TY - JOUR

T1 - 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

AU - Николаев, Дмитрий Михайлович

AU - Миронов, Владимир Николаевич

AU - Метелкина, Екатерина Михайловна

AU - Штыров, Андрей Андреевич

AU - Мерещенко, Андрей Сергеевич

AU - Демидов, Никита Александрович

AU - Вязьмин , Сергей Юрьевич

AU - Тенникова, Татьяна Борисовна

AU - Москаленко, Светлана Евгеньевна

AU - Бондарев, Станислав Александрович

AU - Журавлева, Галина Анатольевна

AU - Васин, Андрей Владимирович

AU - Панов, Максим Сергеевич

AU - Рязанцев, Михаил Николаевич

PY - 2024/7/24

Y1 - 2024/7/24

N2 - 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%).

AB - 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%).

KW - GEVI

KW - fluorescent rhodopsins

KW - genetically encoded voltage indicators

KW - optogenetics

KW - protein design

KW - rhodopsins

UR - https://www.mendeley.com/catalogue/bf95c12a-80e9-3c21-99d4-982d5d5ac59a/

U2 - 10.1021/acsphyschemau.3c00073

DO - 10.1021/acsphyschemau.3c00073

M3 - Article

C2 - 39069984

VL - 4

SP - 347

EP - 362

JO - ACS Physical Chemistry Au

JF - ACS Physical Chemistry Au

SN - 2694-2445

IS - 4

ER -