Experimental characterization ofin silicored-shift predicted iLOVL470T/Q489Kand iLOVV392K/F410V/A426Smutants

Wehler, Pierre; Öztürk, Mehmet Ali

doi:10.1101/2021.03.25.436974

Cited by 2 publications

(2 citation statements)

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“…44 Wehler and co-workers experimentally attempted to prepare these mutants but could not prepare a functional red-shifted FbFP, as they found that a double-point mutant (iLOV-L470T/Q489K) gives a ∼2 nm blue shift and a triple-point mutation (iLOV-V392K/F410V/ A426S) lost the ability to bind the chromophore due to the V392K mutation. 45 Overall, while the strategy of placing a positive charge in the vicinity of flavin's N5 and C4a atoms was theoretically shown to work, most of the amino acids on that side of the protein turned out to be conformationally unstable or essential for chromophore binding. Recently, Rollen and coworkers prepared a red-shifted iLOV with a double-point mutation (iLOV-V392T/Q489K).…”

Section: ■ Introductionmentioning

confidence: 99%

Alternative Strategy for Spectral Tuning of Flavin-Binding Fluorescent Proteins

et al. 2023

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iLOV is an engineered flavin-binding fluorescent protein (FbFP) with applications for in vivo cellular imaging. To expand the range of applications of FbFPs for multicolor imaging and FRET-based biosensing, it is desirable to understand how to modify their absorption and emission wavelengths (i.e., through spectral tuning). There is particular interest in developing FbFPs that absorb and emit light at longer wavelengths, which has proven challenging thus far. Existing spectral tuning strategies that do not involve chemical modification of the flavin cofactor have focused on placing positively charged amino acids near flavin’s C4a and N5 atoms. Guided by previously reported electrostatic spectral tunning maps (ESTMs) of the flavin cofactor and by quantum mechanical/molecular mechanical (QM/MM) calculations reported in this work, we suggest an alternative strategy: placing a negatively charged amino acid near flavin’s N1 atom. We predict that a single-point mutant, iLOV-Q430E, has a slightly red-shifted absorption and fluorescence maximum wavelength relative to iLOV. To validate our theoretical prediction, we experimentally expressed and purified iLOV-Q430E and measured its spectral properties. We found that the Q430E mutation results in a slight change in absorption and a 4–8 nm red shift in the fluorescence relative to iLOV, in good agreement with the computational predictions. Molecular dynamics simulations showed that the carboxylate side chain of the glutamate in iLOV-Q430E points away from the flavin cofactor, which leads to a future expectation that further red shifting may be achieved by bringing the side chain closer to the cofactor.

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Section: ■ Introductionmentioning

confidence: 99%

Alternative Strategy for Spectral Tuning of Flavin-Binding Fluorescent Proteins

et al. 2023

View full text Add to dashboard Cite

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“…(44) Recently, Wehler and coworkers experimentally attempted to prepare these mutants but could not prepare a functional red-shifted FbFP, as they found that a double-point mutant (iLOV-L470T/Q489K) gives ~2 nm blueshift and a triple-point mutation (iLOV-V392K/F410V/A426S) lost the ability to bind the chromophore due to the V392K mutation. (45) Overall, while the strategy of placing a positive charge in the vicinity of flavin's N5 and C4a atoms was theoretically shown to work, most of the amino acids on that side of the protein turned out to be conformationally unstable or essential for chromophore binding.…”

Section: Introductionmentioning

confidence: 99%

An Alternative Strategy for Spectral Tuning of Flavin-binding Fluorescent Proteins

Kabir

Ouedraogo

Orozco‐Gonzalez

et al. 2022

Preprint

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iLOV is an engineered flavin-binding fluorescent protein (FbFP) with applications for in vivo cellular imaging. To expand the range of applications of FbFPs for multicolor imaging and FRET-based biosensing, it is desirable to understand how to modify their absorption and emission wavelengths (i.e., through spectral tuning). There is particular interest in developing FbFPs that absorb and emit light at longer wavelengths, which has proven challenging thus far. Existing spectral tuning strategies that do not involve chemical modification of the flavin cofactor have focused on placing positively charged amino acids near flavin’s C4a and N5 atoms. Guided by previously reported electrostatic spectral tunning maps (ESTMs) of the flavin cofactor and by quantum mechanical/molecular mechanical (QM/MM) calculations reported in this work, we suggest an alternative strategy: placing a negatively charged amino acid near flavin’s N1 atom. We predict that a single-point mutant, iLOV-Q430E, has a slightly red-shifted absorption and fluorescence maximum wavelength relative to iLOV. To validate our theoretical prediction, we experimentally expressed and purified iLOV-Q430E and measured its spectral properties. We found that the Q430E mutation in iLOV results in a slight change in absorption and a 4-8 nm redshift in the fluorescence relative to iLOV, in good agreement with the computational prediction. Molecular dynamics simulations showed that the carboxylate side chain of the glutamate in iLOV-Q430E points away from the flavin cofactor, which leads to a future expectation that further red-shifting may be achieved by bringing the side chain closer to the cofactor.

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Experimental characterization ofin silicored-shift predicted iLOV^L470T/Q489Kand iLOV^{V392K/F410V/A426S}mutants

Cited by 2 publications

References 38 publications

Alternative Strategy for Spectral Tuning of Flavin-Binding Fluorescent Proteins

Alternative Strategy for Spectral Tuning of Flavin-Binding Fluorescent Proteins

An Alternative Strategy for Spectral Tuning of Flavin-binding Fluorescent Proteins

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