de Foresta Béatrice, Tortech Ludovic, Vincent Michel, Gallay Jacques
Section de Biophysique des Protéines et des Membranes, Département de Biologie Cellulaire et Moléculaire et URA 2096 (CNRS), CEA Saclay, 91191 Gif-sur-Yvette Cedex, France.
Eur Biophys J. 2002 Jun;31(3):185-97. doi: 10.1007/s00249-002-0211-9. Epub 2002 Mar 8.
Amphiphilic and hydrophobic peptides play a key role in many biological processes. We have developed a reference system for evaluating the insertion of such peptides bearing Trp fluorescent reporter groups into membrane mimetic systems. This system involves a set of six 25-amino acid synthetic peptides that are models of transmembrane alpha-helices. They are Lys-flanked polyLeu sequences, each containing a single Trp residue at a different position (P i, with i=3, 5, 7, 9, 11 and 13). These peptides were inserted into micelles of a non-ionic detergent, dodecylmaltoside (DM). We analyzed this system by use of circular dichroism and steady-state and time-resolved fluorescence in combination with Trp quenching with two brominated DM analogs. We found significant variations in the Trp emission maximum according to its position in each peptide (from 327 to 313 nm). This is consistent with the radial insertion of the peptides within DM micelles. We observed characteristic patterns of fluorescence quenching of these peptides in mixed micelles of DM, with either 7,8-dibromododecylmaltoside (BrDM) or 10,11-dibromoundecanoylmaltoside (BrUM), that reflect differences in the accessibility of the Trp residue to the bromine atoms located on the detergent acyl chain. In the isotropic reference solvent, methanol, the alpha-helix content was high and identical (approximately 76%) for all peptides. In DM micelles, the alpha-helix content for P9 to P13 was similar to that in methanol, but slightly lower for P3 to P7. The fluorescence intensity decays were heterogeneous and depended upon the position of the Trp. The Trp dynamics of each peptide are described by sub-nanosecond and nanosecond rotational motions that were significantly lower than those observed in methanol. These results, which precisely describe structural, dynamic and microenvironment parameters of peptide Trp in micelles according to its depth, should be useful for describing the interactions of peptides of biological interest with micelles.
两亲性和疏水性肽在许多生物过程中起着关键作用。我们开发了一种参考系统，用于评估带有色氨酸荧光报告基团的此类肽插入模拟膜系统的情况。该系统涉及一组六个25个氨基酸的合成肽，它们是跨膜α-螺旋的模型。它们是赖氨酸侧翼的聚亮氨酸序列，每个序列在不同位置（Pi，i = 3、5、7、9、11和13）含有一个色氨酸残基。这些肽被插入到非离子去污剂十二烷基麦芽糖苷（DM）的胶束中。我们通过圆二色性、稳态和时间分辨荧光分析该系统，并结合使用两种溴化DM类似物进行色氨酸猝灭。我们发现，根据色氨酸在每个肽中的位置，其发射最大值存在显著变化（从327纳米到313纳米）。这与肽在DM胶束中的径向插入一致。我们在DM与7,8-二溴十二烷基麦芽糖苷（BrDM）或10,11-二溴十一烷酰麦芽糖苷（BrUM）的混合胶束中观察到这些肽的特征性荧光猝灭模式，这反映了色氨酸残基与位于去污剂酰基链上的溴原子的可及性差异。在各向同性参考溶剂甲醇中，所有肽的α-螺旋含量都很高且相同（约76%）。在DM胶束中，P9至P13的α-螺旋含量与甲醇中的相似，但P3至P7的略低。荧光强度衰减是不均匀的，并且取决于色氨酸的位置。每个肽的色氨酸动力学由亚纳秒和纳秒旋转运动描述，这些运动明显低于在甲醇中观察到的运动。这些结果根据肽色氨酸在胶束中的深度精确描述了其结构、动力学和微环境参数，对于描述具有生物学意义的肽与胶束的相互作用应该是有用的。
