Helda

Helsingin yliopisto

Helsingfors universitet

University of Helsinki

Browsing by Subject "AGP1"

Now showing items 1-3 of 3

Allosteric effects of chromophore interaction with dimeric near-infrared fluorescent proteins engineered from bacterial phytochromes

Stepanenko, Olesya V.; Baloban, Mikhail; Bublikov, Grigory S.; Shcherbakova, Daria M.; Stepanenko, Olga V.; Turoverov, Konstantin K.; Kuznetsova, Irina M.; Verkhusha, Vladislav Vitaliyevich (2016)

Fluorescent proteins (FPs) engineered from bacterial phytochromes attract attention as probes for in vivo imaging due to their near-infrared (NIR) spectra and use of available in mammalian cells biliverdin (BV) as chromophore. We studied spectral properties of the iRFP670, iRFP682 and iRFP713 proteins and their mutants having Cys residues able to bind BV either in both PAS (Cys15) and GAF (Cys256) domains, in one of these domains, or without these Cys residues. We show that the absorption and fluorescence spectra and the chromophore binding depend on the location of the Cys residues. Compared with NIR FPs in which BV covalently binds to Cys15 or those that incorporate BV noncovalently, the proteins with BV covalently bound to Cys256 have blue-shifted spectra and higher quantum yield. In dimeric NIR FPs without Cys15, the covalent binding of BV to Cys256 in one monomer allosterically inhibits the covalent binding of BV to the other monomer, whereas the presence of Cys15 allosterically promotes BV binding to Cys256 in both monomers. The NIR FPs with both Cys residues have the narrowest blue-shifted spectra and the highest quantum yield. Our analysis resulted in the iRFP713/Val256Cys protein with the highest brightness in mammalian cells among available NIR FPs.
Interaction of Biliverdin Chromophore with Near-Infrared Fluorescent Protein BphP1-FP Engineered from Bacterial Phytochrome

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.
On the (un)coupling of the chromophore, tongue interactions, and overall conformation in a bacterial phytochrome

Takala, Heikki; Lehtivuori, Heli K.; Berntsson, Oskar; Hughes, Ashley; Nanekar, Rahul; Niebling, Stephan; Panman, Matthijs; Henry, Leocadie; Menzel, Andreas; Westenhoff, Sebastian; Ihalainen, Janne A. (2018)

Phytochromes are photoreceptors in plants, fungi, and various microorganisms and cycle between metastable red light-absorbing (Pr) and far-red light-absorbing (Pfr) states. Their light responses are thought to follow a conserved structural mechanism that is triggered by isomerization of the chromophore. Downstream structural changes involve refolding of the so-called tongue extension of the phytochrome-specific GAF-related (PHY) domain of the photoreceptor. The tongue is connected to the chromophore by conserved DIP and PRXSF motifs and a conserved tyrosine, but the role of these residues in signal transduction is not clear. Here, we examine the tongue interactions and their interplay with the chromophore by substituting the conserved tyrosine (Tyr(263)) in the phytochrome from the extremophile bacterium Deinococcus radiodurans with phenylalanine. Using optical and FTIR spectroscopy, X-ray solution scattering, and crystallography of chromophore-binding domain (CBD) and CBD-PHY fragments, we show that the absence of the Tyr(263) hydroxyl destabilizes the -sheet conformation of the tongue. This allowed the phytochrome to adopt an -helical tongue conformation regardless of the chromophore state, hence distorting the activity state of the protein. Our crystal structures further revealed that water interactions are missing in the Y263F mutant, correlating with a decrease of the photoconversion yield and underpinning the functional role of Tyr(263) in phytochrome conformational changes. We propose a model in which isomerization of the chromophore, refolding of the tongue, and globular conformational changes are represented as weakly coupled equilibria. The results also suggest that the phytochromes have several redundant signaling routes.