Protein/Chromphore Interactions via Protein Design: Interrogation and Application

通过蛋白质设计的蛋白质/发色团相互作用：询问和应用

• 批准号: 10438821
• 负责人: BABAK BORHAN
• 金额: $ 30.65万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-20 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10438821
• 关键词: 11 cis Retinal; Active Sites; Aldehydes; Area; Behavior; Binding; Binding Proteins; Biological; Biological Phenomena; Characteristics; Charge; Chemicals; Color Perception; Complex; Crystallization; Development; Electrostatics; Engineering; Environment; Event; Evolution; Family; Fluorescence; Fluorochrome; Goals; Grant; Image; Imines; Investigation; Ions; Isomerism; Kinetics; Light; Lysine; Marriage; Microscopy; Modality; Modeling; Molecular Biology; Mutation; Opsin; Output; Photosynthesis; Pigments; Process; Production; Property; Protein Engineering; Protein Family; Proteins; Proton Pump; Protons; Regulation; Research; Resistance; Retina; Rhodopsin; Schiff Bases; Series; Structure; System; Technology; Translating; Visible Radiation; Vision; Work; absorption; base; chromophore; crystallinity; deprotonation; design; experience; fatty acid-binding proteins; fluorophore; irradiation; live cell imaging; member; optical spectra; photo switch; physical property; programs; protonation; rational design; retinylidene chromophore; sensor; single molecule; spatiotemporal; tool; ultra high resolution

Project Summary We began our program by re-engineering members of the Fatty Acid Binding protein family to be mimics of rhodopsin, through rational design principles. In the process of achieving these goals we became convinced that the characteristics of these fantastically stable proteins rendered them ideal vehicles for a variety of other applications. With their small size, large binding pocket that could accommodate a variety of unrelated structures, high expression yield, resistance to structural misfolding due to mutations, and propensity for crystallization, we found these proteins as ideal tools for sensor and imaging applications. The story of their development into unique fluorescent proteins continues with this proposal as a result of two fundamentally important observations, translated to the two major aims of this grant. First, having suitably engineered the binding pocket, and chosen the appropriate chromophore as a partner, we demonstrate the creation of a protein/chromophore complex as an imine, which upon photo-irradiation experiences Excited State Proton Transfer (ESPT) to generate the iminium. Critical to the design of the chromophore is that iminium protonation generates a highly conjugated system capable of Intramolecular Charge Transfer (ICT). ICT fluorophores are typically red-shifted and highly fluorescent. Thus, photo-irradiation of a blue absorbing complex leads to an excited red- shifting species, which fluoresces with a Large Stokes Shift (LSS). Second, we have realized the ability to create a parallel suite of photo-switchable fluorochromes, where the fluorescence output can be rapidly and photo-chemically switched between `ON' and `OFF' states. Such fluorescent systems are the essential tools required for ultra-high resolution microscopy, a technology that has the potential to revolutionize our understanding of biological phenomena if the proper fluorochromes can be developed. They are also essential in biological imaging applications that require spatio-temporal control. The approach to these goals involves the precise, structure-based design and optimization of both protein and fluorophore to find the ideal system. We will optimize ESPT of a protein/chromophore complex as a photobase, a photoacid, and also in what we suggest to be a dual-ESPT mode, requiring both photoacid and photobase activity during the single photo-excitation event. This would convert a ground state neutral imine to a zwitterionic, highly polarized, conjugated excited state that will emit far in the red from the wavelength of excitation. The design of photo- switching fluorophores married to the appropriate protein environment that supports and promotes the structural change in the chromophore, will optimize the characteristics necessary for obtaining a desired photo-switch, such as red-shifted emission and high brightness. Furthermore, `ON' and `OFF' kinetics will be optimized, as rapid rates are advantageous in many imaging applications.

项目总结

项目成果

Engineering of a Red Fluorogenic Protein/Merocyanine Complex for Live-Cell Imaging.

用于活细胞成像的红色荧光蛋白/部花青复合物的工程。

• DOI: 10.1002/cbic.201900428
• 发表时间: 2020
• 期刊: Chembiochem : a European journal of chemical biology
• 作者: Santos,ElizabethM;Berbasova,Tetyana;Wang,Wenjing;Salmani,RaheleEsmatpour;Sheng,Wei;Vasileiou,Chrysoula;Geiger,JamesH;Borhan,Babak
• 通讯作者: Borhan,Babak

Free-Energy-Based Protein Design: Re-Engineering Cellular Retinoic Acid Binding Protein II Assisted by the Moveable-Type Approach.

基于自由能的蛋白质设计：在可移动型方法的辅助下重新设计细胞视黄酸结合蛋白 II。

• DOI: 10.1021/jacs.7b10368
• 发表时间: 2018
• 期刊: Journal of the American Chemical Society
• 影响因子: 15
• 作者: Zhong,HaizhenA;Santos,ElizabethM;Vasileiou,Chrysoula;Zheng,Zheng;Geiger,JamesH;Borhan,Babak;MerzJr,KennethM
• 通讯作者: MerzJr,KennethM

Steric effects in light-induced solvent proton abstraction.

• DOI: 10.1039/d0cp03037f
• 发表时间: 2020-09-21
• 期刊: Physical chemistry chemical physics : PCCP
• 作者: Lahiri J ;Moemeni M ;Magoulas I ;Yuwono SH ;Kline J ;Borhan B ;Piecuch P ;Jackson JE ;Blanchard GJ ;Dantus M
• 通讯作者: Dantus M

A structural explanation for the mechanism and specificity of plant branching enzymes I and IIb.

• DOI: 10.1016/j.jbc.2021.101395
• 发表时间: 2022-01
• 期刊: The Journal of biological chemistry
• 作者: Gavgani HN;Fawaz R;Ehyaei N;Walls D;Pawlowski K;Fulgos R;Park S;Assar Z;Ghanbarpour A;Geiger JH
• 通讯作者: Geiger JH

Intramolecular Relaxation Dynamics Mediated by Solvent-Solute Interactions of Substituted Fluorene Derivatives. Solute Structural Dependence.

• DOI: 10.1021/acs.jpcb.1c06475
• 发表时间: 2021-11-18
• 期刊: JOURNAL OF PHYSICAL CHEMISTRY B
• 影响因子: 3.3
• 作者: Capistran, Briana A.;Yuwono, Stephen H.;Moemeni, Mehdi;Maity, Soham;Vahdani, Aria;Borhan, Babak;Jackson, James E.;Piecuch, Piotr;Dantus, Marcos;Blanchard, G. J.
• 通讯作者: Blanchard, G. J.