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: 6385798
• 负责人: VIOLA VOGEL
• 金额: $ 26.35万
• 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/6385798
• 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粘附蛋白的识别位点暴露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“生物相容性”材料开辟一条重要道路。这对于具有多个识别位点的粘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之间的定量信息难以获得，这至少部分源于传统分析技术对大量蛋白质的平均处理。因此，我们的方法是在界面上一个接一个地研究单个分子，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构象状态的数量，并探索构象随时间的演变。我们的主要工具将是单分子光谱结合荧光共振能量转移（FRET）测量，以深入了解单个蛋白质上特定位点之间的相对距离。选择纤维连接蛋白作为我们的模型蛋白是因为它具有多种构象和在不同条件下暴露多个识别位点的能力。本文提出的实验辅以计算机模拟，将为界面处纤维连接蛋白的结构和功能提供全新的见解。使能技术已经发展到现在可以应用于生物分子的研究，这在外科和生物工程中是至关重要的。本提案概述了需要做的实验，以探索单分子光谱和定向分子动力学模拟的力量来分析纤维连接蛋白的构象状态。所获得的专业知识将立即适用于其他粘附蛋白的研究，以及一般的蛋白质。同样重要的是，该提案提出的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特性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，对于学习如何设计触发特定愈合反应的生物材料表面涂层至关重要。