Shining Light on Metalloprotein Mechanism: Single Protein Crystal Catalytic Studies Driven by 'Caged' Electron Sources

揭示金属蛋白机制：“笼式”电子源驱动的单蛋白晶体催化研究

• 批准号: EP/V048988/1
• 负责人: Philip Ash
• 金额: $ 23.75万
• 依托单位: University of Leicester
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=EP%2FV048988%2F1
• 关键词: Shining; Light; Metalloprotein; Mechanism; Single

Early reviews of time-resolved crystallography identified the need for generalised ways of triggering reactivity. Roughly 30-50% of proteins are redox proteins, one third of all proteins contain a redox-active metal, and approximately 22% of submissions to the PDB contain a transition metal, so new methods that enable time-resolved study of redox reactions using sub-turnover techniques will have significant academic impact. Pulse radiolysis or X-ray photoreduction are not generally for protein studies, causing primary and secondary radiation damage and leading to structural ambiguity in reduced states. The methods proposed here use lower energy triggers; we anticipate future use of longer wavelength chromophores, further minimising risk of photodamage.The ambitious technical developments in this proposal have the potential to revolutionise biophysical capabilities, enabling studies of redox protein mechanism in exquisite chemical and structural detail. Combining single crystal spectroscopy, electrochemical control, and synchronous reaction initiation using a 'photo-caged' electron source we will build a platform technology with potentially transformative impact on biophysics and structural biology, and provide unprecedented possibilities to exploit time-resolved crystallographic and spectroscopic methods at national and international facilities. Thus far these methods have been largely inaccessible to 'real time' studies of redox proteins, as generalised methods to synchronise redox reactivity in the crystalline state do not exist. The methodology developed here overcomes the challenges of rapid triggering of electrochemical reactions in crystallo, whilst simultaneously allowing in situ infrared spectroscopic monitoring of transient redox species to characterise electrocatalytic reactions on sub-turnover timescales. This cutting-edge enabling technology will allow studies of previously inaccessible catalytic intermediates, driving scientific progress in biophysics, chemical and structural biology, and establishing the UK at the forefront of these unique and exciting scientific developments.

对时间分辨晶体学的早期回顾表明，需要一种触发反应性的通用方法。大约30-50%的蛋白质是氧化还原蛋白，三分之一的蛋白质含有氧化还原活性金属，大约22%的提交给PDB的蛋白质含有过渡金属，因此，使用亚转换技术对氧化还原反应进行时间分辨研究的新方法将产生重大的学术影响。脉冲辐射分解或x射线光还原通常不用于蛋白质研究，导致原发性和继发性辐射损伤，并导致还原状态下的结构模糊。本文提出的方法使用较低能量的触发器；我们预计未来会使用波长更长的发色团，进一步减少光损伤的风险。本提案中雄心勃勃的技术发展有可能彻底改变生物物理能力，使氧化还原蛋白机制的精细化学和结构细节的研究成为可能。结合单晶光谱、电化学控制和使用“光笼”电子源的同步反应引发，我们将建立一个对生物物理学和结构生物学具有潜在变革性影响的平台技术，并为在国家和国际设施中利用时间分辨晶体学和光谱方法提供前所未有的可能性。到目前为止，这些方法在很大程度上无法用于氧化还原蛋白的“实时”研究，因为在晶体状态下同步氧化还原反应的通用方法并不存在。这里开发的方法克服了在晶体中快速触发电化学反应的挑战，同时允许现场红外光谱监测瞬态氧化还原物质，以表征亚周转时间尺度上的电催化反应。这项尖端的技术将使以前无法获得的催化中间体的研究成为可能，推动生物物理学、化学和结构生物学的科学进步，并使英国站在这些独特而令人兴奋的科学发展的前沿。