Hydrogen exchange behavior of [U-15N]-labeled oxidized and reduced iso-1-cytochrome c.
Heteronuclear NMR spectroscopy was used to measure the hydrogen-deuterium exchange rates of backbone amide hydrogens in both oxidized and reduced [U-15N]iso-1-cytochrome c from the yeast Saccharomyces cerevisiae. The exchange data confirm previously reported data [Marmorino et al. (1993) Protein Sci. 2, 1966-1974], resolve several inconsistencies, and provide more thorough coverage of exchange rates throughout the cytochrome c protein in both oxidation states. Combining the data previously collected on unlabeled C102T with the current data collected on [U-15N]C102T, exchange rates for 53 protons in the oxidized state and 52 protons in the reduced state can now be reported. Most significantly, hydrogen exchange measurements on [U-15N]iso-1-cytochrome c allowed the observation of exchange behavior of the secondary structures, such as large loops, that are not extensively hydrogen-bonded. For the helices, the most slowly exchanging protons are found in the middle of the helix, with more rapidly exchanging protons at the helix ends. The observation for the Omega-loops in cytochrome c is just the opposite. In the loops, the ends contain the most slowly exchanging protons and the loop middles allow more rapid exchange. This is found to be true in cytochrome c loops, even though the loop ends are not attached to any regular secondary structures. Some of the exchange data are strikingly inconsistent with data collected on the C102S variant at a different pH, which suggests pH-dependent dynamic differences in the protein structure. This new hydrogen exchange data for loop residues could have implications for the substructure model of eukaryotic cytochrome c folding. Isotopic labeling of variant forms of cytochrome c can now be used to answer many questions about the structure and folding of this model protein.
Baxter
S M
SM
Wadsworth Center, Department of Biomedical Sciences, New York State Department of Health, Albany 12201-0509, USA.
Fetrow
J S
JS
D003545:Cysteine / Q000737:chemistry; D003574:Cytochrome c Group / Q000737:chemistry*; D045304:Cytochromes c; D004795:Enzyme Stability; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D008715:Methionine / Q000737:chemistry; D008958:Models, Molecular; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011522:Protons; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins; D012996:Solutions; D013912:Threonine / Q000737:chemistry
C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D009587:Nitrogen Isotopes; D011522:Protons; D029701:Saccharomyces cerevisiae Proteins; D012996:Solutions; D013912:Threonine; D045304:Cytochromes c; D008715:Methionine; D007501:Iron; D003545:Cysteine