Opportunistic complexation and mesoscopic aggregates in protein solutions

标题：蛋白质溶液中的机会络合和介观聚集体蛋白质聚集是生物物理学和其他生命科学的中心问题。本研究的重点是一种特别令人费解的蛋白质聚集类型，在这种聚集过程中，富含蛋白质的内含物在蛋白质溶液中形成。这些内含物的大小为微米或更小，被称为“介观星团”。尽管它们的体积很小，但这些簇是有序蛋白质固体（如晶体和镰状细胞性贫血纤维）必不可少的成核位点。介观团簇也与热力学的标准概念不一致，热力学的标准概念规定这样的团簇要么要大得多，要么根本不应该存在。了解介观团簇的分子起源将解决热力学的一个主要基本问题。这项研究将验证介观簇是由单个蛋白质分子组成的长寿命复合物形成的假设。将采用先进的实验技术、理论建模和计算机模拟相结合的方法来检验这一假设。了解蛋白质聚集对生物学的各个方面以及生物技术和健康都有影响。在更广泛的影响活动的核心是培训研究生，本科生，特别是高中生。该项目是一项涉及物理、化学、生物等多个学科的跨学科研究，为参与研究的学生提供了进一步学术研究的良好平台。提出的工作的微观假设是介观簇源于瞬态含蛋白复合物的形成。复合物在高蛋白密度下稳定。与散装溶液相比，络合物是团簇内主要的含蛋白质种类。在一个稳态簇中，以单体形式流入的蛋白质与以复合物形式流出的蛋白质正好平衡。复合体的性质取决于蛋白质是单体的，如溶菌酶，还是低聚的，如血红蛋白，通常是四聚体蛋白质。本研究的研究人员假设，对于典型的单体蛋白质，复杂的形成伴随着部分蛋白质展开和可能的结构域交换。在低聚蛋白质的情况下，复合物是含有非典型数量的单个单体的低聚物。该假说的一个核心方面是，这些复合物是机会主义的；它们代表了将单个蛋白质分子短暂结合在一起的非典型方式。研究团队将利用物理化学和生物化学实验技术（动态光散射、布朗显微镜、质谱、酰胺交换核磁共振、纯流）和理论工具（采用粗粒度能量函数和经典密度泛函理论的分子建模）相结合，建立络合的身份和机制。他们将测试星团是否是真正的稳态物体，并探索星团形成有一个缓慢的、不可逆的组成部分的可能性。这项研究的这一部分可能会回答这样一个问题：集群的成熟是根据奥斯特瓦尔德式的情景，还是涉及不同的机制。

Title: Opportunistic complexation and mesoscopic aggregates in protein solutionProtein aggregation is a central problem of biophysics and other life sciences. This investigation focuses on a particularly puzzling type of protein aggregation during which protein-rich inclusions form in protein solutions. These inclusions are a micron or less in size and have been called the "mesoscopic clusters". Despite their small volume, the clusters are essential nucleation sites for ordered protein solids such as crystals and sickle cell anemia fibers. The mesoscopic clusters are also at odds with standard notions of thermodynamics, which dictates that such clusters should be either much larger or should not exist at all. Understanding the molecular origin of the mesoscopic clusters will resolve a major fundamental question of thermodynamics. This research will test the hypothesis that the mesoscopic clusters are caused by formation of long-lived complexes made up of individual protein molecules. A combination of advanced experimental techniques, theoretical modeling, and computer simulations will be employed to test this hypothesis. Understanding protein aggregation has implications in all facets of biology as well as on biotechnology and health. At the core of the broader impact activities is training of graduate, undergraduate, and, in particular, high school students. The project is a multidisciplinary study that spans many topics in physics, chemistry, and biology and represents a great platform for further academic endeavors of the involved students.The microscopic hypothesis underlying the proposed work is that the mesoscopic clusters stem from the formation of transient protein-containing complexes. The complexes are stabilized at high protein densities. In contrast with the bulk solution, the complexes are the dominant protein-containing species inside the clusters. In a steady-state cluster, the influx of protein in the form of monomers is exactly balanced by the outflow of protein in the form of complexes. The nature of the complex depends on whether the protein is monomeric, as is lysozyme, or oligomeric, as is hemoglobin, ordinarily a tetrameric protein. The investigators in this study hypothesize that for typically monomeric proteins, complex formation is accompanied by partial protein unfolding and, possibly, domain swapping. In the case of oligomeric proteins, the complexes are oligomers that contain an untypical number of individual monomers. A core aspect of the hypothesis is that the complexes are opportunistic; they represent untypical ways to transiently bind individual protein molecules together. The research team will establish the identity and mechanisms of the complexation using a combination of physicochemical and biochemical experimental techniques (dynamic light scattering, Brownian microscopy, mass spectroscopy, amide exchange NMR, sheer flow) and theoretical tools (molecular modeling employing coarse-grained energy functions and the classical density functional theory). They will test whether the clusters are truly steady-state objects and explore the possibility that cluster formation has a slow, irreversible component. This part of the proposed study may answer the question whether clusters ripen according to an Ostwald-like scenario, or a different mechanism is involved.