MQ-HNCO-TROSY for the measurement of scalar and residual dipolar couplings in larger
 

Abstract We describe a novel pulse sequence, MQ-HNCO-TROSY, for the measurement of scalar and residual dipolar couplings between amide proton and nitrogen in larger proteins. The experiment utilizes the whole 2TN polarization transfer delay for labeling of 15N chemical shift in a constant time manner, which efficiently doubles the attainable resolution in 15N dimension with respect to the conventional HNCO-TROSY experiment. In addition, the accordion principle is employed for measuring (J + D)NHs, and the multiplet components are selected with the generalized version of the TROSY scheme introduced by Nietlispach (J Biomol NMR 31:161â??166, 2005). Therefore, cross peak overlap is diminished while the time period during which the 15N spin is susceptible to fast transverse relaxation associated with the anti-TROSY transition is minimized per attainable resolution unit. The proposed MQ-HNCO-TROSY scheme was employed for measuring RDCs in high molecular weight protein IgFLNa16-21 of 557 residues, resulting in 431 experimental RDCs. Correlations between experimental and back-calculated RDCs in individual domains gave relatively low Q-factors (0.19â??0.39), indicative of sufficient accuracy that can be obtained with the proposed MQ-HNCO-TROSY experiment in high molecular weight proteins.

• Content Type Journal Article
• DOI 10.1007/s10858-010-9422-z
• Authors
  • Sampo Mäntylahti, University of Helsinki NMR Laboratory, Program in Structural Biology and Biophysics, Institute of Biotechnology/NMR Laboratory P.O. Box 65 00014 Helsinki Finland
  • Outi Koskela, University of Helsinki Laboratory of Organic Chemistry, Department of Chemistry P.O. Box 55 00014 Helsinki Finland
  • Pengju Jiang, University of Oxford Biochemistry Department Oxford OX1 3QU UK
  • Perttu Permi, University of Helsinki NMR Laboratory, Program in Structural Biology and Biophysics, Institute of Biotechnology/NMR Laboratory P.O. Box 65 00014 Helsinki Finland

• • Journal Journal of Biomolecular NMR
  • Online ISSN 1573-5001
  • Print ISSN 0925-2738
  • Journal Volume Volume 47
  • Journal Issue Volume 47, Number 3

Source: Journal of Biomolecular NMR