FLUOR - Fluorinated Surfactants for Membrane-Protein Research

膜蛋白在细胞通讯和运输过程中发挥着重要作用，是大多数药物靶点的代表。尽管如此，我们对膜蛋白结构、动力学和功能的认识和理解进展缓慢，主要是因为它们非常疏水的性质造成了困难。这些蛋白质需要一个模拟膜的环境，以保持它们在体外研究中的溶解和活性，这通常是在洗涤剂的帮助下完成的，洗涤剂是一种表面活性化合物，可以溶解膜蛋白和脂类。然而，许多膜蛋白在洗涤剂中溶解时会失活，也就是说，它们失去了天然的结构和功能。这促使许多人努力用温和的替代品取代传统的洗涤剂，其中氟化表面活性剂似乎特别有希望。由于氟碳化合物对碳氢化合物的亲和力较弱，以及氟碳化合物的体积较大，因此假设含氟表面活性剂的不稳定程度较小，因为它们几乎不会与蛋白质-蛋白质和蛋白质-脂/疏水辅助因子相互作用竞争。事实上，已经多次证明，与氢化洗涤剂相比，各种膜蛋白在氟化表面活性剂中的增溶更稳定。然而，不幸的是，中性氟化表面活性剂不能溶解脂质双层并直接从膜上提取膜蛋白。因此，增溶仍然需要传统的洗涤剂，而氟化表面活性剂只有在不稳定的蛋白质已经遭受不可逆转的损害的较晚阶段才会发挥作用。我们最近已经证明，氟化本身并不排除去污力，因为氟化的辛基麦芽糖苷衍生物保留了温和的洗涤剂类活性。在这个项目中，我们打算利用这一发现来开发、测试和建立氟化洗涤剂，这些洗涤剂(I)可以合成足够的数量和纯度，以便广泛用于膜蛋白研究；(Ii)表现出良好的胶束性质，如小而明确的尺寸；(Iii)以快速、热力学控制的方式分配到膜中、跨膜转移和增溶膜；(Iv)直接从天然或合成膜中溶解膜蛋白，而不需要更苛刻的氢化洗涤剂；以及(V)为这些蛋白提供一个稳定的环境，使其在较长时间内保持其天然结构和功能。这类氟化洗涤剂将为迄今为止逃避详细审查的具有生理和药理意义的膜蛋白的体外研究打开新的可能性。这一高度跨学科的项目应在一个唯一有资格处理合成、物理化学、生物物理、生化和结构生物学方面的联合体内完成。

Membrane proteins play numerous vital roles in cellular communication and transport processes and represent the majority of drug targets. Still, progress in our knowledge and understanding of membrane-protein structure, dynamics, and function is slow, primarily because of difficulties arising from their very hydrophobic nature. These proteins require a membrane-mimetic environment to keep them both soluble and active during in vitro investigations, which is typically accomplished with the aid of detergents, surface-active compounds that solubilise membrane proteins and lipids. However, many membrane proteins are inactivated when solubilised in detergents, that is, they lose their native structures and functions. This has motivated many efforts at replacing conventional detergents by milder alternatives, among which fluorinated surfactants appear particularly promising. Owing to the weak affinity of fluorocarbons for hydrocarbons and to the larger volume of fluorocarbons, fluorinated surfactants are hypothesised to be less destabilising because they hardly compete with protein-protein and protein-lipid/hydrophobic cofactor interactions. Indeed, it has been shown repeatedly that various membrane proteins are more stable when solubilised in fluorinated surfactants as compared with hydrogenated detergents.Unfortunately, though, neutral fluorinated surfactants are not able to solubilise lipid bilayers and extract membrane proteins directly from membranes. Hence, conventional detergents are still required for solubilisation, and fluorinated surfactants come into play only at a later stage, when labile proteins will already have suffered irreversible damage. We have recently demonstrated that fluorination per se does not exclude detergency, as a fluorinated octyl maltoside derivative retains mild detergent-like activity. In this project, we intend to capitalise on this finding to develop, test, and establish fluorinated detergents that (i) can be synthesised in sufficient quantities and purities for widespread use in membrane-protein research; (ii) display favourable micellar properties such as small and well-defined sizes; (iii) partition into, translocate across, and solubilise membranes in a rapid, thermodynamically controlled manner; (iv) solubilise membrane proteins directly from native or synthetic membranes without requiring harsher, hydrogenated detergents; and (v) offer these proteins a stabilising environment that preserves their native structures and functions for extended periods of time. Such fluorinated detergents will open new possibilities for in vitro studies of physiologically and pharmacologically interesting membrane proteins that have thus far evaded detailed scrutiny.This highly interdisciplinary project shall be accomplished within a consortium that is uniquely qualified to address synthetic, physicochemical, biophysical, biochemical, and structural-biological aspects alike.