Protein structure analysis by robust electron spin resonance at the single molecule level.

本项目旨在演示用单分子电子自旋光谱分析蛋白质的结构和动力学。这一雄心勃勃的目标已经通过申请者最近的联合工作得以实现：利用金刚石中附近的氮空位（NV）色中心作为传感器，用稳定的有机电子自旋标记单个蛋白质的ESR检测[F]。施，等。[j].中国科学：自然科学版，2015,33(5)。该项目将克服在这个原理验证实验中遇到的两个主要问题：所涉及的自旋标签的低稳定性和低效检测。这将通过两种方法来实现：低温下NV传感器的单光子谐振和双光子激发；使用更具弹性的标签和纳米光子结构在室温下增强读出。该项目还将制定在传感器表面制备单位点特异性标记蛋白质和通过多维光谱确定结构的协议。它将最终结合所有这些发展，使稳定和有效的光谱单分子的电子自旋共振，特别是三维结构分析多重标记的蛋白质。

The project aims to demonstrate analysis of structure and dynamics of a protein by single-molecule electron spin spectroscopy. This ambitious goal has come within reach by recent joint work of the applicants: ESR detection of a single protein, labeled by a stable organic electron spin, using a nearby Nitrogen-Vacancy (NV) color center in diamond as a sensor [F. Shi, et al., Science 347, 1135 (2015)]. The project will overcome two major problems encountered in this proof-of-principle experiment: low stability of the spin labels involved and inefficient detection. This shall be accomplished by two approaches: resonant single-photon and two-photon excitation of the NV sensor at cryogenic temperature; use of more resilient labels and nano-photonic structures for readout enhancement at room temperature.The project will equally develop protocols for preparation of single site-specifically labeled proteins on the sensor surface and structure determination by multidimensional spectroscopy. It will finally combine all these developments to enable stable and efficient spectroscopy of single molecules by electron spin resonance, in particular three-dimensional structure analysis of multiply labeled proteins.