Solid- and Solution-State NMR Techniques for the Investigation of Local Ordering in Membrane-Derived Solids via Measurement of Scaled Dipolar Couplings.

摘要

In the low-viscosity samples used in solution-state nuclear magnetic resonance (NMR) spectroscopy, molecular tumbling occurs on a much faster timescale than a single NMR experiment. As a result, anisotropic interactions between the nuclei average to zero; and sharp, narrow, isotropic lineshapes are easily obtained. However, in systems with local order, rotational and translation mobility of molecules is significantly reduced. In this solid-state regime, anisotropic interactions are no longer motionally averaged, and three additional effects are observed that can severely complicate NMR spectra: heteronuclear and homonuclear dipolar couplings, and chemical shift anisotropy (CSA). Magic-angle spinning (MAS) is a solid-state NMR (ssNMR) technique that makes it possible to mechanically average these anisotropic effects to zero and acquire sharp, isotropic peaks analogous to those found in solution. However, the presence of dipolar couplings and CSA allows additional structural information about biomolecules to be extracted; so there is demand for NMR techniques that take advantage of anisotropic interactions.;Alignment media, such as bicelle mixtures, are used to introduce a controllable degree of anisotropy for the purpose of collecting scaled dipolar couplings in ssNMRand residual dipolar couplings (RDCs) in solution state, and their composition can be tailored to optimize the degree of order they impose and therefore the degree to which dipolar couplings and other anisotropic interactions are motionally averaged. Altering the composition of an alignment medium can also influence the temperature range over which an aligned phase exists, which is beneficial in dealing with temperature sensitive biomolecules. Switched-angle spinning (SAS) is another NMR method aimed at analyzing anisotropic interactions in the structural studies of oriented biological solids. SAS combines the advantages of the magic angle spinning with the structural information provided by heteronuclear and homonuclear dipolar couplings and CSA by correlating isotropic chemical shifts in one dimension with anisotropic information in a second dimension. Finally, symmetry-based pulse sequences introduce sophisticated spin-space selection rules into MAS experiments that allow for reintroduction of only specific NMR interactions while suppressing others.;Presented herein is current work on three related research efforts: the development and characterization of a new biologically-relevant alignment medium with increased thermal stability ideal for recovery of additional structural constraints from dipolar couplings and CSA in membrane proteins and peptides; simulation work and preliminary experiments laying the foundation for new variable-angle spinning (VAS) and switched-angle spinning (SAS) experiments optimized for structural studies of membrane proteins, peptides, and other biomolecules with local order induced by bicelles or other liquid-crystalline alignment media; and an investigation of an unusual chlorosulfolipid membrane component via a proton-detected local field experiment that utilizes a symmetry-based pulse sequence to selectively recouple heteronuclear dipolar couplings and CSA while prohibiting evolution of J-coupling, chemical shift, and homonuclear dipolar couplings.