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，使表面化学影响吸附蛋白质构象和分子间结合的机制研究成为可能。虽然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，但即使是最耐蛋白质的表面也允许一些蛋白质吸附。因此，本研究将验证邻近表面化学间接影响吸附后蛋白质行为从而影响分子间倾向的假设

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.