HUMAN RECOMBINANT [C22A] FK506-BINDING PROTEIN AMIDE HYDROGEN-EXCHANGE RATES FROM MASS-SPECTROMETRY MATCH AND EXTEND THOSE FROM NMR

Hydrogen/deuterium exchange behavior of human recombinant [C22A] FK506 binding protein (C22A FKBP) has been determined by protein fragmentat ion, combined with electrospray Fourier transform ion cyclotron resona nce mass spectrometry (MS). After a specified period of H/D exchange i n solution, C22A FKBP was digested by pepsin under slow exchange condi tions (pH 2.4, 0 degrees C), and then subjected to on-line HPLC/MS for deuterium analysis of each proteolytic peptide. The hydrogen exchange rate of each individual amide hydrogen was then determined independen tly by heteronuclear two-dimensional NMR on N-15-enriched C22A FKBP. A maximum entropy method (MEM) algorithm makes it possible to derive th e distributions of hydrogen exchange rate constants from the MS-determ ined deuterium exchange-in curves in either the holoprotein or its pro teolytic segments. The MEM-derived rate constant distributions of C22A FKBP and different segments of C22A FKBP are compared to the rate con stants determined by NMR for individual amide protons. The rate consta nt distributions determined by both methods are consistent and complem entary, thereby validating protein fragmentation/mass spectrometry as a reliable measure of hydrogen exchange in proteins.