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，来开启/关闭蛋白质功能。植物、真菌和细菌中的可光切换蛋白质在吸收特定波长的光后会改变其构象。构象变化导致控制细胞运动、生长、发育和其他过程的下游通路的激活/失活。光开关被证明是合成生物学中一种很有吸引力的策略，可以实现对各种细胞过程的精确时空分辨率控制。因此，光开关蛋白的设计和工程在基础科学和合成生物学中的许多应用都引起了极大的兴趣。该项目将开发和测试合理设计最低限度光开关单域蛋白的新方法，这些蛋白仅在一种PSC状态下与特定的目标蛋白特异结合。该项目将培养博士后、研究生和本科生，包括未被充分代表的少数群体成员。该项目旨在克服目前使用的方法的局限性，通过合理地将天然和合成的可光开关发色团(PSC)的内部结合位点设计成各种小的蛋白质结构域，这些小蛋白结构域行为良好，并且可以被设计成与可变靶标相互作用。在这种设计中，PSC的结合部位将被雕刻到一个小的蛋白质结构域的核心，并放置半胱氨酸或另一个亲核氨基酸，以实现邻近增强的PSC-蛋白质共价连接。随着蛋白质核心被配体-蛋白质相互作用稳定，蛋白质结构域的表面残基将进化为与特定的靶蛋白结合。在光的激发和PSC的异构化后，结合结构域会表现出局部或整体的展开和随后的蛋白质-蛋白质相互作用的破坏。将发色团切换回基态将导致恢复原来的构象和结合功能，产生稳定的、可回收的光开关蛋白结合剂。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中的结合功能偶联，而不是在大多数天然的光开关蛋白质中观察到的分离光感知和输出功能的情况。这一美国/以色列合作项目得到了美国国家科学基金会和以色列双国科学基金会的支持。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.