QUANTITATIVE CONFORMATIONAL-ANALYSIS OF CYTOCHROME-C BOUND TO PHOSPHOLIPID-VESICLES STUDIED BY RESONANCE RAMAN-SPECTROSCOPY
QUANTITATIVE CONFORMATIONAL-ANALYSIS OF CYTOCHROME-C BOUND TO PHOSPHOLIPID-VESICLES STUDIED BY RESONANCE RAMAN-SPECTROSCOPY
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DOI：
10.1007/bf02427378
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发表时间：
1990-01-01
影响因子：
2
通讯作者：
MARSH, D
中科院分区：
文献类型：
--
作者：
HILDEBRANDT, P;HEIMBURG, T;MARSH, D
本文记录了铁细胞色素c与二豆油酰基磷脂酰甘油（DMPG）、二油基磷脂酰甘油（DOPG）或二油基磷脂酰甘油-二油基磷脂酰胆碱（DOPG- dopc）（70∶30摩尔/摩尔）组成的磷脂囊泡的共振拉曼光谱。脂质结合在蛋白质分子中引起非常显著的构象变化。共振拉曼光谱中来自蛋白质的两种不同构象I和II以及构象II中血红素的两种不同自旋和配位态的条带含量不同。获得了足够高精度的数据，使得单个物种的光谱可以通过使用单个洛伦兹剖面的约束交互拟合程序进行定量。在高频或标记波段区域（1200 ~ 1700 cm-1），单个波段的频率、半宽度和相对强度可以通过先前吸附在银电极上的细胞色素c的表面增强共振拉曼测量来估计。然后对这些进行进一步优化，以获得不同物种的光谱参数和相对含量。在低频或指纹区（200 ~ 800 cm-1），利用标记带区获得的物种相对比例，将三种不同脂质体系中铁-细胞色素c的光谱进行序列相减，得到各物种的光谱参数。然后通过迭代优化对这些参数进行细化。给出了六协调低自旋态I和II以及五协调高自旋态II在两个频率区域的优化谱参数。II态（血红素缝隙呈开放结构）和五配位高自旋构型的比例在铁细胞色素c与带负电荷的脂质囊泡结合时增加。这种构象变化的程度按DOPG-DOPC的顺序增加
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 interative 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 weren 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. The proportion of state II, in which hence the heme crevice assumes an open structure, and of the five-coordinated high spin configuration, is found to increase on binding ferricytochrome c to negatively charged lipid vesicles. The extent of this conformational change increases in the order: DOPG-DOPC