Assignment of 15N chemical shifts and 15N relaxation measurements for oxidized and reduced iso-1-cytochrome c.
A protocol for complete isotopic labeling of iso-1-cytochrome c from the eukaryote Saccharomyces cerevisiae is reported. Assignments are reported for the vast majority of the 15N amide resonances in both oxidized and reduced states. 15N heteronuclear relaxation experiments were collected to study the picosecond-nanosecond backbone dynamics of this protein. Relaxation rates were computed and fit to spectral density functions by a model-free analysis. Backbone amides in the overlapping loop B/C region are the most flexible on the picosecond-nanosecond time scale in both forms of the protein. The results show that, on average, the protein backbone is slightly more dynamic in the oxidized than the reduced state, though not significantly so. Exchange terms, which suggest significant motion on a time scale at least an order of magnitude slower than the overall correlation time of 5.2 ns, were required for only two residues in the reduced state and 27 residues in the oxidized state. When analyzed on a per-residue basis, the lower order parameters found in the oxidized state were scattered throughout the protein, with a few continuous segments found in loop C and the C-terminal helix, suggesting greater flexibility of these regions in the oxidized state. The results provide dynamic interpretations for previously presented structural and functional data, including redox-dependent changes that occur in the protein. The way is now paved for extensive dynamic analysis of variant cytochromes c.
Fetrow
J S
JS
Wadsworth Center, Department of Biomedical Sciences, New York State Department of Health, Albany 12201-0509, USA.
Baxter
S M
SM
D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D003574:Cytochrome c Group / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D045304:Cytochromes c; D005285:Fermentation; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D008956:Models, Chemical; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins; D013816:Thermodynamics; D013912:Threonine / Q000737:chemistry / Q000235:genetics
C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D007527:Isoenzymes; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D013912:Threonine; D045304:Cytochromes c; D003545:Cysteine