Interactions at the protein-lipid bilayer interface: quantitative characterization via single-molecule force spectroscopy

蛋白质是细胞的重要组成部分，是所有生物的基石。所有进出细胞的东西(如营养素和药物)都必须通过细胞膜。然而，人们对蛋白质和膜如何在分子水平上相互作用知之甚少。为了更好地了解蛋白质与脂质的相互作用，我们将把单个蛋白质连接到原子力显微镜的尖端，这是一种能够在分子尺度上揭示相互作用的极其强大的显微镜。然后，我们将把这些蛋白质带到膜的附近，并使用显微镜记录由此产生的力。测量这些微观层面的相互作用将揭示取决于所使用的特定蛋白质的细节。更广泛地说，这项工作将提供对发生在生物膜上的基本活动的更深入和定量的了解。就更广泛的影响而言，该项目旨在提高STEM教育和公众科学素养。计划开展几项活动来支持这些目标，包括为密苏里州高中物理学生开发和实施一个实验室模块；在密苏里大学哥伦比亚分校实施和完善一个针对非理科专业学生的新的实践本科课程；通过科学咖啡馆与密苏里州中部的普通观众进行直接对话，科学咖啡馆是一个鼓励听众提问的非正式场所。本研究的主要目标是阐明模型亲脂蛋白在通过液体脂双层时的相互作用强度、能量图景和动力学路径，从而改进和定量地理解蛋白质在脂双层膜界面上的分配和折叠。这将通过将基于高精度原子力显微镜(AFM)的力光谱方法与分析建模、计算机模拟和生物化学相结合来实现。中心实验观测(力)将以最先进的时空精度进行测量。该项目解决了膜生物物理学中的基本问题，例如：蛋白质序列和二级结构的出现如何影响蛋白质-脂质双层相互作用的强度？三种不同结构复杂性的模型蛋白质系统将与支持的脂类双层结合使用。具体的研究目标包括：(I)将肽序列与肽-脂双层相互作用的强度、能量分布和动力学途径相关联；(Ii)在多肽-脂双层相互作用的模型中，从一级结构贡献中去卷积蛋白质的二级结构；以及(Iii)定量表征在大肠杆菌中支持蛋白质输出活性的关键的外周膜蛋白-脂双层相互作用。通过直接探测模型蛋白质从脂质双层界面的重复结合和解离，该项目有望提供对膜中蛋白质分配和折叠的更好理解，这是一个基本的生物物理过程。该奖项反映了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。

Proteins are important components of cells, which are the building blocks of all living things. Everything that enters and exits a cell (e.g., nutrients and pharmaceuticals) must go through membranes. Yet, little is known about how proteins and membranes interact with each other at the molecular scale. To better understand protein-lipid interactions, we will attach individual proteins to the sharp tip of an atomic force microscope, which is an extremely powerful microscope that is able to reveal interactions at the molecular scale. We will then bring these proteins into the proximity of membranes and use the microscope to record the resulting forces. Measuring these microscopic-level interactions will reveal details that depend on the specific proteins employed. More generally, the work will provide a deeper and quantitative understanding of fundamental activities that take place at the biological membrane. In terms of broader impacts, the project aims to improve STEM education and public science literacy. Several activities are planned to support these goals including developing and implementing a laboratory module for Missouri high school physics students; implementing and refining a new hands-on undergraduate course at the University of Missouri-Columbia targeted to non-science majors; and engaging in direct dialogue with general audiences in mid-Missouri through the Science Café, which is an informal venue that encourages questions from audience members. The overarching goal of this research project is to elucidate interaction strengths, energy landscapes and kinetic pathways of model lipophilic proteins as they negotiate fluid lipid bilayers and thus to provide an improved and quantitative understanding of protein partitioning and folding at the lipid bilayer membrane interface. This will be achieved by combining high precision atomic force microscopy (AFM)-based force spectroscopy methodology with analytical modeling, computer simulations, and biochemistry. The central experimental observable (force) will be measured with state-of-the-art spatial-temporal precision. The project tackles fundamental questions in membrane biophysics such as: How do the protein sequence and the emergence of secondary structure affect the strength of a protein-lipid bilayer interaction? Three model protein systems of differing structural complexity will be employed in conjunction with supported lipid bilayers. Specific research goals include: (i) correlate peptide sequence with peptide-lipid bilayer interaction strength, energy landscape and kinetic pathway; (ii) deconvolve protein secondary structure from primary structure contributions in a model polypeptide-lipid bilayer interaction; and (iii) quantitatively characterize a critical peripheral membrane protein-lipid bilayer interaction underlying protein export activity in E. coli. By directly probing the repeated association and dissociation of model proteins from the lipid bilayer interface, this project is poised to provide an improved understanding of protein partitioning and folding in membranes, a fundamental biophysical process.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.