The Saupe matrix describing protein alignment in a liquid crystalline medium contains five independent elements, enabling the generation of up to five linearly independent alignment conditions. Measurement of internuclear residual dipolar couplings (RDCs) by NMR spectroscopy under these conditions, orthogonal in five-dimensional alignment space, provides access to the amplitude, asymmetry, and direction of motions of the internuclear vector. We previously demonstrated for the small protein domain GB3 (56 residues) that suitably orthogonal alignment conditions can be generated in a single liquid crystalline medium of Pf1 phage, by generating a series of conservative mutants that have negligible impact on the time-averaged backbone structure of the domain. Mutations involve changes in the charge of several solvent-exposed sidechains, as well as extension of the protein by either an N- or C-terminal His-tag peptide, commonly used for protein purification. These protein mutants map out the five-dimensional alignment space, providing unique insights into the structure and dynamics, and providing access to anisotropic parameters such as the 13C, 15N and 1H chemical shielding tensors. This technology has now been developed further in order to derive site-specific 1H chemical shift anisotropy (CSA) tensors for the well-ordered backbone amide moieties in GB3. Experimental input data include residual chemical shift anisotropy (RCSA), measured in six mutants that align differently relative to the static magnetic field when dissolved in a liquid crystalline Pf1 suspension, and cross-correlated relaxation rates between the 1H(N) CSA tensor and either the 1H-15N, the 1H-13C', or the 1H-13C(alpha) dipolar interactions. Analyses with the assumption that the 1H(N) CSA tensor is symmetric with respect to the peptide plane (three-parameter fit) or without this premise (five-parameter fit) yield very similar results, confirming the robustness of the experimental input data, and that, to a good approximation, one of the principal components orients orthogonal to the peptide plane. 1H(N) CSA tensors are found to deviate strongly from axial symmetry, with the most shielded tensor component roughly parallel to the N-H vector, and the least shielded component orthogonal to the peptide plane. DFT calculations on pairs of N-methyl acetamide and acetamide in H-bonded geometries taken from the GB3 X-ray structure correlate with experimental data and indicate that H-bonding effects dominate variations in the 1H(N) CSA. Using experimentally derived 1H(N) CSA tensors, the optimal relaxation interference effect needed for narrowest 1H(N) TROSY line widths is found at ca 1200 MHz.

Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Zip Code
Chiliveri, Sai Chaitanya; Louis, John M; Ghirlando, Rodolfo et al. (2018) Tilted, Uninterrupted, Monomeric HIV-1 gp41 Transmembrane Helix from Residual Dipolar Couplings. J Am Chem Soc 140:34-37
Marchant, Jan; Bax, Ad; Summers, Michael F (2018) Accurate Measurement of Residual Dipolar Couplings in Large RNAs by Variable Flip Angle NMR. J Am Chem Soc 140:6978-6983
Ying, Jinfa; Delaglio, Frank; Torchia, Dennis A et al. (2017) Sparse multidimensional iterative lineshape-enhanced (SMILE) reconstruction of both non-uniformly sampled and conventional NMR data. J Biomol NMR 68:101-118
Roche, Julien; Shen, Yang; Lee, Jung Ho et al. (2016) Monomeric A?(1-40) and A?(1-42) Peptides in Solution Adopt Very Similar Ramachandran Map Distributions That Closely Resemble Random Coil. Biochemistry 55:762-75
Roche, Julien; Ying, Jinfa; Bax, Ad (2016) Accurate measurement of (3)J(HNH?) couplings in small or disordered proteins from WATERGATE-optimized TROSY spectra. J Biomol NMR 64:1-7
Louis, John M; Baber, James L; Ghirlando, Rodolfo et al. (2016) Insights into the Conformation of the Membrane Proximal Regions Critical to the Trimerization of the HIV-1 gp41 Ectodomain Bound to Dodecyl Phosphocholine Micelles. PLoS One 11:e0160597
Ceccon, Alberto; Tugarinov, Vitali; Bax, Ad et al. (2016) Global Dynamics and Exchange Kinetics of a Protein on the Surface of Nanoparticles Revealed by Relaxation-Based Solution NMR Spectroscopy. J Am Chem Soc 138:5789-92
Lee, Jung Ho; Ying, Jinfa; Bax, Ad (2016) Quantitative evaluation of positive ? angle propensity in flexible regions of proteins from three-bond J couplings. Phys Chem Chem Phys 18:5759-70
Roche, Julien; Ying, Jinfa; Shen, Yang et al. (2016) ARTSY-J: Convenient and precise measurement of (3)JHNH? couplings in medium-size proteins from TROSY-HSQC spectra. J Magn Reson 268:73-81
Li, Jingwen; Wang, Yefei; Chen, Jingfei et al. (2016) Observation of ?-Helical Hydrogen-Bond Cooperativity in an Intact Protein. J Am Chem Soc 138:1824-7

Showing the most recent 10 out of 30 publications