title: Reconstitution of acid-denatured holomyoglobin studied by time-resolved electrospray ionization mass spectrometry.
abstract: Time-resolved electrospray ionization (ESI) mass spectrometry (MS) is a new technique for studying the kinetics of protein folding reactions. It can monitor both changes in the protein conformation and the loss or binding of protein ligands as a function of time. Time-resolved ESI MS was previously used to monitor the acid-induced unfolding of holomyoglobin (hMb). The native form of this protein is characterized by a tightly folded conformation and a heme group that is noncovalently attached to the protein. Acid-induced denaturation induces substantial unfolding of the polypeptide chain and disruption of the heme-protein interactions. In this work, time-resolved ESI MS is used to study the reverse reaction, i.e., reconstitution of acid-denatured hMb. To examine the mechanism and the kinetics of this reaction, a continuous-flow setup with two sequential mixing steps was developed. The data presented in this work show that reconstitution involves the formation of various short-lived intermediates such as tightly folded myoglobin without a heme group and several nativelike forms of the protein that are bound to more than one heme. The occurrence of these transient states is most likely due to the rapid aggregation of free heme in solution.
authors.affiliation: Department of Chemistry, University of Western Ontario, London, Canada.
authors.forename: V W
authors.initials: VW
authors.lastname: Lee
authors.forename: Y L
authors.initials: YL
authors.lastname: Chen
authors.forename: L
authors.initials: L
authors.lastname: Konermann
mesh_terms: D019342:Acetic Acid / Q000737:chemistry / Q000494:pharmacology; D006418:Heme / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D009211:Myoglobin / Q000737:chemistry*; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding
chemical_list: D009211:Myoglobin; D006418:Heme; D019342:Acetic Acid