INSPIRE: Concentrated Dispersions of Equilibrium Protein Nanoclusters that Reversibly Dissociate into Active Monomers

这项INSPIRE奖由工程总局(ENG)化学、生物工程、环境和运输部(CBET)的界面过程和热力学项目颁发给奥斯汀的德克萨斯大学，资金来自CBET-ENG中的颗粒和多相过程；以及数学和物理科学局材料研究部的生物材料项目。基于蛋白质的药物是治疗包括癌症在内的各种疾病的最有前途的疗法之一。皮下注射是首选的给药方法，但目前其有效性受到高剂量时出现的蛋白质聚集和凝胶等不良后果的限制。以前通过改变潜在疗法的氨基酸序列来解决这些问题的尝试都是昂贵的，而且往往不成功。研究人员最近报道了一种新的方法来创建高度集中、低粘度的稳定蛋白质纳米簇分散体，这种方法不仅具有重要的基础意义，而且可以为解决蛋白质疗法中的主要挑战的非传统方法提供基础。然而，目前关于纳米团簇起源的基本问题还缺乏答案。此外，目前还不知道特定的纳米团簇特征和所得到的分散体的物理性质之间的关系。智力价值本提案中要研究的蛋白质纳米团簇分散体代表了一种全新的软凝聚物质形式。该提案的目标是探索和发展对蛋白质纳米簇如何形成、为什么它们稳定蛋白质的折叠状态以及簇对分散体物理性质的影响的基本理解。研究人员将使用实验、统计力学理论和计算机模拟来检验这一假设，即团簇是由于一个平衡的自组装过程而自发形成的，在这个过程中，添加一个小分子？Crowder？分子在蛋白质--S等电点附近通过弱静电斥力来平衡蛋白质之间的引力。在这样做的同时，他们还将解决关键的公开问题，如纳米簇分散的路径依赖性，新型分子聚集器在纳米簇组装中的作用和设计，以及纳米簇的结构和动力学。由于蛋白质纳米簇分散体本身具有多尺度特性(簇、蛋白质、聚集物和溶剂都引入了特征长度尺度)，因此在实验上表征它们是具有挑战性的。PI将确定光散射、低温扫描电子显微镜、中子散射和X射线散射的使用程度，从而提供对纳米簇中自聚集蛋白质的结构、动力学和稳定性的洞察。他们还将研究对这些系统进行建模的适当的多尺度策略。广泛影响如果成功，拟议的研究将为重要的基本问题提供解决方案，这些问题涉及形成大小可调的平衡纳米团簇水分散体的可能性，这些分散体在稀释后分解为单体。预期的成果包括一种创建(和调整)纳米团簇水分散体的一般方法，这些方法适用于从药物输送到生物燃料生产的各种技术，以及对为什么以前使用替代策略的尝试只能产生小、稀薄和短寿命的纳米团簇的理论理解。在这项资助中，本科生和研究生都将有机会从事重要的基础研究，这些研究具有非同寻常的强大的跨学科和技术成分。除其他外展工作外，私人投资促进机构建议通过一系列基于模拟和理论的互动模块，将项目中有关蛋白质稳定性和拥挤的科学纳入生物物理本科课程。

This INSPIRE award to University of Texas at Austin by the Interfacial Processes and Thermodynamics program in the Division of Chemical, Bioengineering, Environmental and Transport (CBET) in the Directorate for Engineering (ENG) is cofunded by the Particulate and Multiphase Processes in the CBET-ENG; and the Biomaterials program in the Division of Materials Research in the Directorate for the Mathematical and Physical Sciences. Protein-based drugs represent some of the most promising therapies for a wide range of diseases, including cancer. Subcutaneous injection is the preferred method of delivery, but its usefulness is currently limited by unwanted outcomes such as protein aggregation and gelation that occur for high doses. Previous attempts to address these problems by modifying the amino-acid sequence of potential therapeutics have been expensive and often unsuccessful. The investigators have recently reported a new method for creating highly concentrated, low-viscosity dispersions of stable protein nanoclusters that are not only of great fundamental interest but also could provide a basis for an unconventional means for solving major challenges in the protein-based therapeutics. However, at present, the answers to basic questions about the origins of the nanoclusters are lacking. Furthermore, the relationships between specific nanocluster characteristics and physical properties of the resulting dispersions are currently unknown.Intellectual MeritThe aqueous protein nanocluster dispersions to be studied in this proposal represent an entirely new form of soft condensed matter. The goal of the proposal is to explore and develop a fundamental understanding of how the protein nanoclusters form, why they stabilize the folded state of the proteins, and the impact of the clusters on the physical properties of dispersions. The investigators will use experiments, statistical mechanical theory, and computer simulations to test the hypothesis that clusters spontaneously form due to an equilibrium, self-assembly process where the addition of a small molecule ?crowder? molecule induces attractions between proteins that are balanced by weak electrostatic repulsions near the protein?s isoelectric point. In doing so, they will also address key open questions about the pathway dependence of nanocluster dispersions, the role and design of novel molecular crowders in nanocluster assembly, and the structure and dynamics of the nanoclusters. Since protein nanocluster dispersions are inherently multiscale (clusters, proteins, crowders, and solvent each introduce characteristic length scales), they are challenging to characterize experimentally. The PIs will determine the extent to which light-scattering, cryo-SEM and TEM, neutron scattering, and x-ray scattering can be used provide insights into the structure, dynamics, and stability of self-crowded proteins in the nanoclusters. They will also investigate appropriate multiscale strategies for modelingthese systems.Broader ImpactsIf successful, the proposed research will provide resolutions to important fundamental questions about the possibility of forming aqueous dispersions of equilibrium nanoclusters with tunable size that dissociate to monomers upon dilution. Anticipated outcomes include a general method for creating (and tuning the properties of) aqueous nanocluster dispersions of interest for technologies that range fromdrug delivery to biofuel production, as well as a theoretical understanding for why previous attempts using alternative strategies were only able to produce nanoclusters that were small, dilute, and short lived. In this grant, both undergraduate and graduate students will have the opportunity to work on important fundamental research with unusually strong interdisciplinary and technological components. In addition to other outreach efforts, the PIs propose to integrate the science on protein stability and crowding from the project into an undergraduate course on biological physics through a series of interactive, simulation- and theory-based modules.