Improved Measurement of 3J(Hαi, Ni+1) Coupling Constants in H2O Dissolved Proteins

✍ Scribed by Frank Löhr; Jürgen M Schmidt; Steffen Maurer; Heinz Rüterjans

• Publisher: Elsevier Science
• Year: 2001
• Tongue: English
• Weight: 183 KB
• Volume: 153
• Category: Article
• ISSN: 1090-7807
• DOI: 10.1006/jmre.2001.2417

✦ Synopsis

A modification to the recently proposed α/β-HN(CO)CA-J TROSY pulse sequence (P. Permi et al., J. Magn. Reson. 146, 255-259 (2000)) makes it possible to determine 3 J(H α i , N i+1 ) coupling constants from a single E.COSY-type cross-peak pattern rather than from two 1 H α spin-state-edited subspectra. Advantages are increased 15 N resolution, critical to extracting accurate 1 H α -15 N coupling constants, and minimized differential relaxation due to nested 13 C α and 15 N evolution periods. Application of the improved pulse sequence to Desulfovibrio vulgaris flavodoxin results in 3 J(H α i , N i+1 ) values being systematically larger than those obtained with the original scheme. Parametrization of the coupling dependence on the protein backbone torsion angle ψ yields the Karplus relation 3 J(H α i , N i+1 ) = -1.00 cos 2 (ψ -120 • ) + 0.65 cos(ψ -120 • ) -0.15 Hz, with a residual root-mean-square difference of 0.13 Hz between measured and back-calculated coupling constants. The curve compares with data derived from ubiquitin (A. C. Wang and A. Bax, J. Am. Chem. Soc. 117, 1810-1813 (1995)), although spanning a slightly larger range of J values in flavodoxin. The orientation of the Ala39/Ser40 peptide link, forming a type-II β-turn in flavodoxin, is twisted against X-ray-derived torsions by approximately 10 • in the NMR structure as evident from the analysis of φand ψ-related 3 J coupling constants. The remaining deviation of some experimental values from the prediction is likely to be due to strong hydrogen bonding, substituent effects, or the additional dependence on the adjacent torsions φ.