Characterizing short-lived protein folding intermediates by top-down hydrogen exchange mass spectrometry.
This work combines pulsed hydrogen/deuterium exchange (HDX) and top-down mass spectrometry for the structural characterization of short-lived protein folding intermediates. A custom-built flow device with three sequential mixing steps is used for (i) triggering protein folding, (ii) pulsed D(2)O labeling, and (iii) acid quenching. The earliest folding time point that can be studied with this system is 10 ms. The mixing device was coupled online to the electrospray source of a Fourier transform mass spectrometer, where intact protein ions are fragmented by electron capture dissociation (ECD). The viability of this experimental strategy is demonstrated by applying it to the refolding of horse apo-myoglobin (aMb), a reaction known to involve a transient intermediate. Cooling of the mixing device to 0 °C reduces the reaction rate such that the folding process occurs within the experimentally accessible time window. Top-down ECD provides an average spatial resolution of ca. 2 residues, surpassing the resolution typically achieved in traditional proteolytic digestion/HDX studies. Amide back exchange is virtually eliminated by the short (∼1 s) duration of the acid quenching step. The aMb folding intermediate exhibits HDX protection in helices G and H, whereas the remainder of the protein is largely unfolded. Marginal protection is seen for helix A. Overall, the top-down ECD approach used here offers insights into the sequence of events leading from the unfolded state to the native conformation, with envisioned future applications in the areas of protein misfolding and aggregation. The time-resolved experiments reported herein represent an extension of our previous work, where HDX/MS with top-down ECD was employed for monitoring "static" protein structures under equilibrium conditions (Pan et al. J. Am. Chem. Soc. 2009, 131, 12801).
Pan
Jingxi
J
Department of Chemistry, The University of Western Ontario, London, Ontario, N6A 5B7, Canada.
Han
Jun
J
Borchers
Christoph H
CH
Konermann
Lars
L
D000818:Animals; D006736:Horses; D006859:Hydrogen / Q000737:chemistry; D007700:Kinetics; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D008958:Models, Molecular; D009211:Myoglobin / Q000032:analysis* / Q000737:chemistry; D017510:Protein Folding; D017434:Protein Structure, Tertiary
D009211:Myoglobin; D006859:Hydrogen