Characterization of transient protein folding intermediates during myoglobin reconstitution by time-resolved electrospray mass spectrometry with on-line isotopic pulse labeling.
A novel technique for studying protein folding kinetics is presented. It is based on a continuous-flow setup that is coupled to an electrospray (ESI) mass spectrometer and allows initiation of a folding reaction, followed by isotopic pulse labeling. The protein is electrosprayed "quasi-instantaneously" after exposure to the deuterated solvent. This approach yields structural information from the ESI charge state distribution and from the H/D exchange levels of individual protein states, while at the same time noncovalent interactions can be monitored. This technique is used to study the reconstitution of holomyoglobin (hMb) from unfolded apomyoglobin (aMb) and free heme. MS/MS is used to establish that a short-lived folding intermediate with two heme groups attached represents a protein-bound heme dimer. This state appears to have a compactness close to that of native hMb; however, isotopic labeling indicates a significantly perturbed structure. Another intermediate is bound to a single heme group and shows a charge state distribution similar to that of unfolded aMb. Exchange levels exhibited by this state are lower than for unfolded aMb, indicating that fewer hydrogens are exposed to the solvent and/or that more of them are involved in hydrogen bonding. Native hMb leads to the formation of low charge state ions (hMb(9+), hMb(8+)) and shows low exchange levels. However, early during reconstitution, a slightly unfolded form of the heme-protein complex contributes to the observed hMb(9+) ions. A peak width analysis reveals that the structural heterogeneity of some of the observed protein species decreases as reconstitution proceeds.
Simmons
Douglas A
DA
Department of Chemistry, The University of Western Ontario, London, Ontario N6A 5B7, Canada.
Konermann
Lars
L
D000818:Animals; D003903:Deuterium; D006418:Heme / Q000737:chemistry; D006736:Horses; D006859:Hydrogen; D006860:Hydrogen Bonding; D066298:In Vitro Techniques; D013058:Mass Spectrometry; D009211:Myoglobin / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding; D021241:Spectrometry, Mass, Electrospray Ionization
D009211:Myoglobin; D006418:Heme; D006859:Hydrogen; D003903:Deuterium