Probing iso-1-cytochrome c structure by site-directed spin labeling and electron paramagnetic resonance techniques.
A cysteine-specific methanethiosulfonate spin label was introduced into yeast iso-1-cytochrome c at three different positions. The modified forms of cytochrome c included: the wild-type protein labeled at naturally occurring C102, and two mutated proteins, S47C and L85C, labeled at positions 47 and 85, respectively (both S47C and L85C derived from the protein in which C102 had been replaced by threonine). All three spin-labeled protein derivatives were characterized using electron paramagnetic resonance (EPR) techniques. The continuous wave (CW) EPR spectrum of spin label attached to L85C differed from those recorded for spin label attached to C102 or S47C, indicating that spin label at position 85 was more immobilized and exhibited more complex tumbling than spin label at two other positions. The temperature dependence of the CW EPR spectra and CW EPR power saturation revealed further differences of spin-labeled L85C. The results were discussed in terms of application of the site-directed spin labeling technique in probing the local dynamic structure of iso-1-cytochrome c.
Pyka
J
J
Department of Biophysics, Institute of Molecular Biology, Jagiellonian University, Kraków, Poland.
Osyczka
A
A
Turyna
B
B
Blicharski
W
W
Froncisz
W
W
D001483:Base Sequence; D003497:Cyclic N-Oxides; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics; D045304:Cytochromes c; D017931:DNA Primers / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D008698:Mesylates; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D013113:Spin Labels; D013816:Thermodynamics
C014059:CYC1 protein, S cerevisiae; D003497:Cyclic N-Oxides; D003574:Cytochrome c Group; D017931:DNA Primers; D005656:Fungal Proteins; D008698:Mesylates; D029701:Saccharomyces cerevisiae Proteins; D013113:Spin Labels; C034248:(1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)methanethiosulfonate; D045304:Cytochromes c