MQ-HNCO-TROSY for the measurement of scalar and residual dipolar couplings in larger proteins: Application to a 557-residue IgFLNa16-21

摘要

We describe a novel pulse sequence, MQHNCO- 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 ~(15)N chemical shift in a constant time manner, which efficiently doubles the attainable resolution in ~(15)N dimension with respect to the conventional HNCO-TROSY experiment. In addition, the accordion principle is employed for measuring (J + D)NH~s, and the multiplet components are selected with the generalized version of the TROSY scheme introduced by Nietlispach (J Biomol NMR 31:161-166, 2005). Therefore, cross peak overlap is diminished while the time period during which the ~(15)N 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 (0.19-0.39), indicative of sufficient accuracy that can be obtained with the proposed MQ-HNCO-TROSY experiment in high molecular weight proteins.