Defining protein ensembles with native-state NH exchange: Kinetics of interconversion and cooperative units from combined NMR and MS analysis

Previous studies of native-state peptide hydrogen atom (NH) exchange in tur key ovomucoid third domain (OMTKY3) yielded the thermodynamics and kinetics of unfolding and folding for the 14 slowest-exchanging peptide hydrogen at oms (NHs). Unfolding rate constants and free energies for nine of the NHs a re very similar, suggesting that these NHs exchange during a single coopera tive unfolding event. Electrospray ionization mass spectrometry (ESI-MS) ha s been used to test this hypothesis. ESI-MS data and MS peak simulations su ggest that this hypothesis is incorrect: in spite of the similarity in thei r unfolding rate constants, only three to five of the nine residues exchang e in a cooperative manner. Thus, residues with similar thermodynamics and k inetics of exchange are probably involved in multiple conformational equili bria. Overall, combined NMR and MS analysis of NH exchange provides a rich and complex picture of the ensemble properties of native proteins. (C) 1999 Academic Press.