Chupin V, Leenhouts J M, de Kroon A I, de Kruijff B
Department of Biochemistry of Membranes, Institute of Biomembranes, Utrecht University, The Netherlands.
Biochemistry. 1996 Mar 12;35(10):3141-6. doi: 10.1021/bi952482a.
In this study the secondary structure and topology of the peptide, corresponding to the presequence of cytochrome oxidase subunit IV (p25) in a negatively charged membrane-mimetic environment, were assessed by circular dichroism and two-dimensional nuclear magnetic resonance. The micelles used consisted of dodecylphosphoglycol (DPG), a mild anionic detergent with a headgroup resembling that of phosphatidylglycerol. The secondary structure was analyzed by interresidue nuclear Overhauser enhancement measurements and chemical shifts of backbone protons. The data revealed alpha-helix formation of the peptide upon interaction with the micelles, both in the N- and in the C-terminal halves, which are separated from each other by the proline residue at position 13. The topology of the peptide was studied by determining the effect of spin-labeled 12-doxylstearate on the line widths of the peptide proton resonances. This method revealed the insertion of hydrophobic residues of both the N- and the C-terminal halves of p25 into the hydrophobic environment of the micelles, demonstrating the orientation of the amphiphilic helix.
在本研究中，通过圆二色光谱法和二维核磁共振技术，评估了在带负电荷的膜模拟环境中，与细胞色素氧化酶亚基IV（p25）前序列相对应的肽段的二级结构和拓扑结构。所用的胶束由十二烷基磷酸甘油（DPG）组成，DPG是一种温和的阴离子去污剂，其头部基团类似于磷脂酰甘油。通过残基间核Overhauser效应测量和主链质子的化学位移分析二级结构。数据显示，肽段与胶束相互作用时，在N端和C端均形成了α螺旋，这两个区域被第13位的脯氨酸残基隔开。通过测定自旋标记的12-硬脂酰氧基硬脂酸对肽段质子共振线宽的影响，研究了肽段的拓扑结构。该方法揭示了p25的N端和C端的疏水残基均插入到胶束的疏水环境中，证明了两亲性螺旋的取向。
