Identification of non-covalent structure in apocytochrome c by hydrogen exchange and mass spectrometry.
Apocytochrome c, the in vivo precursor to active cytochrome c, was analyzed by amide hydrogen exchange and mass spectrometry to search for fixed, non-covalent structure. The protein was incubated in H(2)O at pH 3.3 or 6.7 for various times, then exposed to D(2)O to initiate isotope labeling of unfolded regions. Following acid quenching of hydrogen exchange, the labeled apocytochrome c was digested with pepsin into fragments that were analyzed by directly coupled high-performance liquid chromatography/electrospray ionization mass spectrometry. The intermolecular distribution of deuterium and the deuterium levels in structurally distinctive populations were determined from the mass spectra of the peptic fragments. Spectra of peptic fragments derived from apocytochrome c incubated at pH 3.3 had single envelopes of isotope peaks with masses indicating that all of the amide hydrogens had been replaced with deuterium. These results showed that apocytochrome c at pH 3.3 offered little resistance to hydrogen exchange, indicating that it was unfolded with little fixed structure. However, mass spectra of peptic fragments including residues 81-94 of apocytochrome c incubated at pH 6.7 had two envelopes of isotope peaks, indicating that one population was unfolded and the other population was highly structured in this region. Mass spectra of peptic fragments including residues N-terminal to residue 81 indicated that this region of the protein remained unfolded with little fixed structure at pH 6.7.
Raza
A S
AS
Department of Chemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 68588-0304, USA.
Dharmasiri
K
K
Smith
D L
DL
D000818:Animals; D001059:Apoproteins / Q000737:chemistry*; D003574:Cytochrome c Group / Q000737:chemistry*; D045304:Cytochromes c; D006736:Horses; D006859:Hydrogen; D013058:Mass Spectrometry
D001059:Apoproteins; D003574:Cytochrome c Group; D006859:Hydrogen; D045304:Cytochromes c