Protein structures and protein-solvent interactions : development and application of methods in high-resloultion nuclear magnetic resonance spectroscopy

Abstract

Nuclear magnetic resonance (NMR) spectroscopy in solution was used for the structural characterization of the proteins glutaredoxin-3 and flavodoxin from E. coli. A new NMR technique for the measurement of three-bond a proton-amide proton scalar coupling constants was developed. An improved method for the study of protein-solvent interactions was devised.

Two- and three-dimensional NMR spectra of uniformly 15N enriched glutaredoxin-3, a glutathione-disulfide oxidoreductase of 82 amino acid residues, were recorded and the resonances assigned in the native reduced and in the acid denatured state. These investigations led to the secondary structure determination of the native protein. The acid-denatured form revealed no significant population of residual structure except perhaps for a short helical segment in the middle of the amino acid sequence.

The 176 residue protein flavodoxin was investigated in its oxidized form using three-dimensional NMR techniques. Cloned and overexpressed flavodoxin was uniformly enriched with the stable isotopes 13C and 15N to resolve spectral overlap. Nearly complete 1H, 15N and 13C resonance assignments were obtained and the secondary structure of the protein as well as the binding site of the flavinmononucleotide (FMN) cofactor were determined. Hydration sites in the protein interior were identified by intermolecular water-protein nuclear Overhauser effects (NOEs).

Triggered by the studies of flavodoxin, a new two-dimensional NMR method was developed for the measurement of the three-bond scalar coupling constants between the a and amide protons of the protein backbone. The method was demonstrated to yield reliable results by comparison to two established techniques. The new method requires only 15N enriched protein and has the advantage of simple data evaluation. For molecules of molecular weights larger than about 15 kDa, up to ten times shorter experimental times can be used than with conventional techniques.

Protein-solvent interactions were studied for proteins in binary mixtures of water and organic co-solvents. A new technique was developed for the selective and simultaneous observation of intermolecular solvent-protein NOEs involving different solvent resonances. The technique employs a diffusion filter for the suppression of the dominant solvent signals in combination with a relaxation filter to prevent interference from intramolecular protein-protein NOEs. With Iysozyme in aqueous solution of N,N-dimethylformamide (DMF), the observed interactions could be modeled by a single DMF molecule binding to site C which is the specificity-determining binding site for the natural substrate. Such studies are expected to provide clues about preferential solvation of the protein surface at atomic resolution, with implications for non-aqueous enzymology, protein stability and for the rational design of ligands.