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

时间分辨晶体学的早期回顾确定了触发反应性的一般方法的需要。大约30 - 50%的蛋白质是氧化还原蛋白质，三分之一的蛋白质含有氧化还原活性金属，大约22%的PDB提交含有过渡金属，因此使用亚周转技术进行氧化还原反应时间分辨研究的新方法将产生重大的学术影响。脉冲辐解或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，并导致还原状态的结构模糊。这里提出的方法使用较低的能量触发器;我们预计未来使用较长波长的发色团，进一步降低光损伤的风险。这一提议中雄心勃勃的技术发展有可能彻底改变生物物理能力，使氧化还原蛋白质机制的研究在精致的化学和结构细节。结合单晶光谱学，电化学控制和使用“光笼”电子源的同步反应引发，我们将建立一个平台技术，对生物物理学和结构生物学具有潜在的变革性影响，并提供前所未有的可能性，在国家和国际设施中利用时间分辨晶体学和光谱学方法。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质的"真实的时间"研究，因为不存在使结晶状态下的氧化还原反应性同步的通用方法。在这里开发的方法克服了快速触发的电化学反应在crystallo的挑战，同时允许在原位红外光谱监测的瞬态氧化还原物种，以避免在子营业额时间尺度上的电催化反应。这种尖端的使能技术将允许对以前无法获得的催化中间体进行研究，推动生物物理学，化学和结构生物学的科学进步，并使英国处于这些独特而令人兴奋的科学发展的最前沿。

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.