| Investigating the folding, unfolding, and conformational motions of proteins currently represents a major area of research in biophysical chemistry. The static structures provided by conventional nuclear magnetic resonance spectroscopy and x-ray crystallography are not sufficient for describing the dynamic nature of proteins in solution. Consequently, there is great incentive for the development of new methodologies to provide structural characterization of short-lived species encountered during protein folding and unfolding, and for elucidating the complex structural fluctuations of proteins at equilibrium. In the last several years, electrospray mass spectrometry (ESI-MS) has gained acceptance as a powerful tool for protein studies. In addition to facilitating detection of proteins with high mass accuracy, ESI-MS serves as a probe of protein structure through conformationally sensitive protonation, and though the ability to directly observed protein-ligand interactions. In this work, ESI-MS is combined with continuous-flow rapid on-line mixing, and isotopic labeling to enable time-resolved studies ("time-resolved ESI-MS") on protein folding, equilibrium conformational dynamics, and multi-protein complex disassembly.; A novel on-line pulse-labeling technique is introduced and applied to investigate the reconstitution of holo-myoglobin from apoprotein and free heme. This technique permits the intercorrelation of hydrogen/deuterium exchange behavior, ESI charge state, and ligand-binding state, allowing for a multidimensional structural analysis of short-lived intermediate species. Time-resolved ESI-MS is also used to examine the equilibrium conformational dynamics of partially denatured myoglobin by continuous-labeling hydrogen/deuterium exchange. Isotopic labeling times upward from several milliseconds are accessible with this methodology, thereby enabling the elucidation of rapid conformational fluctuations. Finally, the unfolding and subunit disassembly kinetics of hemoglobin are investigated. Time-resolved ESI-MS is used to monitor the decay and formation of numerous oligomerization and heme-binding states. This allows for a detailed picture of the disassembly of tetrameric hemoglobin into its monomeric constituents. |
