Kinetics of cytochrome c folding examined by hydrogen exchange and mass spectrometry.
Pulsed hydrogen exchange/mass spectrometry, a new method for studying protein folding, has been used to investigate folding of cytochrome c on the 5 ms to 15 s time scale. Cytochrome c, unfolded in guanidine hydrochloride/D2O, was allowed to refold in a high-speed quenched-flow apparatus and pulse-labeled with protium to identify unfolded regions. Intact, labeled cytochrome c was digested into fragments which were analyzed by HPLC electrospray ionization mass spectrometry to determine the level of deuterium in each fragment. Bimodal distributions of deuterium were found for most segments, indicating that regions represented by these segments were either unfolded or completely folded in the intact polypeptide prior to labeling. This behavior is consistent with cooperative, localized folding which occurs in less than 10 ms in individual molecules. Deuterium levels found in the fragments were normalized to levels found in the same fragments derived from folded cytochrome c, pulse-labeled in the same manner, to indicate the percentage of cytochrome c that was folded. These results show that the N/C-terminal regions fold cooperatively on a time scale extending from less than the mixing time of the apparatus (5 ms) to as long as 15 s, and that the other regions also fold cooperatively. However, these regions do not begin to fold until 30 ms after mixing. In addition to providing new information on cytochrome c folding, these results demonstrate that pulse-hydrogen exchange/mass spectrometry is complementary to NMR in some respects and advantageous in others. Results of this study form the foundation required to extend the pulsed hydrogen exchange approach to folding studies of proteins too large to be analyzed by NMR.
Yang
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Department of Chemistry, University of Nebraska-Lincoln 68588-0304, USA.
Smith
D L
DL
D003574:Cytochrome c Group / Q000737:chemistry*; D003903:Deuterium; D017022:Flow Injection Analysis; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D006859:Hydrogen; D006860:Hydrogen Bonding; D007700:Kinetics; D008956:Models, Chemical; D019906:Nuclear Magnetic Resonance, Biomolecular; D011489:Protein Denaturation; D017510:Protein Folding; D013997:Time Factors
D003574:Cytochrome c Group; D006859:Hydrogen; D003903:Deuterium