:4621–4626ADSCrossRefGoogle Scholar
- Gullion T, Schaefer J (1989) Rotational echo double resonance NMR. J Magn Reson 81:196–200Google Scholar
- Heinig M, Frishman D (2004) STRIDE: a web server for secondary structure assignment from known atomic coordinates of proteins. Nucleic Acids Res 32, Web Server IssueGoogle Scholar
- Schmidt-Rohr K, Spiess I HW (1994) Multidimensional solid-state NMR and polymers. Academic Press, San DiegoGoogle Scholar