Junrui Zhang

Phytochromes are red/far red light photochromic photoreceptors that direct many photosensory behaviors in the bacterial, fungal, and plant kingdoms. They consist of an N-terminal domain that covalently binds a bilin chromophore and a C-terminal region that transmits the light signal, often through a histidine kinase relay. Using x-ray crystallography, we recently solved the first three-dimensional structure of a phytochrome, using the chromophore-binding domain of Deinococcus radiodurans bacterial phytochrome assembled with its chromophore, biliverdin IXalpha. Now, by engineering the crystallization interface, we have achieved a significantly higher resolution model. This 1.45A resolution structure helps identify an extensive buried surface between crystal symmetry mates that may promote dimerization in vivo. It also reveals that upon ligation of the C3(2) carbon of biliverdin to Cys(24), the chromophore A-ring assumes a chiral center at C2, thus becoming 2(R),3(E)-phytochromobilin, a chemistry more similar to that proposed for the attached chromophores of cyanobacterial and plant phytochromes than previously appreciated. The evolution of bacterial phytochromes to those found in cyanobacteria and higher plants must have involved greater fitness using more reduced bilins, such as phycocyanobilin, combined with a switch of the attachment site from a cysteine near the N terminus to one conserved within the cGMP phosphodiesterase/adenyl cyclase/FhlA domain. From analysis of site-directed mutants in the D. radiodurans phytochrome, we show that this bilin preference was partially driven by the change in binding site, which ultimately may have helped photosynthetic organisms optimize shade detection. Collectively, these three-dimensional structural results better clarify bilin/protein interactions and help explain how higher plant phytochromes evolved from prokaryotic progenitors.

The ability of phytochromes (Phy) to act as photointerconvertible light switches in plants and microorganisms depends on key interactions between the bilin chromophore and the apoprotein that promote bilin attachment and photointerconversion between the spectrally distinct red light-absorbing Pr conformer and far red light-absorbing Pfr conformer. Using structurally guided site-directed mutagenesis combined with several spectroscopic methods, we examined the roles of conserved amino acids within the bilin-binding domain of Deinococcus radiodurans bacteriophytochrome with respect to chromophore ligation and Pr/Pfr photoconversion. Incorporation of biliverdin IXα (BV), its structure in the Pr state, and its ability to photoisomerize to the first photocycle intermediate are insensitive to most single mutations, implying that these properties are robust with respect to small structural/electrostatic alterations in the binding pocket. In contrast, photoconversion to Pfr is highly sensitive to the chromophore environment. Many of the variants form spectrally bleached Meta-type intermediates in red light that do not relax to Pfr. Particularly important are Asp-207 and His-260, which are invariant within the Phy superfamily and participate in a unique hydrogen bond matrix involving the A, B, and C pyrrole ring nitrogens of BV and their associated pyrrole water. Resonance Raman spectroscopy demonstrates that substitutions of these residues disrupt the Pr to Pfr protonation cycle of BV with the chromophore locked in a deprotonated Meta-Rc-like photoconversion intermediate after red light irradiation. Collectively, the data show that a number of contacts contribute to the unique photochromicity of Phy-type photoreceptors. These include residues that fix the bilin in the pocket, coordinate the pyrrole water, and possibly promote the proton exchange cycle during photoconversion. The ability of phytochromes (Phy) to act as photointerconvertible light switches in plants and microorganisms depends on key interactions between the bilin chromophore and the apoprotein that promote bilin attachment and photointerconversion between the spectrally distinct red light-absorbing Pr conformer and far red light-absorbing Pfr conformer. Using structurally guided site-directed mutagenesis combined with several spectroscopic methods, we examined the roles of conserved amino acids within the bilin-binding domain of Deinococcus radiodurans bacteriophytochrome with respect to chromophore ligation and Pr/Pfr photoconversion. Incorporation of biliverdin IXα (BV), its structure in the Pr state, and its ability to photoisomerize to the first photocycle intermediate are insensitive to most single mutations, implying that these properties are robust with respect to small structural/electrostatic alterations in the binding pocket. In contrast, photoconversion to Pfr is highly sensitive to the chromophore environment. Many of the variants form spectrally bleached Meta-type intermediates in red light that do not relax to Pfr. Particularly important are Asp-207 and His-260, which are invariant within the Phy superfamily and participate in a unique hydrogen bond matrix involving the A, B, and C pyrrole ring nitrogens of BV and their associated pyrrole water. Resonance Raman spectroscopy demonstrates that substitutions of these residues disrupt the Pr to Pfr protonation cycle of BV with the chromophore locked in a deprotonated Meta-Rc-like photoconversion intermediate after red light irradiation. Collectively, the data show that a number of contacts contribute to the unique photochromicity of Phy-type photoreceptors. These include residues that fix the bilin in the pocket, coordinate the pyrrole water, and possibly promote the proton exchange cycle during photoconversion. The phytochrome (Phy) 5The abbreviations used are: Phy, phytochrome; BphP, bacteriophytochrome; BV, biliverdin IXα; CBD, chromophore-binding domain; GAF, cGMP phosphodiesterase/adenylcyclase/FhlA; HO, heme oxygenase; ip, in-plane bending; PAS, Per/Arndt/Sim; PCB, phycocyanobilin; PΦB, phytochromobilin; PHY, phytochrome domain; PPIXa, protoporphyrin IXa; Pr, red light-absorbing state of phytochrome; Pfr, far red light-absorbing state of phytochrome; RR, resonance Raman. superfamily encompasses a large and diverse set of photoreceptors present in the plant, fungal, and bacterial kingdoms where they play critical roles in various light-regulated processes (1Rockwell N.C. Su Y.S. Lagarias J.C. Annu. Rev. Plant Biol. 2006; 57: 837-858Crossref PubMed Scopus (844) Google Scholar, 2Vierstra R.D., and Karniol, B. (2005) in Handbook of Photosensory Receptors (Briggs, W. R., and Spudich, Scholar, Rev. Biol. 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Cortisol is a hormone released in response to stress and is a major glucocorticoid produced by adrenal glands. Here, we report a wearable sensory electronic chip using label-free detection, based on a platinum/graphene aptamer extended gate field effect transistor (EG-FET) for the recognition of cortisol in biological buffers within the Debye screening length. The device shows promising experimental features for real-time monitoring of the circadian rhythm of cortisol in human sweat. We report a hysteresis-free EG-FET with a voltage sensitivity of the order of 14 mV/decade and current sensitivity up to 80% over the four decades of cortisol concentration. The detection limit is 0.2 nM over a wide range, between 1 nM and 10 µM, of cortisol concentrations in physiological fluid, with negligible drift over time and high selectivity. The dynamic range fully covers those in human sweat. We propose a comprehensive analysis and a unified, predictive analytical mapping of current sensitivity in all regimes of operation.