Vié V, Legardinier S, Chieze L, Le Bihan O, Qin Y, Sarkis J, Hubert J-F, Renault A, Desbat B, Le Rumeur E
Université de Rennes 1, Institut de Physique UMR CNRS 6251, Rennes, France.
Biochim Biophys Acta. 2010 Aug;1798(8):1503-11. doi: 10.1016/j.bbamem.2010.04.005. Epub 2010 Apr 14.
Dystrophin rod repeats 1-3 sub-domain binds to acidic phosphatidylserine in a small vesicle binding assay, while the repeats 20-24 sub-domain does not. In the present work, we studied the adsorption behaviour of both sub-domains at the air/liquid interface and at the air/lipid interface in a Langmuir trough in order to highlight differences in interfacial properties. The adsorption behaviour of the two proteins at the air/liquid interface shows that they display surface activity while maintaining their alpha-helical secondary structure as shown by PM-IRRAS. Strikingly, R20-24 needs to be highly hydrated even at the interface, while this is not the case for R1-3, indicating that the surface activity is dramatically higher for R1-3 than R20-24. Surface-pressure measurements, atomic force microscopy and PM-IRRAS are used in a Langmuir experiment with DOPC-DOPS monolayers at two different surface pressures, 20 mN/m and 30 mN/m. At the lower surface pressure, the proteins are adsorbed at the lipid film interface while maintaining its alpha-helical structure. After an increase of the surface pressure, R1-3 subsequently produces a stable film, while R20-24 induces a reorganization of the lipid film with a subsequent decrease of the surface pressure close to the initial value. AFM and PM-IRRAS show that R1-3 is present in high amounts at the interface, being arranged in clusters representing 3.3% of the surface at low pressure. By contrast, R20-24 is present at the interface in small amounts bound only by a few electrostatic residues to the lipid film while the major part of the molecule remains floating in the sub-phase. Then for R1-3, the electrostatic interaction between the proteins and the film is enhanced by hydrophobic interactions. At higher surface pressure, the number of protein clusters increases and becomes closer in both cases implying the electrostatic character of the binding. These results indicate that even if the repeats exhibit large structural similarities, their interfacial properties are highly contrasted by their differential anchor mode in the membrane. Our work provides strong support for distinct physiological roles for the spectrin-like repeats and may partly explain the effects of therapeutic replacement of dystrophin deficiency by minidystrophins.
在小泡结合实验中，肌营养不良蛋白杆状重复序列1 - 3亚结构域与酸性磷脂酰丝氨酸结合，而重复序列20 - 24亚结构域则不结合。在本研究中，我们在Langmuir槽中研究了这两个亚结构域在气/液界面和气/脂界面的吸附行为，以突出界面性质的差异。两种蛋白质在气/液界面的吸附行为表明，它们表现出表面活性，同时如PM - IRRAS所示保持其α - 螺旋二级结构。引人注目的是，即使在界面处，R20 - 24也需要高度水合，而R1 - 3则不然，这表明R1 - 3的表面活性比R20 - 24高得多。表面压力测量、原子力显微镜和PM - IRRAS用于在Langmuir实验中研究DOPC - DOPS单层在两种不同表面压力（20 mN/m和30 mN/m）下的情况。在较低表面压力下，蛋白质吸附在脂质膜界面，同时保持其α - 螺旋结构。表面压力增加后，R1 - 3随后形成稳定的膜，而R20 - 24则诱导脂质膜重组，随后表面压力降低至接近初始值。原子力显微镜和PM - IRRAS表明，R1 - 3在界面处大量存在，在低压下以簇状排列，占表面的3.3%。相比之下，R20 - 24在界面处少量存在，仅通过少数静电残基与脂质膜结合，而分子的大部分仍漂浮在亚相中。对于R1 - 3，蛋白质与膜之间的静电相互作用通过疏水相互作用增强。在较高表面压力下，两种情况下蛋白质簇的数量都增加且靠得更近，这意味着结合具有静电性质。这些结果表明，即使这些重复序列表现出很大的结构相似性，它们在膜中的不同锚定模式使其界面性质形成了强烈对比。我们的工作为血影蛋白样重复序列的不同生理作用提供了有力支持，并可能部分解释了微小肌营养不良蛋白治疗性替代肌营养不良蛋白缺陷的效果。
