Single-Molecule Optical Probes of Protein Biophysics

蛋白质生物物理学的单分子光学探针

• 批准号: 0212503
• 负责人: William Moerner
• 金额: $ 33万
• 依托单位: Stanford University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2005-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0212503&HistoricalAwards=false
• 关键词: Single; Molecule; Optical; Probes; Protein

The objective of this project is to study protein and enzyme dynamics and function using optical single-molecule detection and spectroscopy with in vitro and in vivo techniques. The central goals of this research are to utilize diffraction-limited confocal, total internal reflection, and wide-field epifluorescence optical microscopy and spectroscopy to attack three primary thrust areas: (a) characterization and understanding of the photophysical behavior of several red-emitting autofluorescent proteins at the single-molecule level, proteins which are key elements in a wide array of in vivo cellular labeling and gene expression studies, (b) development of reliable and reproducible methods for specific surface attachment of functional proteins to transparent surfaces, so that unwanted proteins do not stick to the surface in single-molecule studies, and (c) extension of recent success in observing single transmembrane proteins in living cells to establish new methods and protocols for observing single molecules in vivo. In all of these studies, the fact that individual copies of proteins will be studied, one at a time, provides an additional critical source of information about the presence and extent of heterogeneity in these complex systems.To accomplish these ends, physical, chemical, and optical expertise will be combined with the molecular biological and biochemical expertise of several collaborators. Owing to its multidisciplinary organization, the fundamental research in this program will not only generate new knowledge about the biophysical properties of important protein systems, but, in addition the advances in instrumentation will provide novel groundwork for technology transfer to other relevant disciplines. Furthermore, the increased knowledge about both optically driven and spontaneous changes in the emissive state of fluorescent proteins may lead to design of mutants with improved properties for application in biology and other fields. At the same time, the broadly-based multidisciplinary environment which has been assembled to perform this research will provide excellent training and education opportunities for the next generation of biophysical scientists. This project is supported by the Molecular Biophysics Program in the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences and the Divisions of Physics and Chemistry in the Mathematical and Physical Sciences Directorate.

本项目的目的是利用光学单分子检测和光谱学以及体外和体内技术研究蛋白质和酶的动力学和功能。 这项研究的中心目标是利用衍射限制共焦，全内反射和宽场落射荧光光学显微镜和光谱学来攻击三个主要的推力领域：（a）在单分子水平上表征和理解几种发红光的自体荧光蛋白的生物物理行为，蛋白质，其是广泛的体内细胞标记和基因表达研究中的关键元件，（B）开发用于将功能蛋白质特异性表面附着到透明表面的可靠和可重复的方法，因此，在单分子研究中，不需要的蛋白质不会粘附在表面上，以及（c）扩大最近在观察活细胞中的单跨膜蛋白质方面的成功，以建立在体内观察单分子的新方法和方案。 在所有这些研究中，蛋白质的单个拷贝将被研究，一次一个，这一事实为这些复杂系统中异质性的存在和程度提供了额外的关键信息来源。为了实现这些目标，物理，化学和光学专业知识将与几个合作者的分子生物学和生物化学专业知识相结合。由于其多学科组织，该计划的基础研究不仅将产生有关重要蛋白质系统的生物物理特性的新知识，而且仪器的进步将为技术转移到其他相关学科提供新的基础。此外，增加知识的光驱动和自发变化的荧光蛋白的发射状态可能会导致设计的突变体具有改善的性能，在生物学和其他领域的应用。 与此同时，为开展这项研究而建立的基础广泛的多学科环境将为下一代生物物理科学家提供极好的培训和教育机会。该项目得到了生物科学理事会分子和细胞生物科学部分子生物物理学计划以及数学和物理科学理事会物理和化学部的支持。