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. We have developed and demonstrated new methods for obtaining experimental data sets that correspond to purely orthogonal alignment tensors, thereby removing the intrinsic correlation present between RDC values measured under a variety of alignment conditions. Application to the fusion peptide of hemagglutinin provided unique insights into its structure and internal dynamics.

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Roche, Julien; Ying, Jinfa; Shen, Yang et al. (2016) ARTSY-J: Convenient and precise measurement of (3)JHNHα couplings in medium-size proteins from TROSY-HSQC spectra. J Magn Reson 268:73-81
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Roche, Julien; Shen, Yang; Lee, Jung Ho et al. (2016) Monomeric Aβ(1-40) and Aβ(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(HNHα) couplings in small or disordered proteins from WATERGATE-optimized TROSY spectra. J Biomol NMR 64:1-7
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Maltsev, Alexander S; Grishaev, Alexander; Roche, Julien et al. (2014) Improved cross validation of a static ubiquitin structure derived from high precision residual dipolar couplings measured in a drug-based liquid crystalline phase. J Am Chem Soc 136:3752-5
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