Elucidation of Membrane Fusion Mechanisms Using a Combined Simulation and Experimental Approach

[666666]智力价值：生命进化的决定性步骤是将生物分子封装在脂质膜内。然而，为了生长、愈合、分裂、运输和分泌，细胞膜需要经历裂变和融合。基本的生命过程，如神经传递、激素和酶的分泌，都需要膜融合。因此，了解膜融合的分子机制不仅有助于理解上述过程，而且有助于设计和开发生物传感器、阻断感染的药物以及有毒工业化学品暴露的治疗技术。此外，从对膜融合的理解，可以开发出高效的药物递送系统，用于专门和靶向递送和持续释放。虽然已经发现了不同的膜蛋白，如电视- snare（可溶性NSF附着蛋白受体，其中NSF = n -乙基-马酰亚胺敏感融合蛋白），以促进和确认细胞中膜融合的特异性，但原子水平上该过程的化学性质仍不清楚。因此，这项工作的总体目标是在原子水平上理解膜融合。具体来说，pi试图确定Ca2+触发tv-SNARE介导的融合反应的机制，以及膜融合蛋白synaptotagmin-I在膜融合调节中的作用。在这个假设驱动的工作中，分子动力学模拟被用来定量地确定Ca2+在囊泡之间的释放是如何通过计算溶剂化力、平均力势、界面张力和弹性模量来改变相对脂质囊泡之间的相互作用的，这是[Ca2+]、双层组成和温度的函数。分子动力学模拟用于研究Ca2+浓度梯度存在下水孔的形成，以及孔形成对膜融合速率的影响。分子动力学模拟被用来提供洞察synaptotagmin- 1在调节膜融合中的作用。在所有情况下，使用原子力显微镜（AFM），光学镊子，光散射，zeta电位测量和x射线散射进行互补实验。研究的新颖性：实验已被广泛用于研究Ca2+和膜融合蛋白与脂质囊泡的相互作用；然而，在原子水平上探测动态非均质流体系统中的特定相互作用是极其困难的。因此，膜融合在体内的分子机制仍然是一个相当有争议的主题。分子模拟提供了在原子水平上研究复杂生物系统的另一种方法，然而，最近的膜融合模拟在很大程度上忽略了触发这一事件的核心机制：Ca2+和各种膜蛋白，如电视- snare和synaptotagmin- 1。因此，拟议的研究解决了该领域的两个关键限制。模拟用于研究Ca2+，脂质和膜融合蛋白（如synaptotagmin-I和tv-SNARE）之间的相互作用，原子尺度分辨率无法通过实验实现。更广泛的影响：拟议的工作是变革性的，因为我们所寻求的对聚变事件的基本原子水平的理解将促进广泛的医学进步的发展，其中包括基于膜的智能生物传感器、药物输送装置和有毒工业化学品暴露的治疗技术。提高对膜融合这一基本生命过程的理解，将使从疾病检测到治疗的广泛领域取得进展。作为提议工作的一部分，pi将使用开放式课件模型开发化学和生物系统的分子模拟和建模课程。课堂讲稿和习题将发布在我们的小组网站上，而整个课程的视频将发布在YouTube上。鉴于模拟在研究中的应用越来越多，开放课程模式为化学工程、物理、化学和生物医学科学领域的数千名学生提供了机会，从而产生了广泛的影响。本科研究经验将用于招募和指导来自代表性不足群体的学生。

1066661PotoffIntellectual Merit: The defining step in evolution of life was the encapsulation of bio-molecules within a lipid membrane. However, to grow, heal, divide, transport, and secrete, cellular membranes need to undergo fission and fusion. Fundamental life processes such as neurotransmission, or the secretion of hormones and enzymes, require membrane fusion. Therefore, understanding the molecular mechanism of membrane fusion will not only enable an understanding of the above mentioned processes, but help in the design and development of biosensors, drugs to block infections, and the development of treatment technologies for exposure to toxic industrial chemicals. Furthermore, from this understanding of membrane fusion, efficient drug delivery systems could be developed for specialized and targeted delivery, and sustained release. Although distinct membrane proteins, such as tv-SNARE (soluble NSF attachment protein receptor, where NSF = N-ethyl-maleimide-sensitive fusion protein), have been identified to facilitate and confirm specificity to membrane fusion in cells, the chemistry of the process at the atomic level is still unclear. Therefore the overall objective of this work is to understand membrane fusion at the atomic level. Specifically, the PIs seek to identify the mechanism through which Ca2+ triggers the tv-SNARE mediated fusion reaction, and the role of the membrane fusion protein synaptotagmin-I on the regulation of membrane fusion. In this hypothesis driven work, molecular dynamics simulations are used to determine quantitatively how the release of Ca2+ between vesicles alters the interactions between apposed lipid vesicles through the calculation of solvation force, the potential of mean force, interfacial tension and elastic modulus, as a function of [Ca2+], bilayer composition, and temperature. Molecular dynamics simulations are used to investigate the formation of water pores in the presence of a Ca2+ concentration gradient, and the effect of pore formation on the rate of membrane fusion. Molecular dynamics simulations are used to provide insight into the role of synaptotagmin-I on the regulation of membrane fusion. In all cases, complimentary experiments are performed using atomic force microscopy (AFM), optical tweezers, light scattering, zeta potential measurements and x-ray scattering.Novelty of the proposed research: Experiments have been used extensively to study the Ca2+ and membrane fusion protein interactions with lipid vesicles; however, probing specific interactions in dynamic, heterogeneous fluid systems at the atomic level is extremely difficult. As a result, the molecular mechanism of membrane fusion in vivo is still the subject of considerable debate. Molecular simulation provides an alternative means to study complex biological systems at the atomic-level, however, recent simulations of membrane fusion have largely ignored the core machinery responsible for triggering the event: Ca2+ and various membrane proteins, such as tv-SNARE and synaptotagmin-I. The proposed research therefore addresses two key limitations in the field. Simulations are used to study interactions between Ca2+, lipids and membrane fusion proteins, such as synaptotagmin-I and tv-SNARE, with atomic scale resolution not possible with experiments. Broader Impacts: The proposed work is transformative because the fundamental atomic level understanding of the fusion event that we seek will enable the development of a wide range of medical advances, which include smart membrane-based bio-sensors, drug delivery devices and treatment technologies for exposure to toxic industrial chemicals. Improved understanding of membrane fusion, a fundamental life process, will enable advances in a wide array of fields from disease detection to treatment. As part of the proposed work, the PIs will develop courses on molecular simulation and modeling of chemical and biological systems using the OpenCourseWare model. Lecture notes and problem sets will be posted on our group website, while videos of lectures for the entire course will be posted on YouTube. Given the increasing use of simulation in research, the OpenCourseWare model provides an opportunity to reach thousands of students in Chemical Engineering, Physics, Chemistry and the Biomedical Sciences, thereby providing a broad impact. The undergraduate research experience will be used to recruit and mentor students from under-represented groups.