Abstract: The present invention provides methods of structure prediction and/or determination of a protein of interest of unknown structure. Invention methods compare calculated rates of amide hydrogen exchange for a set of predicted possible structures for said protein with experimental hydrogen exchange analysis of said protein to identify valid protein structures based on similarities between hydrogen exchange profiles. In preferred methods, hydrogen exchange analysis is performed by determining the quantity of isotope and/or rate of exchange of peptide amide hydrogen(s) with isotope on a labeled protein, by generating a population of sequence-overlapping endopeptidase fragments of a protein labeled with a hydrogen isotope other than 1H under conditions of slowed hydrogen exchange, and then deconvoluting fragmentation data acquired from said population of sequence-overlapping endopeptidase-generated fragments.

Abstract: The present invention provides methods for crystallographic structure determination employing hydrogen exchange analysis. Hydrogen exchange analysis is used to identify unstructured regions of a protein, and then hydrogen exchange analysis repeated after the protein is admixed with candidate agents and/or conditions that may induce structure in said identified unstructured regions. Agents that induce desired structure in the protein are then employed, admixed with the protein, in co-crystallization studies for structure determination. Hydrogen exchange analysis is performed by determining the quantity of isotope and/or rate of exchange of peptide amide hydrogen(s) with isotope on a labeled protein. Proteins with agent-induced decreases in unstructured regions, and thus improved hydrogen exchange structural maps, are optimal for high quality crystallization and structure determination.

Abstract: The present invention provides methods of determining, at a resolution of about 1-5 amino acid residues, the position of a peptide amide hydrogen that has been labeled with an isotope of hydrogen other than 1H by determining the quantity of isotope and/or rate of exchange of peptide amide hydrogen(s) with isotope. Invention methods comprise generating a population of sequence-overlapping endopeptidase fragments of the labeled protein under conditions of slowed hydrogen exchange and then deconvoluting fragmentation data. Invention methods allow for the localization of labeled peptide amide positions in an amino acid-specific manner, thereby providing information on residues involved in binding sites, surface conformation and accessibility of residues, and conformational changes at different times or conditions, for example.