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

这项INSPIRE奖由美国工程局化学、生物工程、环境和运输部门（CBET）的界面过程和热力学项目授予德克萨斯大学奥斯汀分校，由CBET-ENG的颗粒和多相过程项目共同资助；以及数学和物理科学理事会材料研究部的生物材料项目。以蛋白质为基础的药物代表了包括癌症在内的一系列疾病的一些最有希望的治疗方法。皮下注射是首选的给药方法，但其有效性目前受到诸如高剂量时发生的蛋白质聚集和凝胶化等不良后果的限制。以前试图通过修改潜在治疗药物的氨基酸序列来解决这些问题的尝试是昂贵的，而且往往不成功。研究人员最近报告了一种新方法，用于制造高度浓缩、低粘度的稳定蛋白质纳米团簇分散体，这不仅具有重大的基础意义，而且可以为解决基于蛋白质的治疗中的主要挑战提供一种非常规方法的基础。然而，目前，关于纳米团簇起源的基本问题还缺乏答案。此外，特定的纳米团簇特征与所产生的分散体的物理性质之间的关系目前尚不清楚。本文所研究的水性蛋白质纳米团簇分散体代表了一种全新形式的软凝聚态物质。该提案的目标是探索和发展对蛋白质纳米团簇如何形成的基本理解，为什么它们稳定蛋白质的折叠状态，以及团簇对分散体物理性质的影响。研究人员将使用实验、统计力学理论和计算机模拟来验证这一假设，即簇的自发形成是由于一个平衡的自组装过程，在这个过程中，一个小分子的加入？分子诱导蛋白质之间的吸引力，通过蛋白质附近的弱静电排斥来平衡。S等电点。在此过程中，他们还将解决一些关键的开放性问题，如纳米团簇分散的途径依赖性、新型分子聚集剂在纳米团簇组装中的作用和设计，以及纳米团簇的结构和动力学。由于蛋白质纳米团簇分散本身是多尺度的（团簇、蛋白质、聚合体和溶剂各自引入特征长度尺度），因此在实验上对它们进行表征具有挑战性。pi将决定光散射、低温扫描电镜和透射电镜、中子散射和x射线散射的使用程度，这些方法可以深入了解纳米团簇中自拥挤蛋白质的结构、动力学和稳定性。他们还将研究适当的多尺度策略来模拟这些系统。更广泛的影响：如果成功，拟议的研究将解决有关形成具有可调尺寸的平衡纳米团簇的水分散体的可能性的重要基本问题，这些平衡纳米团簇在稀释后解离成单体。预期的结果包括创造（和调整）水纳米团簇分散体的一般方法，用于从药物输送到生物燃料生产的各种技术，以及为什么以前使用替代策略的尝试只能产生小的、稀释的、短寿命的纳米团簇的理论理解。在这项资助下，本科生和研究生都将有机会从事具有不同寻常的跨学科和技术成分的重要基础研究。除了其他拓展工作外，pi还建议通过一系列互动、模拟和基于理论的模块，将项目中关于蛋白质稳定性和拥挤的科学整合到生物物理学的本科课程中。

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.