Experimental Characterization of In Silico Red-Shift-Predicted iLOVL470T/Q489K and iLOVV392K/F410V/A426S Mutants

Wehler, Pierre; Armbruster, Daniel; Günter, Andreas; Schleicher, Erik; Ventura, Barbara Di; Öztürk, Mehmet Ali

doi:10.1021/acsomega.2c01283

Cited by 2 publications

(3 citation statements)

References 31 publications

(55 reference statements)

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“…The maps indicate that a positive charge near the C4a or N5 flavin atoms would lead to a red-shifted absorption/emission; this has been the strategy proposed by Khrenova et al and the ensuing computational and experimental work. 23 , 41 − 45 A second strategy, not yet explored in FbFPs, would be to introduce a negatively charged amino acid in the vicinity of the N1 atom of flavin. Here, we pursue this strategy with Q430E.…”

Section: Resultsmentioning

confidence: 99%

“…In a follow-up QM/MM study, Khrenova et al proposed additional mutations to stabilize the lysine side chain close to the N5 and C4a atoms of the chromophore . 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 . 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%

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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: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Alternative Strategy for Spectral Tuning of Flavin-Binding Fluorescent Proteins

et al. 2023

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“…Extensive screening of variants with random mutations of A85 in CagFbFP (corresponding to the conserved cysteine in LOV domains) revealed either red-shifted A85Q variant or variants with essentially unaltered spectra (Nikolaev et al, 2023). Variants with multiple mutations, theoretically predicted to display larger shifts (Khrenova et al, 2017), could not be obtained in experiments (Wehler et al, 2022). Structural information presented in this work could be used for rational engineering of new CagFbFP variants with Q85 better stabilized near the flavin, both in the ground and excited states, or for transferring the analyzed mutations to other FbFPs.…”

Section: Resultsmentioning

confidence: 98%

Two distinct mechanisms of flavoprotein spectral tuning revealed by low‐temperature and time‐dependent spectroscopy

Nikolaev,

Tropina,

Boldyrev

et al. 2023

Protein Science

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Flavins such as flavin mononucleotide or flavin adenine dinucleotide are bound by diverse proteins, yet have very similar spectra when in the oxidized state. Recently, we developed new variants of flavin‐binding protein CagFbFP exhibiting notable blue (Q148V) or red (I52V A85Q) shifts of fluorescence emission maxima. Here, we use time‐resolved and low‐temperature spectroscopy to show that whereas the chromophore environment is static in Q148V, an additional protein‐flavin hydrogen bond is formed upon photoexcitation in the I52V A85Q variant. Consequently, in Q148V, excitation, emission, and phosphorescence spectra are shifted, whereas in I52V A85Q, excitation and low‐temperature phosphorescence spectra are relatively unchanged, while emission spectrum is altered. We also determine the x‐ray structures of the two variants to reveal the flavin environment and complement the spectroscopy data. Our findings illustrate two distinct color‐tuning mechanisms of flavin‐binding proteins and could be helpful for the engineering of new variants with improved optical properties.

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Experimental Characterization of In Silico Red-Shift-Predicted iLOV^L470T/Q489K and iLOV^{V392K/F410V/A426S} Mutants

Cited by 2 publications

References 31 publications

Alternative Strategy for Spectral Tuning of Flavin-Binding Fluorescent Proteins

Alternative Strategy for Spectral Tuning of Flavin-Binding Fluorescent Proteins

Two distinct mechanisms of flavoprotein spectral tuning revealed by low‐temperature and time‐dependent spectroscopy

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