Stabilization of structure in near-infrared fluorescent proteins by binding of biliverdin chromophore

Kuznetsova

Shcherbakova

et al. 2017

IJMS

Near-infrared (NIR) fluorescent proteins (FPs) designed from PAS (Per-ARNT-Sim repeats) and GAF (cGMP phosphodiesterase/adenylate cyclase/FhlA transcriptional activator) domains of bacterial phytochromes covalently bind biliverdin (BV) chromophore via one or two Cys residues. We studied BV interaction with a series of NIR FP variants derived from the recently reported BphP1-FP protein. The latter was engineered from a bacterial phytochrome RpBphP1, and has two reactive Cys residues (Cys15 in the PAS domain and Cys256 in the GAF domain), whereas its mutants contain single Cys residues either in the PAS domain or in the GAF domain, or no Cys residues. We characterized BphP1-FP and its mutants biochemically and spectroscopically in the absence and in the presence of denaturant. We found that all BphP1-FP variants are monomers. We revealed that spectral properties of the BphP1-FP variants containing either Cys15 or Cys256, or both, are determined by the covalently bound BV chromophore only. Consequently, this suggests an involvement of the inter-monomeric allosteric effects in the BV interaction with monomers in dimeric NIR FPs, such as iRFPs. Likely, insertion of the Cys15 residue, in addition to the Cys256 residue, in dimeric NIR FPs influences BV binding by promoting the BV chromophore covalent cross-linking to both PAS and GAF domains.

Section: Resultsmentioning

confidence: 99%

Interaction of Biliverdin Chromophore with Near-Infrared Fluorescent Protein BphP1-FP Engineered from Bacterial Phytochrome

Kuznetsova

Shcherbakova

et al. 2017

IJMS

“…The tryptophan fluorescence spectra of the analyzed proteins assembled with PCB and iRFP713/BV normalized to unity at the maximum were superimposed well in the short-wave spectral region but differed in the long-wave spectral edge (Supplementary Figure S2). We have previously shown that an effective energy transfer from the tryptophan residue Trp281 of iRFP713 to BV takes place [37]. Based on this, the observed difference in the tryptophan fluorescence spectra of PCB-bound iRFP713 variants and BV-bound iRFP713 can be explained by the higher efficiency of nonradiative energy transfer from Trp281 to PCB than to BV.…”

Section: Resultsmentioning

confidence: 90%

Near-Infrared Markers based on Bacterial Phytochromes with Phycocyanobilin as a Chromophore

Shpironok

et al. 2019

IJMS

Self Cite

Biomarkers engineered on the basis of bacterial phytochromes with biliverdin IXα (BV) cofactor as a chromophore are increasingly used in cell biology and biomedicine, since their absorption and fluorescence spectra lie within the so-called optical “transparency window” of biological tissues. However, the quantum yield of BV fluorescence in these biomarkers does not exceed 0.145. The task of generating biomarkers with a higher fluorescence quantum yield remains relevant. To address the problem, we proposed the use of phycocyanobilin (PCB) as a chromophore of biomarkers derived from bacterial phytochromes. In this work, we characterized the complexes of iRFP713 evolved from RpBphP2 and its mutant variants with different location of cysteine residues capable of covalent tetrapyrrole attachment with the PCB cofactor. All analyzed proteins assembled with PCB were shown to have a higher fluorescence quantum yield than the proteins assembled with BV. The iRFP713/V256C and iRFP713/C15S/V256C assembled with PCB have a particularly high quantum yield of 0.5 and 0.45, which exceeds the quantum yield of all currently available near-infrared biomarkers. Moreover, PCB has 4 times greater affinity for iRFP713/V256C and iRFP713/C15S/V256C proteins compared to BV. These data establish iRFP713/V256C and iRFP713/C15S/V256C assembled with the PCB chromophore as promising biomarkers for application in vivo. The analysis of the spectral properties of the tested biomarkers allowed for suggesting that the high-fluorescence quantum yield of the PCB chromophore can be attributed to the lower mobility of the D-ring of PCB compared to BV.

“…The increased fluorescence quantum yield of the tryptophan residues W281 and W311 of iRFP713 at the intermediate state is explained by the absence of the conditions for the effective nonradiative energy transfer from tryptophan residues of the protein to the chromophore (Stepanenko et al, 2014; Stepanenko et al, 2017). The fact that the formation of the intermediate state was not accompanied by any increase in fluorescence intensity of iRFP713-W109 may be connected with less efficient nonradiative energy transfer from this residue to the chromophore compared to the other tryptophan residues, as W109 is the most distant tryptophan residue from the pocket of the GAF domain of iRFP713 (Stepanenko et al, 2017). An increase in the parameter A of iRFP713-W311 in the holoform, which was observed in the range of GdnHCl concentrations from 1.5 to 2.3–2.5 M, is obviously related with the re-shielding of this tryptophan residue in the intermediate state of iRFP713 (Fig.…”

Section: Resultsmentioning

confidence: 99%

The unfolding of iRFP713 in a crowded milieu

Kuznetsova

Turoverov

2019

PeerJ

Self Cite

The exploring of biological processes in vitro under conditions of macromolecular crowding is a way to achieve an understanding of how these processes occur in vivo. In this work, we study the unfolding of the fluorescent probe iRFP713 in crowded environment in vitro. Previously, we showed that the unfolding of the dimeric iRFP713 is accompanied by the formation of a compact monomer and an intermediate state of the protein. In the intermediate state, the macromolecules of iRFP713 have hydrophobic clusters exposed to the surface of the protein and are prone to aggregation. Concentrated solutions of polyethylene glycol (PEG-8000), Dextran-40 and Dextran-70 with a molecular mass of 8000, 40000 and 70000 Da, respectively, were used to model the conditions for macromolecular crowding. A limited available space provided by all the crowding agents used favors to the enhanced aggregation of iRFP713 in the intermediate state at the concentration of guanidine hydrochloride (GdnHCl), at which the charge of protein surface is neutralized by the guanidine cations. This is in line with the theory of the excluded volume. In concentrated solutions of the crowding agents (240–300 mg/ml), the stabilization of the structure of iRFP713 in the intermediate state is observed. PEG-8000 also enhances the stability of iRFP713 in the monomeric compact state, whereas in concentrated solutions of Dextran-40 and Dextran-70 the resistance of the protein in the monomeric state against GdnHCl-induced unfolding decreases. The obtained data argues for the excluded volume effect being not the only factor that contributes the behavior of biological molecules in a crowded milieu. Crowding agents do not affect the structure of the native dimer of iRFP713, which excludes the direct interactions between the target protein and the crowding agents. PEGs of different molecular mass and Dextran-40/Dextran-70 are known to influence the solvent properties of water. The solvent dipolarity/polarizability and basicity/acidity in aqueous solutions of these crowding agents vary in different ways. The change of the solvent properties in aqueous solutions of crowding agents might impact the functioning of a target protein.