Understanding Highly Heterogeneous Biological Membranes

生物膜是高度复杂的环境，在细胞间的通讯和能量生产等广泛的细胞功能中发挥着核心作用。膜含有数百种在纳米尺度上组织的独特的脂质，这种组织对细胞功能至关重要。膜含有大量的脂类，它们具有特定的功能。例如，某些脂质物种是作为信号分子存在的，以调节某些生化过程。脂质分布异常被认为与人类疾病有关。由于研究膜结构的工具严重有限，脂膜成分的具体生物学作用还没有完全被了解。不同成分之间的微观相互作用，在分子对分子的基础上，决定了脂质的微观分布和局部膜结构。该项目将推出基于时间分辨光谱和单分子成像的尖端实验工具，以研究与膜信号相关的特定环境和分子几何结构。此外，该团队将开发多尺度模拟方法，以获得膜的原子学视图，并帮助解释实验。具体地说，该项目将专注于了解某些带负电荷的脂类如何改变信号蛋白的结合亲和力。这一合作项目将提供一个平台，用于培训不同研究小组的来访学生，并增加德克萨斯大学奥斯汀分校化学和神经科学研究生项目招收的人数不足的学生人数。在这个项目中，研究小组将结合使用生化方法、荧光标记的脂类、振动光谱、单分子显微镜和增强采样分子动力学模拟来研究脂膜环境和信号脂类的作用。具体地说，该团队将专注于从哺乳动物细胞系中获取完整的质膜，并将使用荧光显微镜和尖端增强红外光谱对其进行成像。该项目旨在建立脂-脂和脂-蛋白质相互作用的分子水平的观点，以告知膜组成的重要性。膜的以下方面将是该项目的主要重点：1.该团队将表征模型跨膜螺旋如何在与其他脂质的混合物中采样不同范围的局部环境，并特别强调蛋白质序列的序列。2.该团队将使用模型螺旋多肽来确定蛋白质如何在不同的双层中分配，这些螺旋多肽在疏水和亲水残基之间具有明确的平衡。该团队将测量嵌入到收获的质膜中的单个跨膜螺旋的红外光谱。3.该团队将通过捕捉膜块中PIP2信号脂类和其他阴离子脂类之间的相互作用来探索膜作为“信号脂库”的作用，以告知这些相互作用如何驱动信号功能。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Biological membranes are highly complex environments that play a central role in a wide range of cellular functions such as cell-cell communication and energy production. Membranes contain hundreds of unique lipids organized on a nanometer length scale, and this organization is critical for cellular function. Membranes contain a large number of lipids, which serve specific functions. For example, certain lipid species are present as signaling molecules to regulate certain biochemical processes. Abnormal lipid distributions have been linked to human diseases. The specific biological roles of lipid membrane composition are not fully understood since the tools to investigate membrane architectures are severely limited. Microscopic interactions among the different components, on a molecule-to-molecule basis, determine the microscopic distribution of lipids and local membrane structure. This project will bring forth cutting-edge experimental tools based on time-resolved spectroscopy and single-molecule imaging to investigate the specific environment and the molecular geometries associated with membrane signaling. In addition, the team will develop multiscale simulation methods to obtain an atomistic view of membranes and aid in the interpretation of experiments. Specifically, the project will focus on understanding how certain negatively-charged lipid species can alter the binding affinities of signaling proteins. This collaborative project will provide a platform for training visiting students across research groups and to increase the number of underrepresented students enrolled in the Chemistry and Neuroscience graduate programs at UT-Austin. In this project, the research team will investigate lipid membrane environments and the role of signaling lipids using a combination of biochemical methods, fluorescently-labeled lipids, vibrational spectroscopy, single-molecule microscopy, and enhanced-sampling molecular dynamics simulations. Specifically, the team will focus on harvesting intact plasma membranes from mammalian cell lines that will be imaged using fluorescence microscopy and tip-enhanced infrared spectroscopy. This project seeks to establish a molecular-level view of lipid-lipid and lipid-protein interactions to inform the importance of membrane composition. The following aspects of membranes will be the primary focus of the project: 1. The team will characterize how model transmembrane helices sample a different range of local environments in a mixture with other lipids, with specific emphasis on the sequence of the protein sequence. 2. The team will determine how proteins become partitioned in heterogeneous bilayers using model helical peptides with a well-defined balance between hydrophobic and hydrophilic residues. The team will measure IR spectra of single transmembrane helices embedded in harvested plasma membranes. 3. The team will explore the role of the membrane as a “signaling lipid reservoir” by capturing the interactions between PIP2 signaling lipids and other anionic lipids within the membrane bulk to inform how these interactions drive signaling functions.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.