MQ-HNCO-TROSY for the measurement of scalar and residual dipolar couplings in larger proteins: application to a 557-residue IgFLNa16-21.
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 2T(N) 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.
Mäntylahti
Sampo
S
NMR Laboratory, Program in Structural Biology and Biophysics, Institute of Biotechnology/NMR Laboratory, University of Helsinki, P.O. Box 65, 00014, Helsinki, Finland.
Koskela
Outi
O
Jiang
Pengju
P
Permi
Perttu
P
D001426:Bacterial Proteins / Q000737:chemistry; D002247:Carbon Isotopes / Q000737:chemistry; D003285:Contractile Proteins / Q000737:chemistry; D003903:Deuterium / Q000737:chemistry; D064448:Filamins; D005583:Fourier Analysis; D006801:Humans; D008840:Microfilament Proteins / Q000737:chemistry; D009587:Nitrogen Isotopes / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D025801:Ubiquitin / Q000737:chemistry; D014867:Water / Q000737:chemistry
D001426:Bacterial Proteins; D002247:Carbon Isotopes; D003285:Contractile Proteins; D064448:Filamins; C034284:IgG Fc-binding protein, Streptococcus; D008840:Microfilament Proteins; D009587:Nitrogen Isotopes; D011506:Proteins; D025801:Ubiquitin; D014867:Water; D003903:Deuterium
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