Simultaneous measurement of protein one-bond residual dipolar couplings without increased resonance overlap.
A NMR strategy designed to measure simultaneously and without increased resonance overlap scalar and dipolar couplings (RDCs) in (13)C-, (15)N-labeled proteins is presented. Contrary to common schemes for simultaneous measurement of RDCs, a single reference experiment is used for the extraction of more than one type of coupling, thereby reducing the required measurement time. This is accomplished by a common reference spectrum followed by a series of interleaved experiments, in which a particular coupling dependent parameter is varied according to the quantitative J-correlation method or using accordion spectroscopy. To illustrate this idea, we have modified the 3D TROSY-HNCO and the 3D CBCA(CO)NH experiment allowing efficient measurement of one-bond (1)D(NH), (1)D(C'N), (1)D(CalphaHalpha), (1)D(CbetaHbeta), and (1)D(CalphaC') couplings in small to medium sized proteins. In addition, the experiments are expected to be useful for largely unfolded proteins, which show strong resonance overlap but have very favorable relaxation properties. Measurement of RDCs is demonstrated on uniformly (15)N-(13)C-labeled ubiquitin and on the sensory domain of the membraneous two-component fumarate sensor DcuS of Escherichia coli (17 kDa). DcuS was found to be unstable and to precipitate in one to two weeks. RDCs obtained from these experiments are in good agreement with the 1.8A X-ray structure of ubiquitin.
Vijayan
Vinesh
V
Department for NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
Zweckstetter
Markus
M
D002247:Carbon Isotopes; D029968:Escherichia coli Proteins / Q000737:chemistry*; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011494:Protein Kinases / Q000737:chemistry*; D012815:Signal Processing, Computer-Assisted; D025801:Ubiquitin / Q000737:chemistry*
D002247:Carbon Isotopes; D029968:Escherichia coli Proteins; D009587:Nitrogen Isotopes; D025801:Ubiquitin; D011494:Protein Kinases; C469359:DcuS protein, E coli