First Principles Studies and Design of Photoswitchable Fluorescent Proteins

光开关荧光蛋白的第一原理研究和设计

• 批准号: 7911287
• 负责人: Christine Isborn
• 金额: $ 4.56万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7911287
• 关键词: Binding; Biological; Cells; Color; Engineering; Fluorescence; Goals; Image; Imaging Techniques; Knowledge; Lead; Life; Light; Lighting; Methods; Microscopy; Modeling; Process; Proteins; Research Design; Research Project Grants; Resolution; Solvents; Specimen; Structure; Techniques; Tissues; Work; absorption; design; improved; molecular mechanics; nanoscale; public health relevance; quantum; simulation

DESCRIPTION (provided by applicant): The goal of the proposed research project is to provide understanding of the mechanism of photoswitching in fluorescent proteins and to aid in the rational design of new and improved fluorescent proteins. This class of proteins has the ability to be reversibly switched between the 'on' fluorescent bright state and the 'off' dark state via the application of light. These photoswitchable fluorescent proteins have led to revolutionary new imaging techniques that can resolve biological structures on the nanometer scale. Because a low level of illumination is required, these techniques are also much less phototoxic than conventional fluorescence methods, and thus show significant promise for imaging live cells and tissues. For these superresolution microscopy methods to become a reality for imaging living cells, there is a great need for engineering new photoswitchable FPs that emit in a range of colors, are bright, and have tunable switching rates. However, the current understanding of how photoswitching occurs in these proteins is limited. Detailed knowledge of the photoswitching mechanism will undoubtedly aid in the rational design and optimization of new bright photoswitches with a variety of emission colors and switching rates. The proposed work will use quantum dynamic and molecular mechanics simulations of the protein and solvent to model the fluorescent 'on' and 'off' states, and the process of switching between these states. Upon understanding the common mechanistic motif in the known photoswitches, simulations with changes to the protein will be studied to predict possible new photoswitchable fluorescent proteins with shifted absorption/emission maxima, quantum yield, and/or switching rate. PUBLIC HEALTH RELEVANCE: With improved photoswitchable fluorescent proteins, new, noninvasive imaging microscopy techniques will be able to visualize processes in live cells and tissues with unprecedented resolution. This will lead to a deeper understanding of dynamic molecular interactions in biological specimens.

描述（由申请人提供）：拟议研究项目的目标是提供对荧光蛋白中光开关机制的理解，并帮助合理设计新的和改进的荧光蛋白。这类蛋白质具有通过施加光在“开”荧光亮态和“关”暗态之间可逆切换的能力。这些可光转换的荧光蛋白导致了革命性的新成像技术，可以在纳米尺度上解析生物结构。由于需要低水平的照明，这些技术也比传统的荧光方法少得多的光毒性，因此显示出对活细胞和组织成像的显著前景。为了使这些超分辨率显微镜方法成为活细胞成像的现实，非常需要设计新的光可切换FP，这些FP发射一系列颜色，明亮，并且具有可调的切换速率。然而，目前对这些蛋白质中光开关如何发生的理解是有限的。详细的知识的光开关机制无疑将有助于合理设计和优化新的明亮的光开关与各种发射颜色和开关速率。拟议的工作将使用蛋白质和溶剂的量子动力学和分子力学模拟来模拟荧光“开”和“关”状态，以及这些状态之间的切换过程。在了解已知的光开关中的常见机制基序后，将研究具有蛋白质变化的模拟，以预测具有偏移的吸收/发射最大值、量子产率和/或切换速率的可能的新的光可切换荧光蛋白。 公共卫生关系：随着光开关荧光蛋白的改进，新的非侵入性成像显微镜技术将能够以前所未有的分辨率可视化活细胞和组织中的过程。这将导致更深入地了解生物标本中的动态分子相互作用。