Long-distance REDOR NMR measurements for determination of secondary structure and conformational heterogeneity in peptides

Abstract

High-resolution NMR spectroscopy of liquids has become a standard method for conformational studies in biochemistry. This method provides information on the secondary and tertiary structure of macromolecules and on intermolecular interactions such as ligand receptor binding. These determinations are based mainly on interatomic distance measurements by quantitative analysis of NOESY spectra and, to a lesser degree, on torsion angle measurements based on homoor heteronuclear scalar coupling constants. As with liquids, internuclear distances in solids are reflected in dipole-dipole coupling constants between the nuclear magnetic dipoles. Rotational-echo double resonance (REDOR) spectroscopy has been employed to determine heteronuclear dipole-dipole coupling constants [1,2] and to study internuclear distances in peptides, proteins and bound ligands. An important target for the REDOR experiment is an amorphous solid. For biologically important amorphous solids that cannot be obtained in crystalline form, NMR spectroscopy is the only method for direct investigation of conformation at the atomic level. In the present study we examine the REDOR of selectively labeled peptide hormone in lyophilized powders. In order to understand the contribution of naturalabundance nuclei to the REDOR data at long evolution times, we have used a three-body calculation [3,4] and assumed a spherical distance distribution for natural abundance corrections. In addition we have evaluated the REDOR results in terms of a superposition of different distances with appropriate weighting factors. These approaches allow us to fit the experimental data on the to a high degree of accuracy and to conclude that this peptide has a tendency to be bent in a lyophilized powder.