Thermodynamic and Structural Characterisation of Membrane Peptides and Proteins

生物膜是由蛋白质和其他分子嵌入双分子层脂质分子组成的复杂系统。然而，脂质性质和蛋白质结构的相互作用仍然知之甚少，特别是在分子水平上。这种相互作用对参与调节细胞活性的许多蛋白质的功能以及已知在与膜结合后具有活性的大量抗菌肽的活性具有重要影响。因此，提高我们对这些分子如何发挥作用的认识将有助于设计针对膜蛋白和新型抗生素的改进或新型药物。本研究将利用互补策略解决关于蛋白质-脂质系统内分子相互作用的两个关键问题。首先，我们将探索在高分辨率下表征膜相关肽和蛋白质结构的方法。其次，我们将尝试解决芳香侧链残基对蛋白质-脂质结合热力学的熵和焓贡献。电子显微镜将用于表征结合细胞膜的病毒蛋白的螺旋排列。分析将使用电子衍射和图像重建方法进行。合成肽将通过固态核磁共振波谱进行研究，以表征肽主链和关键氨基酸侧链残基相对于膜平面的排列。这些实验的关键将是在肽中引入同位素标签，以便于化学位移各向异性的测量。一个不同的多肽家族将被用来量化与脂质膜结合的热力学。将使用一系列类似物，在关键氨基酸侧链的关键位置上进行单次取代。这些肽与膜的结合将通过量热法和更传统的膜-水和辛醇-水分配方法来检测。对数据的检查将使我们能够得出关于焓和熵对肽脂结合的相对贡献的结论。这个项目的数据将使我们了解肽和蛋白质结构之间的关系以及蛋白质与膜结合的热力学。从长远来看，从这个项目中发展出来的合作以及与之相关的新技术将使我们能够更严格地在这一领域进行研究。

Biological membranes are complex systems composed of proteins and other molecules embedded in a bilayer of lipid molecules. However, the interplay of lipid properties and protein structure is still poorly understood, especially at the molecular level. This interplay has important consequences for the function of a number of proteins that are involved in regulating cell activity, as well as the activity of a large number of antimicrobial peptides that are known to be active following binding to the membrane. Improving our knowledge of how these molecules function will therefore facilitate the design of improved or novel drugs targeted towards membrane proteins and new classes of antibiotics. This research will address two key issues concerning the molecular interactions within protein-lipid systems using complementary strategies. Firstly, we will explore methods for characterising the structure of membrane-associated peptides and proteins at high resolution. Secondly, we will attempt to resolve the entropic and enthalpic contributions of aromatic side chain residues the thermodynamics of protein-lipid binding.Electron microscopy will be used to characterise helical arrays of a viral protein that binds to cell membranes. Analysis will be performed using electron diffraction and image reconstruction approaches. Synthetic peptides will be investigated by solid-state NMR spectroscopy in order to characterise the alignment of the peptide backbone and key amino acid side chain residues with respect to the plane of the membrane. The key to these experiments will be the introduction of isotopic labels into the peptide in order to facilitate measurements of chemical shift anisotropy. A different family of peptides will be used to quantify thermodynamics of binding to lipid membranes. A series of analogues, related by single substitutions at a critical position in a key amino acid side chain, will be used. The binding of these peptides to membrane will be examined by calorimetry and more conventional membrane-water and octanol-water partitioning methods. Examination of the data will enable us to reach conclusions regarding the relative contributions of enthalpy and entropy to peptide-lipid binding.Data from this project will enable us to understand the relationship between peptide and protein structure and the thermodynamics of protein association with membranes. In the long term, the collaborations that develop from this project and the new techniques associated with them will enable us to pursue research in this area more rigorously.