Kinetics of Cytochrome c Folding Examined by Hydrogen Exchange and Mass Spectrometry

Abstract

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.

Rate and Equilibrium Constants for Protein Unfolding and Refolding Determined by Hydrogen Exchange-Mass Spectrometry

“…Due to limitations in current NMR technology, similar unfolding studies of large proteins (M r Ͼ 30 kDa) are not possible at this time. However, if more denaturant is added to increase further the population of unfolded forms, they can be detected by H/D exchange and mass spectrometry (12,15,18,20,21).…”

“…The solvent-accessible surfaces of intermediates have been used to track the progress made by the protein as it traverses the folding energy surface (4 -9). Experimental methods, including fluorescence (8 -11), circular dichroism (12)(13)(14), hydrogen exchange (15)(16)(17)(18), and molecular biology (19), have been used to determine the energies of structures intermediate between native and unfolded forms of several small proteins. However, extension of these methods to studies of large proteins has been less fruitful.…”

Unfolding dynamics of aβ-sheet protein studied by mass spectrometry

“…This situation would give rise to a bimodal distribution of mass spectral peaks, since for every opening event all of the amides become exposed to solvent simultaneously and exchange for protons. 10 Under most conditions, however, refolding rates and intrinsic exchange rates are of a similar order of magnitude, so the measured rate of exchange is second order, termed EX 2 , and governed by the rate equation k meas D K op k int , where k meas is the measured exchange rate, K op is the unfolding equilibrium constant and k int is the intrinsic amide exchange rate. This gives rise to a gradual mass shift with increased exchange time and peak broadening in the mass spectrum, as each amide will exchange to a varying extent depending on its individual intrinsic exchange rate.…”

“…For instance, quenched flow techniques in conjunction with NMR or ESI-MS allow the detection of structure formation during kinetic refolding since those amides which are hydrogen bonded become protected against exchange with bulk solvent. 9,10 Whereas NMR gives detailed information as to the average extent of exchange at each individual backbone amide, mass spectrometry as a complementary technique allows the measurement of the cooperativity of these exchange events. 9,11 This invaluable feature allows us to probe the multiplicity of folding pathways and the energy landscape of folding funnels in the 'new view' of protein folding.…”

From Small-Molecule Reactions to Protein Folding: Studying Biochemical Kinetics by Stopped-Flow Electrospray Mass Spectrometry

“…This step abruptly stops the reaction. Afterward the reaction mixture is analyzed off-line using methods such as chromatography, NMR, or mass spectrometry (MS) (13)(14)(15)(16)(17). Quenchflow experiments require that the quenched reaction mixture be stable during the off-line analysis which often represents a problem.…”