Quantitative conformational analysis of cytochrome c bound to phospholipid vesicles studied by resonance Raman spectroscopy.
Resonance Raman spectra have been recorded from ferri-cytochrome c bound to phospholipid vesicles composed of dimyristoyl phosphatidylglycerol (DMPG), dioleoyl phosphatidylglycerol (DOPG) or dioleoyl phosphatidylglycerol-dioleoyl phosphatidylcholine (DOPG-DOPC) (70:30 mole/mole). Lipid binding induces very significant conformational changes in the protein molecule. The resonance Raman spectra differ in their content of bands originating from two different conformational species, I and II, of the protein, and from two different spin and coordination states of the heme in conformation II. Data of sufficiently high precision were obtained that the spectra of the individual species could be quantitated by a constraint interactive fitting routine using single Lorentzian profiles. In the high frequency, or marker band region (1200 to 1700 cm-1), the frequencies, half widths and relative intensities of the individual bands could be estimated from previous surface enhanced resonance Raman measurements on cytochrome c adsorbed on a silver electrode. These were then further optimized to yield both the spectral parameters and relative contents of the different species. In the low frequency, or fingerprint, region (200 to 800 cm-1), the spectral parameters of the individual species were obtained from difference spectra derived by sequential subtraction between the spectra of ferri-cytochrome c in the three different lipid systems, using the relative proportions of the species derived from the marker band region. These parameters were then subsequently refined by iterative optimization. The optimized spectral parameters in both frequency regions for the six-coordinated low spin states I and II, and for the five-coordinated high spin state II are presented.(ABSTRACT TRUNCATED AT 250 WORDS)
Hildebrandt
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P
Max-Planck-Institut für Biophysikalische Chemie, Abteilung Spektroskopie, Göttingen, Federal Republic of Germany.
Heimburg
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T
Marsh
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D
D000818:Animals; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003574:Cytochrome c Group / Q000378:metabolism; D010084:Oxidation-Reduction; D010713:Phosphatidylcholines / Q000378:metabolism; D010715:Phosphatidylglycerols / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D011487:Protein Conformation; D013059:Spectrum Analysis, Raman
D003574:Cytochrome c Group; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D010743:Phospholipids; C051388:1,2-dioleoyl-sn-glycero-3-phosphoglycerol; C002773:dimyristoylphosphatidylglycerol; C017251:1,2-oleoylphosphatidylcholine