Nuclear Magnetic Resonance--new Methods And Molecular St

Diabetes, Digestive, Kidney Diseases

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Abstract

We have extended our technology for studying macromolecular structure in solution under weakly aligning conditions. New developments focus on the assembly of protein structure from dipolar coupling data with a minimum requirement for 1H-1H distance restraints from NOEs. A robust procedure has been developed that searches the crystallographic database for small fragments that match experimentally observed dipolar couplings. This so-called molecular fragment replacement (MFR) method was used to determine the solution structure of gamma-S crystalline, which was shown to have its two homologous domains tightly anchored to one another, despite flexibility in the peptide sequence linking the two domains. A procedure has been developed that permits direct incorporation of small angle X-ray scattering data into NMR structure determination. Although computationally expensive, use of a ?glob? approach, which treats certain peptide groups as fixed single point units, accelerates the method by three orders of magnitude over a full atom calculation. Application to gamma-S crystallin showed a considerably better fit to homologous X-ray structure upon incorporation of experimental SAXS data in the structure refinement. Use of SAXS data is anticipated to be particularly useful for the study of molecular complexes and for studying quaternary structure of complex systems under solution conditions. Study of alpha-synuclein in the presence of detergent micelles shows that this protein adopts an alpha-helical conformation from residue 10 through 92, with a turn region between residues 40 and 44, and a dynamically disordered tail region for residues 99-140. The U-shaped conformation seen when bound to a detergent micelle may or may not be present when bound to lower curved phospholipids vesicles or synapse surfaces, although the structured features of the turn region imply a possible structural role. Analysis of two mutants, A30P and A56T, both implicated in Parkinson?s disease show essentially no structural change for A56T compared to wild type, but an unraveling of the first helix of A30P, starting from residue 28, and no change in the second helix. The change in the first helix decreases its curvature and thereby impacts the detergent micelle shape, which is sensed by the second helix in terms of very small 1H chemical shift changes.