NANOSCALE: Structural Changes in Fibronectin Binding Domains upon Adsorption to Well-Defined Surface Chemistries

细胞对吸附在材料表面的蛋白质的粘附性对许多生物医学和生物技术应用是至关重要的，包括生物材料、组织工程和体外细胞培养系统。细胞与胞外蛋白的黏附主要是由细胞表面受体整合素家族介导的。整合素与细胞外基质成分中特定的氨基酸序列结合。例如，整合素a5b1与纤维连接蛋白(FN)中的PHSRN和RGD结构域结合。这种相互作用由这两个结构域的相对结构取向控制，这两个结构域之间的距离为3-4 nm。每个结构域单独对结合的贡献很小，但结合在一起，它们协同结合到受体上，产生显著的粘附性增加。此外，当FN吸附到表面时，FN经历了改变5b1结合的结构变化，这意味着这些结合域的相对取向的变化。本项目重点是模拟FN(FnIII7-10)的一个特征良好的片段，它包含PHSRN和RGD结合位点，在表现出明确的表面化学性质(CH3，NH2，COOH和OH)的烷硫醇自组装单分子膜上的吸附。具体地说，创新的实验和计算方法被整合在一起来模拟PHSRN和RGD结构域的相对取向的变化，以及这些结构变化和整合素结合之间的关系。首先，将使用单抗和从分子模拟获得的结构和热力学信息来表征吸附的FnIII7-10的构象。然后分析整合素a5b1与吸附的FnIII7-10的功能结合，特征是结合强度和相对结合亲和力，并与结构参数相关联。通过将实验和计算方法相结合，这种分析将提供对蛋白质-表面相互作用和吸附诱导的蛋白质功能结构变化的作用的机制的理解。最后，建议的研究将提供一个适用于其他蛋白质/表面的分析框架。

Cell adhesion to proteins adsorbed onto material surfaces is critical to numerous biomedical and biotechnological applications, including biomaterials, tissue engineering, and in vitro cell culture systems. Cell attachment to extracellular proteins is primarily mediated by the integrin family of cell surface receptors. Integrins bind to specific amino acid sequences in extracellular matrix components. For example, integrin a5b1 binds to the PHSRN and RGD domains in fibronectin (Fn). This interaction is controlled by the relative structural orientation of these two domains, which are 3-4 nm apart. Each domain independently contributes little to binding, but in combination, they synergistically bind to the receptor to produce significant increases in adhesion. Moreover, upon adsorption to surfaces, Fn undergoes structural changes that modify a5b1 binding, suggesting changes in the relative orientation of these binding domains.This project focuses on modeling the adsorption of a well-characterized fragment of Fn (FnIII7-10), which contains the PHSRN and RGD binding sites, onto self-assembled monolayers of alkanethiols presenting well-defined surface chemistries (CH3, NH2, COOH, and OH). Specifically, innovative experimental and computational approaches are integrated to model changes in the relative orientation of the PHSRN and RGD domains and the relationship between these structural changes and integrin binding. First, the conformation of adsorbed FnIII7-10 will be characterized using monoclonal antibodies and structural and thermodynamic information obtained from molecular simulations. The functional binding of integrin a5b1 to adsorbed FnIII7-10, characterized by adhesion strength and relative binding affinity, will then be analyzed and correlated to the structural parameters. By integrating experimental and computational approaches, this analysis will provide a mechanistic understanding of protein-surface interactions and the role of adsorption-induced structural changes in protein function. Finally, the proposed research will provide an analytical framework that is applicable to other protein/surface