Single-Molecule Optical Probes of Protein Biophysics

本项目的目标是利用光学单分子检测和光谱分析技术，结合体外和体内技术，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受限共聚焦、全内反射和广域荧光光学显微镜和光谱学来攻击三个主要方面：(A)在单分子水平上表征和了解几种红色发射的自体荧光蛋白质的光物理行为，这些蛋白质是广泛的体内细胞标记和基因表达研究的关键元素；(B)开发可靠和可重复性的方法，将功能蛋白质特定的表面附着到透明表面，从而使不需要的蛋白质在单分子研究中不会粘在表面上，以及(C)扩大最近在观察活细胞中单一跨膜蛋白方面的成功，以建立观察体内单一分子的新方法和新方案。在所有这些研究中，蛋白质的单个拷贝将被一次研究，这一事实提供了关于这些复杂系统中异质性的存在和程度的额外的关键信息来源。为了实现这些目的，物理、化学和光学专业知识将与几个合作者的分子生物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关于重要蛋白质系统的生物物理性质的新知识，而且仪器方面的进展将为技术转移到其他相关学科提供新的基础。此外，对荧光蛋白发射状态的光驱动和自发变化的了解的增加可能会导致设计出具有更好性能的突变体，应用于生物学和其他领域。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学家提供极好的培训和教育机会。该项目得到了生物科学局分子和细胞生物科学部的分子生物物理学计划以及数学和物理科学局的物理和化学部的支持。

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.