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 turkey ovomucoid third domain (OMTKY3) yielded the thermodynamics and kinetics of unfolding and folding for the 14 slowest-exchanging peptide hydrogen atoms (NHs). Unfolding rate constants and free energies for nine of the NHs are very similar, suggesting that these NHs exchange during a single cooperative unfolding event. Electrospray ionization mass spectrometry (ESI-MS) has been used to test this hypothesis. ESI-MS data and MS peak simulations suggest that this hypothesis is incorrect: in spite of the similarity in their unfolding rate constants, only three to five of the nine residues exchange in a cooperative manner. Thus, residues with similar thermodynamics and kinetics of exchange are probably involved in multiple conformational equilibria. Overall, combined NMR and MS analysis of NH exchange provides a rich and complex picture of the ensemble properties of native proteins.
Arrington
C B
CB
Department of Biochemistry, University of Iowa, Iowa City, 52242, USA.
Teesch
L M
LM
Robertson
A D
AD
D000818:Animals; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008958:Models, Molecular; D010059:Ovomucin / Q000737:chemistry*; D017510:Protein Folding; D013816:Thermodynamics; D014422:Turkeys
D010059:Ovomucin; D006859:Hydrogen