Browsing by Subject "bacterial phytochrome"

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  • Stepanenko, Olesya V.; Stepanenko, Olga V.; Kuznetsova, Irina M.; Shcherbakova, Daria M.; Verkhusha, Vladislav; Turoverov, Konstantin K. (2017)
    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.
  • Li, Lei; Hsu, Hsun-Chia; Verkhusha, Vladislav V.; Wang, Lihong V.; Shcherbakova, Daria M. (2021)
    Photoacoustic tomography (PAT) with genetically encoded near-infrared probes enables visualization of specific cell populations in vivo at high resolution deeply in biological tissues. However, because of a lack of proper probes, PAT of cellular dynamics remains unexplored. Here, the authors report a near-infrared Forster resonance energy transfer (FRET) biosensor based on a miRFP670-iRFP720 pair of the near-infrared fluorescent proteins, which enables dynamic functional imaging of active biological processes in deep tissues. By photoacoustically detecting the changes in the optical absorption of the miRFP670 FRET-donor, they monitored cell apoptosis in deep tissue at high spatiotemporal resolution using PAT. Specifically, they detected apoptosis in single cells at a resolution of approximate to 3 mu m in a mouse ear tumor, and in deep brain tumors (>3 mm beneath the scalp) of living mice at a spatial resolution of approximate to 150 mu m with a 20 Hz frame rate. These results open the way for high-resolution photoacoustic imaging of dynamic biological processes in deep tissues using NIR biosensors and PAT.