Toolkit for High-Resolution Structure and Dynamics of Functional Lipids

项目摘要 膜蛋白在真核细胞中含量丰富，在许多生物学过程中起着重要作用， 从细胞粘附和识别到能量产生再到信号级联。 此外，膜蛋白占目前批准的药物靶点的约60%， 强调了它们与人类疾病的相关性。虽然许多高分辨率的结构 膜蛋白的结构已经被解决，我们缺乏方法，也将使我们能够解决的结构， 与膜包埋蛋白、外周膜蛋白和其他蛋白相互作用的膜脂质， 配体。这是尽管事实上，特定的膜脂质在生物学中发挥关键的调节作用。我们称 这些“功能性脂质”，因为除了它们在膜中众所周知的结构作用外， 脂质是调节和/或直接进行必需的生物学功能的效应分子， 功能协调发展的对功能性脂质进行的相互作用的原子尺度理解是重要的 与人类健康和疾病直接相关的未实现目标。因此，尽管现在存在用于 要以非常高的分辨率解析蛋白质（包括膜蛋白）的结构，该领域缺乏工具 这是以高分辨率解决膜的脂质部分结构所必需的。这个雄心勃勃的项目旨在 开发一个创新的高分辨率方法“工具包”，供科学界用于解决 调节膜生物功能的脂质结构。我们的方法需要协同和 （1）各种磷脂的具有成本效益的位点特异性同位素标记， 甾醇;（2）将标记的脂质组装成纳米级脂质双层系统， 相关配体;（3）核磁共振（NMR）方法，主要是高场魔角 旋转固态NMR（SSNMR），以获得有关脂质与 配体;（4）尖端的计算方法，采用分子动力学（MD）模拟脂质 与双层或双层模拟物中的配体相互作用;和（5）通过以下方法求解脂质结构的新方法： 结合计算NMR和MD方法，以解决解释中固有的独特挑战 以及理解从含有标记脂质的重复拷贝的平面双层获得的光谱数据 除了它们的特定配体之外，还与邻近的脂质相互作用。随着研究的进展，我们建议 将此工具包应用于生物学中的典型问题，包括血液凝固、抗菌肽作用 和甾醇识别。

Project Summary Membrane proteins are abundant in eukaryotic cells and play important roles in a great many biological processes ranging from cell adhesion and recognition to energy production to signaling cascades. Furthermore, membrane proteins make up about 60% of the targets for currently approved drugs, which underscores their relevance to human disease. Although very high resolution structures of a number of membrane proteins have now been solved, we lack methods that will also allow us to resolve the structures of the membrane lipids that interact with membrane-embedded proteins, peripheral membrane proteins and other ligands. This is in spite of the fact that specific membrane lipids play key regulatory roles in biology. We term these “functional lipids” because, in addition to their well-known structural roles in membranes, it is becoming increasingly clear that lipids are effector molecules that modulate and/or directly carry out essential biological functions. An atomic-scale understanding of the interactions carried out by functional lipids is an important unmet goal with direct relevance to human health and disease. Thus, although excellent methods now exist for solving the structures of proteins—including membrane proteins—at very high resolution, the field lacks tools necessary to solve the structures of the lipid part of membranes at high resolution. This ambitious project aims to develop an innovative “toolkit” of high-resolution methods for the scientific community to use in solving the structures of lipids that regulate the biological functions of membranes. Our approach requires synergistic and coordinated efforts throughout: (1) cost-effective, site-specific isotopic labeling of a variety of phospholipids and sterols; (2) assembling labeled lipids into nanoscale lipid bilayer systems together with their biologically relevant ligands; (3) nuclear magnetic resonance (NMR) approaches, principally high-field magic-angle spinning solid-state NMR (SSNMR), to obtain detailed structural information about the lipids interacting with ligands; (4) cutting-edge computational methods employing molecular dynamics (MD) simulations of lipids interacting with ligands in bilayers or bilayer mimetics; and (5) new methods for solving lipid structures by marrying computational NMR and MD approaches to address the unique challenges inherent in interpreting and understanding spectral data obtained from planar bilayers that contain repeating copies of labeled lipids interacting with neighboring lipids in addition to their specific ligands. As our studies progress, we propose to apply this toolkit to exemplary problems in biology, including blood coagulation, antimicrobial peptide action and sterol recognition.