Interactions of membrane proteins with lipids, water, and other proteins in biological membranes

背景 膜蛋白约占所有蛋白质的三分之一。它们参与许多重要的生理过程，几乎代表了已知药物靶点的一半。膜蛋白在天然的脂质双层环境中的研究尤其具有挑战性，因为两种常见的结构方法，X射线结晶学和溶液状态核磁共振(NMR)，都依赖于晶体或洗涤剂胶束，这与脂类的结构不同。固体核磁共振为蛋白质结构的确定提供了一种新的方法，也为研究双层环境中的膜蛋白质提供了新的途径。 目标 在这一应用中，我们将追求旨在提高SS核磁共振表征蛋白质结构和蛋白质相互作用能力的方法学目标，并回答与膜蛋白质构象对环境变化的响应相关的基本生物物理问题。我们的目标是了解膜蛋白与其环境之间的相互作用如何塑造蛋白质结构。具体来说，我们将重点关注以下三种类型的效果： (I)蛋白质-水相互作用。水分子在决定蛋白质的结构、稳定性、动力学和功能方面起着重要作用。埋藏水与主链和侧链原子之间广泛存在氢键，稳定了二级结构和三级结构，发挥了功能作用。由于蛋白质-水的相互作用具有内在的动态性，核磁共振非常适合于研究这些相互作用。我们感兴趣的是确定结合水分子的位置，并表征它们的动力学。 (2)蛋白质-脂质相互作用。我们感兴趣的是表征特定和非特定相互作用的影响，例如，紧密结合的非环状脂类如何影响蛋白质结构，以及双层性质的变化如何影响膜蛋白质结构、齐聚和动力学。 (Iii)细胞膜中膜蛋白的结构。研究细胞环境中的生物分子是分子生物学的终极目标。SS核磁共振提供了一个很好的工具来解释由复杂的环境引起的膜蛋白的结构和动态变化，其中存在许多额外的内源分子成分。我们努力回答的基本问题是，重建的双层膜的理想化环境在多大程度上类似于真正的膜。 意义 越来越多的脂质包埋的膜蛋白的SS核磁共振结构突显了固态核磁共振作为一种强有力的方法在类天然双层环境中研究膜蛋白的潜力。本提案的目的是超越结构，研究膜蛋白与水和脂之间在功能和结构上的重要相互作用。我们将开发新的SS核磁共振方法来全面了解膜蛋白的结构和动力学，包括结合水和脂分子，并将在长期存在的环境对膜蛋白功能组装、结构和动力学影响的问题上提供详细的结构信息。

BACKGROUND Membrane proteins constitute approximately a third of all proteins. They are involved in many important physiological processes, and represent almost a half of known drug targets. Studying membrane proteins in their native lipid bilayer environment is particularly challenging, as the two common structural methods, X-ray crystallography and solution-state Nuclear Magnetic Resonance (NMR), rely on crystals or detergent micelles, which are different from that of lipids. Solid-state NMR (SSNMR) provides an alternative approach to protein structure determination, and offers ways to study membrane proteins in the bilayer environment. OBJECTIVES In this application, we will pursue both the methodological objectives aimed at improving SSNMR ability to characterize protein structures and protein interactions, and to answer the fundamental biophysical questions related to the response of membrane protein conformations to changes in the environment. Our goal is to undestand how interactions between membrane proteins and their environment shape protein structure. Specifically, we will focuss on the following three types of effects: (i) Protein-water interactions. Water molecules play important roles in defining the structure, stability, dynamics and function of proteins. Buried waters are extensively hydrogen-bonded with backbone and side chain atoms, both stabilizing the secondary and tertiary structures, and playing functional roles. As protein-water interactions are inherently dynamic, NMR is ideally suited to study these interactions. We are interested in determining the location of bound water molecules, and in characterizing their dynamics. (ii) Protein-lipid interactions. We are interested in characterizing the effects of both specific and non-specific interactions, e.g., how tightly bound non-annular lipids affect the protein structure, and how variation of the bilayer properties affect membrane proteins structure, oligomerization and dynamics. (iii) Structure of membrane proteins in cellular membranes. Studying biomolecules in the cellular environment is the ultimate goal of molecular biology. SSNMR provides an excellent tool to elucidate the structural and dynamic changes in membrane proteins induced by the complex environment of E. coli membranes with many additional endogenous molecular components present. The fundamental question we strive to answer is to what extent the idealized environment of a reconstituted bilayer resembles that of a real membrane. SIGNIFICANCE A growing number of SSNMR structures of lipid-embedded membrane proteins underscores the potential of solid-state NMR as a powerful method to investigate membrane proteins in the native-like bilayer environment. The present proposal aims to go beyond the structure and to study functionally and structurally important interactions between membrane proteins, and water and lipids. We will develop new SSNMR methodologies for obtaining comprehensive view of membrane protein structure and dynamics that include bound water and lipid molecules, and will contribute detailed structural information on the long-standing questions of the effects of environment on the membrane protein functional assembly, structure, and dynamics.