Engineering photosensory proteins through the better understanding and control of proton-coupled electron transfer reactions

通过更好地理解和控制质子耦合电子转移反应来工程光感蛋白

• 批准号: 2129728
• 负责人: Brian Crane
• 金额: $ 100万
• 依托单位: Cornell University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2129728&HistoricalAwards=false
• 关键词: Engineering; photosensory; proteins; through; better

A wide range of biological processes make extensive use of electron transfer (ET) reactions, which underlie much of energy transduction and metabolism in the cell. Biological systems are well adapted to direct the movement of electrons among macromolecules while retaining the free energies necessary to drive chemical transformations. ET is often a multistep process in that reactive protein residues act as way stations to harbor electrons or their vacancies during long-range charge migration. Moreover, proton movement is often coupled to ET and the acids and bases in the vicinity of the centers undergoing reduction and oxidation (redox) can exert substantial influence over net reactivity. This project will undertake the study of proton-coupled electron transfer (PCET) in a well-developed model system of protein partners to better understand key parameters of energy transduction and metabolism in biology. Stabilizing charge separation is relevant to energy harvesting strategies, sensing technologies, and the production of stable radical pairs, the latter of which have been implicated in the sensing of Earth’s magnetic field. Better understanding of multi-step PCET in proteins will be exploited in the design of genetically encoded probe molecules and actuators. Control of cofactor redox state with light will enable the application of small fusion proteins as structural probes for cellular studies. The project will couple to an on-going program to advance the success of underserved students and engage them in scientific research. This project aims to study proton-coupled electron transfer (PCET) reactions that involve reactive protein side chains (i.e. hopping sites) to both better understand these essential processes of life and to enable the design of new photosensory proteins to be used as tools in cell and neurobiology. The investigators will apply a photoactive model system composed of zinc-porphyrin-substituted cytochrome c peroxidase and cytochrome c to investigate how management of protonation reactions impact the ability of radical-forming sites to accelerate and steer electron transfer (ET). Kinetic and structural studies will explore the reactivity of tryptophan and tyrosine residues that have been substituted by unnatural derivatives and altered by changes to interacting residues. The properties of the redox partners will be further modulated by controlling their association and their structures and reactivities will be characterized through a variety of spectroscopic and structural approaches. Experiments involving crystals at high-pressure will potentially reveal the subtle dependence of PCET on detailed bonding networks. Through both rational and selection methods the investigators will optimize photoreduction of flavin-binding Light- Oxygen- Voltage (LOV) photoreceptors to generate new genetically encoded probes for in vivo electron-spin resonance spectroscopy applications. Control of flavin PCET will also enable the design of new cryptochrome proteins that sense in altered regions of the spectrum and drive interactions that can be adopted for optogenetics. In exploring these systems, the investigators will take a comprehensive mechanistic approach that includes enzymology, spectroscopy, structural biology and computation.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

许多生物学过程都需要电子转移（ET）反应，这是细胞能量传递和代谢的基础。生物系统很好地适应于引导大分子之间的电子运动，同时保留驱动化学转化所需的自由能。ET通常是一个多步骤的过程，因为反应性蛋白残基在长距离电荷迁移期间充当容纳电子或其空位的中途站。此外，质子运动通常与ET偶联，并且在经历还原和氧化（氧化还原）的中心附近的酸和碱可以对净反应性施加实质性影响。该项目将在一个成熟的蛋白质伴侣模型系统中进行质子耦合电子转移（PCET）的研究，以更好地了解生物学中能量转导和代谢的关键参数。稳定电荷分离与能量收集策略、传感技术和稳定自由基对的产生有关，后者与地球磁场的传感有关。更好地了解蛋白质中的多步PCET将被利用在遗传编码的探针分子和致动器的设计。控制辅因子的氧化还原状态与光将使小的融合蛋白作为结构探针的细胞研究的应用。该项目将结合一个正在进行的计划，以促进服务不足的学生的成功，并让他们参与科学研究。该项目旨在研究涉及反应蛋白侧链（即跳跃位点）的质子耦合电子转移（PCET）反应，以更好地理解这些生命的基本过程，并使新的光敏蛋白的设计能够用作细胞和神经生物学的工具。 研究人员将应用由锌卟啉取代的细胞色素c过氧化物酶和细胞色素c组成的光敏模型系统，以研究质子化反应的管理如何影响自由基形成位点加速和引导电子转移（ET）的能力。动力学和结构研究将探索色氨酸和酪氨酸残基的反应性，这些残基已被非天然衍生物取代，并因相互作用残基的变化而改变。氧化还原伙伴的性质将通过控制它们的关联来进一步调节，并且它们的结构和反应性将通过各种光谱和结构方法来表征。 涉及晶体在高压下的实验将可能揭示PCET对详细的键合网络的微妙依赖。 通过理性和选择方法，研究人员将优化黄素结合光-氧-电压（LOV）光受体的光还原，以产生新的遗传编码探针，用于体内电子自旋共振光谱应用。控制黄素PCET还将使新的隐花色素蛋白的设计成为可能，这些隐花色素蛋白在光谱的改变区域中感知并驱动可用于光遗传学的相互作用。在探索这些系统的过程中，研究人员将采取一种综合的机械方法，包括酶学、光谱学、结构生物学和计算。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。