Tools to Connect Protein Conformations, Dynamics, and Associations

描述（由申请人提供）：这项工作的总体目标是开发新的单分子显微镜方法，这将使表面化学影响吸附蛋白质构象和分子间缔合的方式的机理研究。尽管对蛋白质吸附的抗性通常被认为是特定应用（生物传感、生物相容性等）所必需的，即使最抗蛋白质的表面也允许一些蛋白质吸附。因此，这项工作将测试的假设，邻位表面化学间接影响吸附后的蛋白质行为，以影响分子间的倾向， 关联（绑定、聚合等）。对吸附后蛋白质行为和表面介导该行为的能力的机械理解将最终导致用于各种生物医学技术的更好的表面涂层。作为一个相关的和方便的模型系统，荧光标记的纤连蛋白（Fn）将研究模型生物相容性表面以及表面，专门探测静电，亲水和疏水相互作用。这项工作将使用单分子荧光显微镜技术在固-水界面，能够同时测量单个Fn分子的分子构象，并跟踪其对动态过程的影响，如吸附，扩散，解吸，聚集和受体结合。Fn标记之间的共振能量转移将用于探测分子构象。第一个具体的目标将检查不同的表面的能力，影响Fn构象和随后的影响，这对Fn表面的亲和力和扩散。基于这种理解，第二个目标将解决蛋白质-蛋白质相互作用形成稳定蛋白质膜的倾向。预计表面通过蛋白质在表面上的流动性及其簇形成的倾向间接影响膜形成，这些性质可能取决于Fn构象。 公共卫生相关性：表面和蛋白质之间的相互作用与人类健康具有广泛的相关性，影响医学诊断、治疗性蛋白质稳定性、疫苗功效/安全性和生物材料设计。一个共同的技术目标涉及制备“抗蛋白质”表面。然而，蛋白质抵抗的机制仍然难以捉摸，即使是最生物相容的表面也不能完全防止粘连。为了解决这种不一致性，这项工作将开发新的显微镜技术来测试这一假设，即生物相容性表面允许蛋白质粘附，这些蛋白质不太可能引发不良反应，也可能影响它们的粘附后行为，以防止它们采取不必要的行为。对表面在生物相容性中的作用的更全面的理解将导致生物传感器、植入装置和生物实验设备的改进表面的设计。

DESCRIPTION (provided by applicant): The overall objective of this work is to develop novel single molecule microscopy methods that will enable mechanistic studies of the ways in which surface chemistry affects adsorbed protein conformation and intermolecular associations. Although resistance to protein adsorption is often cited as necessary for a particular application (biosensing, biocompatibility, etc.), even the most protein- resistant surfaces permit some protein adsorption. Therefore, this work will test the hypothesis that vicinal surface chemistry indirectly affects protein behavior after adsorption to influence the propensity for intermolecular associations (binding, aggregation, etc.). A mechanistic understanding of post- adsorptive protein behavior and the ability of the surface to mediate this behavior will ultimately lead to better surface coatings for a variety of biomedical technologies. As a relevant and convenient model system, fluorescently-labeled fibronectin (Fn) will be studied on model biocompatible surfaces as well as surfaces that specifically probe electrostatic, hydrophilic and hydrophobic interactions. This work will use single-molecule fluorescence microscopy techniques at the solid-aqueous interface that are capable of simultaneously measuring the molecular conformation of an individual Fn molecule and tracking its effect on dynamic processes such as adsorption, diffusion, desorption, aggregation, and receptor binding. Resonance energy transfer between Fn labels will be used to probe molecular conformation. The first specific aim will examine the ability of different surfaces to influence Fn conformation and the subsequent effect this has on Fn surface affinity and diffusion. Building on this understanding, the second aim will address protein-protein interactions for their propensity to form a stable protein film. The surfac is expected to indirectly influence film formation through the mobility of proteins on the surface and their propensity for cluster formation, properties that likely depend on Fn conformation. PUBLIC HEALTH RELEVANCE: Interactions between surfaces and proteins have widespread relevance to human health, affecting medical diagnostics, therapeutic protein stability, vaccine efficacy/safety, and biomaterial design. A common technological goal involves the preparation of "protein- resistant" surfaces. However, the mechanisms of protein resistance remain elusive, and even the most biocompatible surfaces fail to prevent adhesion entirely. To resolve this inconsistency, this work will develop new microscopy techniques to test the hypothesis that biocompatible surfaces permit adhesion of proteins that are unlikely to trigger an adverse response and may also influence their post-adhesion behavior to prevent them from adopting unwanted behavior. A more complete understanding of the role of the surface in biocompatibility will lead to the design of improved surfaces for biosensors, implanted devices and equipment for biological experimentation.