NSF/MCB-BSF: De novo design of minimalistic light-switchable protein binding domains

自然界经常使用外部信号，如光，来打开/关闭蛋白质功能。植物、真菌和细菌中的光开关蛋白质在吸收特定波长的光时改变其构象。构象变化导致控制细胞运动、生长、发育和其他过程的下游途径的激活/失活。光开关被证明是一个有吸引力的策略，在合成生物学实现控制的各种细胞过程与精确的空间和时间分辨率。因此，光开关蛋白的设计和工程化对于基础科学和合成生物学中的许多应用都具有极大的兴趣。该项目将开发和测试新的方法，用于合理设计极简的光可切换单结构域蛋白，这些蛋白仅在一种PSC状态下特异性结合特定的靶蛋白。该项目将培养博士后，研究生和本科生，包括代表性不足的少数群体的成员。该项目旨在克服目前使用的方法的局限性，通过合理设计天然和合成的光可转换发色团（PSC）的内部结合位点到各种小蛋白质结构域中，这些小蛋白质结构域表现良好，并且可以被工程化以与可变靶标相互作用。在这样的设计中，PSC的结合位点将被雕刻到小蛋白质结构域的核心中，其中放置半胱氨酸或另一种亲核氨基酸以实现邻近增强的PSC-蛋白质共价连接。随着蛋白质核心通过配体-蛋白质相互作用稳定，蛋白质结构域的表面残基将进化为结合特定的靶蛋白。在光激发和PSC异构化后，结合结构域将表现出局部或整体解折叠，随后破坏蛋白质-蛋白质相互作用。将发色团切换回基态将导致原始构象和结合功能的恢复，产生稳定且可回收的光可切换蛋白质结合剂。此外，该项目将探索由蛋白质核心编码的光开关功能是否可以与单个蛋白质结构域内的结合功能相耦合，与大多数分离光传感和输出功能的天然光开关蛋白中观察到的情况相反。这个美国/以色列合作项目得到了美国国家科学基金会和以色列两国科学基金会的支持。这个奖项反映了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。

Nature frequently uses an external signal, such as light, to switch on/off protein function. Photoswitchable proteins in plants, fungi, and bacteria change their conformation upon absorption of a particular wavelength of light. The conformational change results in activation/deactivation of the downstream pathways that control cell movement, growth, development, and other processes. Photoswitching proved to be an attractive strategy in synthetic biology to achieve control of various cellular processes with precise spatial and temporal resolution. Thus, design and engineering of photoswitchable proteins present great interest for both basic science and many applications in synthetic biology. This project will develop and test new methodology for rational design of minimalistic light switchable single-domain proteins that bind specifically to a particular target protein in only one PSC state. This project will train postdoctoral, graduate, and undergraduate students including members of underrepresented minority groups. This project aims to overcome the limitations of currently used approaches by rationally designing interior binding sites for native and synthetic photoswitchable chromophores (PSCs) into various small protein domains that are well-behaved and are could be engineered to interact with variable targets. In such designs, a binding site for a PSC will be carved into the core of a small protein domain, with a cysteine or another nucleophilic amino acid placed to enable proximity enhanced covalent PSC-protein linkage. With protein core stabilized by ligand-protein interactions, the surface residues of the protein domain will be evolved to bind to a particular target protein. Upon excitation by light and isomerization of the PSC, the binding domain would exhibit local or global unfolding and subsequent disruption of the protein-protein interaction. Switching the chromophore back to the ground state would result in restoration of the original conformation and the binding function, producing a stable and recyclable light-switchable protein binder. Moreover, this project will explore whether light switching function encoded by protein core can be coupled to the binding function within a single protein domain, in contrast to what is observed in the majority of native light-switchable proteins that separate light sensing and output functions. This collaborative US/Israel project is supported by the US National Science Foundation and the Israeli Binational Science Foundation.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.