SOLID STATE AND SOLUTION NMR STUDIES OF MODEL SYSTEMS FOR PARTS OF MEMBRANE PROTEINS

Detailed atomic scale structural information on proteins and other biomolecules is needed to understand biological mechanisms. However, many biologically important macromolecules such as large soluble proteins or membrane proteins normally cannot be studied directly by powerful new methods of liquid state NMR due to broad lines inherent in slowly tumbling large molecules. Paramagnetic shift reagents were investigated for use in solid state NMR of macromolecules and EDTA was used as a model for detecting sidechain carboxyls that bind metal ions in proteins. The ς22 tensor from the chemical shift anisotropy (CSA) of the carbonyl carbons was used to distinguish the OCO angle in metal ion complexes in the solid state. Liquid state 2D NMR was used to study a potential metal ion binding site in bacteriorhodopsin (BR); A peptide fragment that is homologous to an EF-hand metal ion binding loop between the C&D helices in BR. This peptide bound La+3 and showed secondary structure in solution which fit into the BR helix structure and the EF-hand coordination could be completed by 161Glu on the EF loop of BR. Protein-protein interactions, important in biological processes, are difficult to study by solution NMR methods because of the high molecular weight of the complexes. We used Tr-NOESY to study the protein bound conformation of a peptide ligand in exchange with a receptor. The p47-phox bound structure of a carboxyl terminal peptide from gp91-phox was studied since this peptide blocks superoxide generation by binding to the p47-phox control protein. The gp91-phox carboxyl terminal peptide is in fast exchange with p47-phox relative to cross relaxation and binds to p47-phox in an extended open loop conformation involving strong interaction of certain peptide sidechains