Dmitrii M. Nikolaev, Vladimir N. Mironov, Ekaterina M. Metelkina, Andrey A. Shtyrov, Andrey S. Mereshchenko, Nikita A. Demidov, Sergey Yu. Vyazmin, Tatiana B. Tennikova, Svetlana E. Moskalenko, Stanislav A. Bondarev, Galina A. Zhouravleva, Andrey V. Vasin, Maxim S. Panov, Mikhail N. Ryazantsev
{"title":"基于远红弓形虫核素-3 的荧光基因编码电压指示器的合理设计:从阐明弓形虫的荧光机制到新型红移变体","authors":"Dmitrii M. Nikolaev, Vladimir N. Mironov, Ekaterina M. Metelkina, Andrey A. Shtyrov, Andrey S. Mereshchenko, Nikita A. Demidov, Sergey Yu. Vyazmin, Tatiana B. Tennikova, Svetlana E. Moskalenko, Stanislav A. Bondarev, Galina A. Zhouravleva, Andrey V. Vasin, Maxim S. Panov, Mikhail N. Ryazantsev","doi":"10.1021/acsphyschemau.3c00073","DOIUrl":null,"url":null,"abstract":"Genetically encoded voltage indicators (GEVIs) have found wide applications as molecular tools for visualization of changes in cell membrane potential. Among others, several classes of archaerhodopsin-3-based GEVIs have been developed and have proved themselves promising in various molecular imaging studies. To expand the application range for this type of GEVIs, new variants with absorption band maxima shifted toward the first biological window and enhanced fluorescence signal are required. Here, we integrate computational and experimental strategies to reveal structural factors that distinguish far-red bright archaerhodopsin-3-based GEVIs, Archers, obtained by directed evolution in a previous study (McIsaac et al., <i>PNAS,</i> 2014) and the wild-type archaerhodopsin-3 with an extremely dim fluorescence signal, aiming to use the obtained information in subsequent rational design. We found that the fluorescence can be enhanced by stabilization of a certain conformation of the protein, which, in turn, can be achieved by tuning the p<i>K</i><sub>a</sub> value of two titratable residues. These findings were supported further by introducing mutations into wild-type archeorhodopsin-3 and detecting the enhancement of the fluorescence signal. Finally, we came up with a rational design and proposed previously unknown Archers variants with red-shifted absorption bands (λ<sub>max</sub> up to 640 nm) and potential-dependent bright fluorescence (quantum yield up to 0.97%).","PeriodicalId":29796,"journal":{"name":"ACS Physical Chemistry Au","volume":"76 1","pages":""},"PeriodicalIF":3.7000,"publicationDate":"2024-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Rational Design of Far-Red Archaerhodopsin-3-Based Fluorescent Genetically Encoded Voltage Indicators: from Elucidation of the Fluorescence Mechanism in Archers to Novel Red-Shifted Variants\",\"authors\":\"Dmitrii M. Nikolaev, Vladimir N. Mironov, Ekaterina M. Metelkina, Andrey A. Shtyrov, Andrey S. Mereshchenko, Nikita A. Demidov, Sergey Yu. Vyazmin, Tatiana B. Tennikova, Svetlana E. Moskalenko, Stanislav A. Bondarev, Galina A. Zhouravleva, Andrey V. Vasin, Maxim S. Panov, Mikhail N. Ryazantsev\",\"doi\":\"10.1021/acsphyschemau.3c00073\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"Genetically encoded voltage indicators (GEVIs) have found wide applications as molecular tools for visualization of changes in cell membrane potential. Among others, several classes of archaerhodopsin-3-based GEVIs have been developed and have proved themselves promising in various molecular imaging studies. To expand the application range for this type of GEVIs, new variants with absorption band maxima shifted toward the first biological window and enhanced fluorescence signal are required. Here, we integrate computational and experimental strategies to reveal structural factors that distinguish far-red bright archaerhodopsin-3-based GEVIs, Archers, obtained by directed evolution in a previous study (McIsaac et al., <i>PNAS,</i> 2014) and the wild-type archaerhodopsin-3 with an extremely dim fluorescence signal, aiming to use the obtained information in subsequent rational design. We found that the fluorescence can be enhanced by stabilization of a certain conformation of the protein, which, in turn, can be achieved by tuning the p<i>K</i><sub>a</sub> value of two titratable residues. These findings were supported further by introducing mutations into wild-type archeorhodopsin-3 and detecting the enhancement of the fluorescence signal. Finally, we came up with a rational design and proposed previously unknown Archers variants with red-shifted absorption bands (λ<sub>max</sub> up to 640 nm) and potential-dependent bright fluorescence (quantum yield up to 0.97%).\",\"PeriodicalId\":29796,\"journal\":{\"name\":\"ACS Physical Chemistry Au\",\"volume\":\"76 1\",\"pages\":\"\"},\"PeriodicalIF\":3.7000,\"publicationDate\":\"2024-04-29\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"ACS Physical Chemistry Au\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.1021/acsphyschemau.3c00073\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"CHEMISTRY, PHYSICAL\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"ACS Physical Chemistry Au","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1021/acsphyschemau.3c00073","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"CHEMISTRY, PHYSICAL","Score":null,"Total":0}
Rational Design of Far-Red Archaerhodopsin-3-Based Fluorescent Genetically Encoded Voltage Indicators: from Elucidation of the Fluorescence Mechanism in Archers to Novel Red-Shifted Variants
Genetically encoded voltage indicators (GEVIs) have found wide applications as molecular tools for visualization of changes in cell membrane potential. Among others, several classes of archaerhodopsin-3-based GEVIs have been developed and have proved themselves promising in various molecular imaging studies. To expand the application range for this type of GEVIs, new variants with absorption band maxima shifted toward the first biological window and enhanced fluorescence signal are required. Here, we integrate computational and experimental strategies to reveal structural factors that distinguish far-red bright archaerhodopsin-3-based GEVIs, Archers, obtained by directed evolution in a previous study (McIsaac et al., PNAS, 2014) and the wild-type archaerhodopsin-3 with an extremely dim fluorescence signal, aiming to use the obtained information in subsequent rational design. We found that the fluorescence can be enhanced by stabilization of a certain conformation of the protein, which, in turn, can be achieved by tuning the pKa value of two titratable residues. These findings were supported further by introducing mutations into wild-type archeorhodopsin-3 and detecting the enhancement of the fluorescence signal. Finally, we came up with a rational design and proposed previously unknown Archers variants with red-shifted absorption bands (λmax up to 640 nm) and potential-dependent bright fluorescence (quantum yield up to 0.97%).
期刊介绍:
ACS Physical Chemistry Au is an open access journal which publishes original fundamental and applied research on all aspects of physical chemistry. The journal publishes new and original experimental computational and theoretical research of interest to physical chemists biophysical chemists chemical physicists physicists material scientists and engineers. An essential criterion for acceptance is that the manuscript provides new physical insight or develops new tools and methods of general interest. Some major topical areas include:Molecules Clusters and Aerosols; Biophysics Biomaterials Liquids and Soft Matter; Energy Materials and Catalysis