Source: Dissertation Abstracts International, Volume: 61-10, Section: B, page: 5193.

Director: James H. Prestegard.

This dissertation describes the development and application of Nuclear Magnetic Resonance (NMR) methods, that rely on the measurement of residual dipolar couplings in partially aligned systems for studying protein structure, protein-ligand binding and internal dynamics.

We present a procedure, based on an order matrix analysis of two independent sets of residual dipolar data, that allows rapid determination of relative molecular fragment orientation, without the need for any other long-range structural constraints. The relative geometry of two molecular fragments in the protein rubredoxin determined using this procedure is in excellent agreement with the available X-ray structure. We also introduce residual dipolar coupling NMR methodology for determining the relative orientation of domains in homomultimeric systems possessing certain types of molecular symmetry.

Residual dipolar coupling methodology has also been developed for determining bound conformations of protein ligands in exchanging systems, with specific application to protein-carbohydrate interactions. The relative geometry of 13C-isotropically enriched (alpha-methyl-mannose bound to unlabelled, 54 kDa homotrimeric Mannose Binding Protein (MBP) has been determined using residual dipolar couplings measured in alpha-methylmannose and considerations of the 3-fold rotational molecular symmetry in MBP. The determined geometry deviates from that determined using available solid-state models and molecular modeling by ∼40°. Possible reasons for this deviation are discussed, and the methodology extended for future application to unlabelled ligands.

We have examined, both theoretically, and experimentally, the effects of internal motion on residual dipolar couplings and the ramifications of these effects on structural analysis. We demonstrate that an order matrix analysis of dipolar data measured in rigid molecular fragments can be used to characterize both the anisotropy, and the amplitude, of internal motions that exist between fragments. At the limit of moderate amplitude, this order matrix analysis can also be used for assembling fragments into a meaningful average structure. Application of this protocol on residual dipolar couplings measured in cyanometinyoglobin indicates that helices in cyanometmyoglobin undergo variable levels of slow internal motions (ms-mus timescales), with helix F undergoing extensive isotropic internal motions, and helix E being largely rigid. The protein fold determined from dipolar data using this protocol is in excellent agreement with the X-ray structure, despite the presence of internal motions.