Hydrogen–deuterium exchange strategy for delineation of contact sites in protein complexes

AbstractWe use NMR spectra to determine protein–protein contact sites by observing differences in amide proton hydrogen–deuterium exchange in the complex compared to the free protein in solution. Aprotic organic solvents are used to preserve H/D labeling patterns that would be scrambled in water solutions. The binding site between the mammalian co-chaperone Aha1 with the middle domain of the chaperone Hsp90 obtained by our H/D exchange method corresponds well with that in the X-ray crystal structure of the homologous complex from yeast, even to the observation of a secondary binding site. This method can potentially provide data for complexes with unknown structure and for large or dynamic complexes inaccessible via NMR and X-ray methods