PROTEIN BEHAVIOR AT SILICONE--A SINGLE MOLECULE STUDY

界面的化学和物理性质与暴露表面吸附的黏附蛋白识别位点的分子途径之间的关系尚不清楚。揭示这种关系将为开发真正的“生物相容”材料开辟一条重要的道路。这对于具有多个识别位点的黏附蛋白来说尤其具有挑战性，众所周知，这些位点的暴露是通过构象变化来调节的。我们的假设是，长期以来困扰生物材料界的蛋白质构象与细胞行为相关的定量信息的难以获得，至少部分源于这样一个事实，即传统的分析技术平均适用于大量蛋白质。因此，我们的方法是逐个研究界面上的单个分子，而不是同时探测大量的蛋白质。这将使我们能够深入了解共存构象状态的数量，并探索作为时间函数的构象演化。我们的主要工具将是单分子光谱学与荧光共振能量转移(FRET)测量相结合，以深入了解单个蛋白质上特定位置之间的相对距离。纤维连接蛋白被选为我们的模型蛋白，因为它具有在不同条件下呈现多种构象和揭示多个识别位点的能力。拟议中的实验加上计算机模拟，将为纤维连接蛋白在界面上的结构和功能提供全新的见解。使能技术已经发展到可以应用于生物分子研究的地步，而生物分子在外科和生物工程中具有基本的重要性。这项建议概述了需要进行的实验，以探索单分子光谱学和引导分子动力学模拟分析纤维连接蛋白构象状态的能力。所获得的专业知识将立即适用于其他黏附蛋白和一般蛋白质的研究。同样重要的是，这项提案列出了一些实验，这些实验提供了关于纤维连接蛋白结合位点的暴露如何受到表面性质和机械张力调节的新信息。拟议的实验和计算工具的开发以及所获得的信息，对于学习如何为触发特定愈合反应的生物材料设计表面涂层将是至关重要的。

The relationship between the chemical and physical nature of an interface and the molecular pathway by which recognition sites of surface adsorbed adhesion proteins are exposed remains unknown. Shedding light on this relationship will open an important path for the development of truly "biocompatible" materials. This is particularly challenging for adhesion proteins, which have multiple recognition sites, and it is well known hat exposure of these sites is regulated via conformational changes. Our hypothesis is that the difficulty in deriving t quantitative information how protein conformation relates to cell behavior, which has troubled the biomaterials community for a long time, originates at least in part from the fact that conventional analytical techniques average over a large population of proteins. Therefore, our approach is to study individual molecules at interfaces one by one rather than probing a large population of proteins simultaneously. This will allow us to gain insight into the number of coexisting conformational states and to probe the conformational evolution as function of time. Our major tool will be single molecule spectroscopy combined with fluorescent resonant energy transfer (FRET) measurements to gain insight into relative distances between specific sites on a single protein. Fibronectin has been chosen as our model protein due to its ability to assume numerous conformations and to expose multiple recognition sites under different conditions. The proposed experiments complemented by computer simulations will provide completely new insights into the structure and function of fibronectin at interfaces. Enabling technology has advanced to the point that it can now be applied to the study of biomolecules that are of fundamental importance in surgery and bioengineering. This proposal outlines the experiments that need to be done to explore the power of single molecule spectroscopy and of steered molecular dynamics simulations to the analysis the conformational states of fibronectin. The expertise gained will be immediately applicable to the study of other adhesion proteins, and proteins in general. Equally important, this proposal lays out experiments that provide new information about how the exposure of fibronectin's binding sites is regulated by surface properties and mechanical tension. The development of the proposed experimental and computational tools, as well as the information derived, will be crucial for learning how to design surface coating for biomaterials that trigger specific healing responses.