Extreme dependence of Chloroflexus aggregans LOV domain thermo- and photostability on the bound flavin species

Smolentseva, Anastasia; Goncharov, Ivan M.; Yudenko, Anna; Bogorodskiy, Andrey; Semenov, Oleg; Nazarenko, Vera V.; Borshchevskiy, Valentin; Fonin, Alexander V.; Remeeva, Alina; Jaeger, Karl‐Erich; Krauß, Ulrich; Gordeliy, Valentin; Gushchin, Ivan

doi:10.1007/s43630-021-00138-3

Cited by 6 publications

(14 citation statements)

References 48 publications

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“…Given that protein stability may be important in some applications, we measured decay of NeuroFbFP fluorescence upon thermal denaturation (Figure 3b). We observed that NeuroFbFP_A purified from E. coli displays two transitions at 67 and 74°C, which most probably indicates presence of protein species bound to two different chromophores, riboflavin and FMN, as in the case of CagFbFP (Smolentseva et al, 2021). NeuroFbFP_B purified from E. coli displayed gradual loss of fluorescence in the range from 45 to 75°C (Supporting Figure 4), while the sample additionally incubated with FMN displayed a clear peak at ~71°C (Figure 3b).…”

Section: Resultsmentioning

confidence: 92%

Reengineering of a flavin‐binding fluorescent protein using ProteinMPNN

Nikolaev,

Kuzmin,

Markeeva

et al. 2024

Protein Science

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Recent advances in machine learning techniques have led to development of a number of protein design and engineering approaches. One of them, ProteinMPNN, predicts an amino acid sequence that would fold and match user‐defined backbone structure. Its performance was previously tested for proteins composed of standard amino acids, as well as for peptide‐ and protein‐binding proteins. In this short report, we test whether ProteinMPNN can be used to reengineer a non‐proteinaceous ligand‐binding protein, flavin‐based fluorescent protein CagFbFP. We fixed the native backbone conformation and the identity of 20 amino acids interacting with the chromophore (flavin mononucleotide, FMN) while letting ProteinMPNN predict the rest of the sequence. The software package suggested replacing 36–48 out of the remaining 86 amino acids so that the resulting sequences are 55%–66% identical to the original one. The three designs that we tested experimentally displayed different expression levels, yet all were able to bind FMN and displayed fluorescence, thermal stability, and other properties similar to those of CagFbFP. Our results demonstrate that ProteinMPNN can be used to generate diverging unnatural variants of fluorescent proteins, and, more generally, to reengineer proteins without losing their ligand‐binding capabilities.

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Section: Resultsmentioning

confidence: 92%

Reengineering of a flavin‐binding fluorescent protein using ProteinMPNN

Nikolaev,

Kuzmin,

Markeeva

et al. 2024

Protein Science

Self Cite

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“…When expressed in E. coli , CagFbFP displays two melting transitions, corresponding to the protein molecules bound to FMN, and those bound to RF/FAD (20). Most mutants identified in this study also show two transitions.…”

Section: Discussionmentioning

confidence: 99%

“…Given that QY of CagFbFP depends on the chromophore composition, which varies with expression conditions (20), all samples were refolded with FMN prior to measurements. The protein in the apo form prepared as described in ref.…”

Section: Methodsmentioning

confidence: 99%

“…The protein in the apo form prepared as described in ref. (20) was mixed with 4 mM FMN (Sigma-Aldrich, USA) to reach 3:5 FMN:protein molar ratio. After incubation for 10 min, the samples were transferred into the buffer containing 50 mM Tris pH 8.0, 300 mM NaCl by dialysis.…”

Section: Methodsmentioning

confidence: 99%

“…They absorb UV and blue light via noncovalently bound FMN or FAD in oxidized state with the characteristic absorption peak around 450 nm. They may also bind RF, which results in altered thermal stability and photobleaching kinetics, but does not affect spectral properties (20, 21). Most of the natural LOVs have a conserved cysteine near the flavin isoalloxazine moiety.…”

Section: Introductionmentioning

confidence: 99%

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Fine spectral tuning of a flavin-binding fluorescent protein for multicolor imaging

Николаев

Yudenko

Smolentseva

et al. 2022

Preprint

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Flavin-binding fluorescent proteins (FbFPs) are promising genetically encoded tags for microscopy. However, spectral properties of their chromophores (riboflavin, flavin mononucleotide and flavin adenine dinucleotide) are notoriously similar even between different protein families, which limits applications of flavoproteins in multicolor imaging. Here, we present a palette of twenty-two finely tuned fluorescent tags based on the thermostable LOV domain from Chloroflexus aggregans (CagFbFP). We performed site saturation mutagenesis of three amino acid positions in the flavin-binding pocket, including the photoactive cysteine, to obtain variants with fluorescence emission maxima uniformly covering the wavelength range from 486 to 512 nm. We demonstrate three-color imaging based on spectral separation and two-color fluorescence lifetime imaging using the proteins from the palette. These results highlight the possibility of fine spectral tuning of flavoproteins and pave the way for further applications of FbFPs in fluorescence microscopy.

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