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

NSF/MCB-BSF：简约光可切换蛋白结合域的从头设计

• 批准号: 2306190
• 负责人: William DeGrado
• 金额: $ 74.08万
• 依托单位: University of California-San Francisco
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2306190&HistoricalAwards=false
• 关键词: NSF; MCB; BSF; De; novo

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.

大自然经常使用外部信号（例如光）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途径的激活/失活。光开关被证明是合成生物学中一种有吸引力的策略，可以以精确的空间和时间分辨率实现对各种细胞过程的控制。因此，光开关蛋白质的设计和工程对基础科学和合成生物学的许多应用都产生了极大的兴趣。该项目将开发和测试合理设计简约光可切换单域蛋白的新方法，这些蛋白仅在一种 PSC 状态下与特定靶蛋白特异性结合。该项目将培训博士后、研究生和本科生，包括代表性不足的少数群体成员。该项目旨在通过合理地将天然和合成光可切换发色团（PSC）的内部结合位点设计到各种小蛋白质结构域中，这些蛋白质结构域表现良好，并且可以被设计为与可变靶点相互作用，从而克服当前使用方法的局限性。在此类设计中，PSC 的结合位点将被刻入小蛋白质结构域的核心，并放置半胱氨酸或其他亲核氨基酸以实现邻近增强的共价 PSC-蛋白质连接。通过配体-蛋白质相互作用稳定蛋白质核心，蛋白质结构域的表面残基将进化为与特定的靶蛋白质结合。在光激发和 PSC 异构化后，结合域将表现出局部或整体解折叠以及随后蛋白质-蛋白质相互作用的破坏。将发色团切换回基态将导致原始构象和结合​​功能的恢复，产生稳定且可回收的光开关蛋白结合剂。此外，该项目将探索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偶联，这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。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。