Methods for the high-resolution identification of solvent-accessible amide hydrogens in protein binding sites

A method of characterizing a binding site of a binding protein of known or determinable amino acid sequence, said method comprising the steps of:

• (a) selectively labeling peptide amide groups in a binding site of the binding protein so as to obtain a selectively labeled binding protein wherein either (i) the solvent inaccessible peptide amide groups of the binding protein comprise a heavy hydrogen and the solvent accessible peptide amide groups comprise a normal hydrogen or (ii) the solvent inaccessible peptide amide groups of the binding protein comprise a normal hydrogen and the solvent accessible peptide amide groups comprise a heavy hydrogen, and wherein the solvent inaccessible peptide amide groups have been determined to be those groups that are substantially inaccessible to solvent in a situation where the binding protein binds its binding partner;
• (b) fragmenting the selectively labeled binding protein into a plurality of fragments under conditions of slow hydrogen exchange;
• (c) determining which fragments are labeled with heavy hydrogen;
• (d) progressively degrading, under conditions of slow hydrogen exchange, each of the heavy hydrogen-labeled fragments to obtain at least one series of subfragments, wherein each subfragment of each of said series is shorter than the preceding subfragment in the series by about 1-5 fewer amino acid residues at one terminus;
• (e) quantifying an amount of heavy hydrogen on each subfragment;
• and
• (f) correlating the amount of heavy hydrogen on the subfragments with the amino acid sequences of the fragments from which the subfragments were generated, thereby localizing the positions of the fragment that had been labeled with heavy hydrogen to a resolution about 1-5 amino acid residues, and thus characterizing the binding site of the binding protein.

A method of determining, at a resolution of about 1-5 amino acid residues, the position of a peptide amide group labeled with a heavy hydrogen in a binding site of a binding protein of known or determinable amino acid sequence, said method comprising the steps of:

• (a) selectively labeling peptide amide groups in a binding site of the binding protein that have been determined to be substantially protected from exchange with solvent hydrogens in a situation where the binding protein binds its binding partner;
• (b) progressively degrading, under conditions of slow hydrogen exchange, said binding protein into a mixture of subfragments, wherein said mixture comprises at least one series of subfragments, such that each subfragment in each of said series is shorter than the preceding subfragment in the series by about 1-5 amino acid residues at one terminus and such that the first subfragment in the series is derived from and composed of about 1-5 fewer amino acid residues than said protein and such that the last subfragment in the series is about 1-5 amino acid residues in length;
• and,
• when said mixture of subfragments comprises an analytically sufficient quantity of each subfragment in each of said series,
• (c) quantifying an amount of heavy hydrogen label on each subfragment;
• and
• (d) correlating the amount of heavy hydrogen label of each subfragment with the amino acid sequence of the binding protein, thereby localizing the position of the labeled peptide amide group in the binding site of the binding protein to within about 1-5 amino acid residues.