PROTEIN BEHAVIOR AT SILICONE--A SINGLE MOLECULE STUDY

蛋白质在有机硅中的行为——单分子研究

• 批准号: 6180263
• 负责人: VIOLA VOGEL
• 金额: $ 26.81万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-01-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6180263
• 关键词: binding sites; bioengineering /biomedical engineering; biological signal transduction; biomaterial interface interaction; cell adhesion molecules; computer simulation; conformation; fibronectins; fluorescence resonance energy transfer; fluorescent dye /probe; ionophores; mathematical model; molecular dynamics; molecular site; nanotechnology; protein sequence; protein structure; protein structure function

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.

界面的化学和物理性质与表面吸附的粘附蛋白的识别位点暴露的分子途径之间的关系仍然未知。阐明这种关系将为开发真正的“生物相容性”材料开辟一条重要的道路。 这对于具有多个识别位点的粘附蛋白而言尤其具有挑战性，并且众所周知，这些位点的暴露通过构象变化来调节。 我们的假设是，在推导t蛋白质构象如何与细胞行为相关的定量信息方面的困难，这已经困扰了生物材料界很长一段时间，至少部分源于这样一个事实，即传统的分析技术平均超过了大量的蛋白质。 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蛋白质。 这将使我们能够深入了解共存的构象状态的数量，并探讨作为时间的函数的构象演变。我们的主要工具将是单分子光谱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点之间的相对距离。 纤连蛋白被选为我们的模型蛋白，因为它能够呈现多种构象，并在不同条件下暴露多个识别位点。 所提出的实验辅以计算机模拟将提供全新的见解纤连蛋白的结构和功能的接口。 使能技术已经发展到现在可以应用于研究在外科手术和生物工程中具有根本重要性的生物分子。 该提案概述了需要进行的实验，以探索单分子光谱学和操纵分子动力学模拟在分析纤连蛋白构象状态中的作用。 所获得的专业知识将立即适用于其他粘附蛋白和一般蛋白质的研究。同样重要的是，该提案列出了实验，提供了关于纤维连接蛋白结合位点的暴露如何受表面性质和机械张力调节的新信息。 所提出的实验和计算工具的发展，以及所获得的信息，将是至关重要的学习如何设计表面涂层的生物材料，触发特定的愈合反应。