Phytochromes (Phys) comprise a large and diverse superfamily of photoreceptors that utilize a linear tetrapyrrole bilin chromophore for light detection. Canonical Phys consist of a red-absorbing ground state (Pr) and, upon photoexcitation with red light (R), photoconvert to their often biologically active far-red light absorbing state, or Pfr (for review see Rockwell et al., 2006, Annu. Rev. Plant Biol. 57: 837-858). First discovered in plants, Phys play an integral role in regulating many important aspects of the plant life cycle, such as shade avoidance, senescence, flowering time, photoperiod, and fruit ripening. These photoreceptors maintain a conserved domain architecture consisting of an N-terminal “Chromophore Binding Domain” (CBD), composed of a Per/Arndt/Sim (PAS) domain followed by a cGMP phosphodiesterase/adenyl cyclase/FhlA (GAF) domain. The CBD is tethered together by a knotted structure, whereby a conserved “lasso loop” found within the GAF domain weaves its way through the PAS domain to form a figure-of-eight configuration. The GAF domain cradles the bilin and includes the conserved cysteine (Cys)-histidine (His) motif, where an intrinsic lyase activity is responsible for forming a covalent thioether linkage to the linear tetrapyrrole phytochromobilin PφB at the C31 carbon. This bilin drives the central event responsible for Pr to Pfr (Pr→Pfr) photoconversion by undergoing a photoisomerization of C14/15 double bond between the A and B rings of the chromophore. Immediately downstream of the CBD is the Phytochrome (PHY) domain that helps stabilize the Pfr conformation.
Although Phys were first discovered in higher plants, these receptors have since been found in lower plants, algae, fungi, cyanobacteria, and numerous probacteria. Unlike plant Phys, the cyanobacterial phytochromes (Cphs) prefer phycocyanobilin (PCB) as the chromophore that is also used as an accessory pigment for photosynthesis. PCB is synthesized in a similar manner to POB in two-step enzymatic process from heme involving a heme oxygenase and biliverdin reductase. In contrast, eubacterial Phys, or Bacterio-phytochromes (BphPs) as well as fungal phytochromes (Fphs) predominantly use biliverdin (BV) as the chromophore, made from a more simplified one-step catalyzed cleavage of heme by a heme oxygenase. In contrast to POB and PCB that form a linkage with the GAF domain, BV chromophores attach to the protein at a conserved Cys upstream of the PAS domain. Both Cph and BphP C-terminal domains have been shown to act as bona-fide HKs, whereas plant Phys appear to have diverged to acquire Ser-Thr kinase activity.
Cyanobacteria have been shown to harbor diverse members of the Phy superfamily, or Phy-like proteins, with some more divergent ones missing several key residues required for phototransformation in canonical Phys, (for reviews see Vierstra and Karniol, 2005, In: Handbook of Photosensory Receptors, Briggs, W. R. and Spudich, J. L., eds, Wiley-VCH Press, Weinheim, Germany, pp 171-196). One of the first of these Phy-like proteins to be described in detail, PixJ, is involved in a blue-light mediated phototaxis response in the mesophilic cyanobacterium Synechocystis sp. PCC6803 (Syn-PixJ; see Yoshihara et al., 2004, Plant Cell Physiol. 45: 1729-1737) and thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 (TePixJ; see Ishizuka et al., 2006, Plant Cell Physiol. 47: 1251-1261). Renamed cyanochromes (Cycs), subsequent work has shown that these chromoproteins might harbor a less-conjugated PCB variant phycoviolobilin (PVB), contributing to their blue-shifted ground state absorbance peak absorbance of 430 nm, or Pb (Ishizuka et al., 2007, Plant Cell Physiol. 48: 1385-1390). Chemically identical to PCB save a missing double bond at the A-B bridge, PVB is a natural chromophore found in the phycoerythrocyanin (PEC) component of the light harvesting phycobilisomes in several cyanobacteria. Similar to PCB and POB with respect to Phys, PVB is proposed to attach to the PEC polypeptide through a C31 thioether linkage as well as undergo a Z, E photoisomerization of the C15=C16 double bond at the D-ring of the chromophore. The attachment of the bilin to PEC is assisted by two enzymes, PecE and PecF, which are responsible for the isomerization of PCB to POB and the lyase step involving the covalent attachment to the C31 Cys carbon. PVB is speculated to attach to cyanochromes in a similar manner, but through an intrinsic lyase activity similar to canonical red-absorbing Phys. However, after the proposed initial C31 thioether ligation, the events responsible for isomerization of the A-:B methine bridge of the bilin that yield the final PVB product remain enigmatic. Ishizuka et al. propose a model whereby after attachment to the C31 carbon, a basic amino acid proton donor facilitates the deconjugation of the A-B bridge (Ishizuka et al., 2007, Plant Cell Physiol. 48: 1385-1390).
Upon photoexcitation with blue light, cyanochromes photoconvert to a green-absorbing exited state, or Pg, which may be, in turn, reversed by subsequent green light irradiation. Unlike canonical red-absorbing Phys, cyanochromes do not require a Phy domain to undergo photoconversion to their excited state. In fact, only the GAF domain alone from TePixJ is needed for transformation to Pg. A recent report has demonstrated that a conserved Cys (TePixJ Cys-494) is actually responsible for the shift in absorbance from a red to blue absorbing chromoprotein, and that this additional Cys is required for phototransformation (Rockwell et al., 2008, Biochemistry 47: 7304-7316). This second Cys is hypothesized to covalently attach via a thioether linkage to the PCB bilin at the B-C methine bridge, and that upon blue light excitation, this transient bond is broken to yield a more conjugated green-absorbing photoproduct. However, biochemical evidence to support these claims is lacking, including hard evidence of a covalent bond provided by the second Cys to PCB. See Ulijasz et al. (2009) Journal of Biological Chemistry, Vol. 284, pp. 29757-29772.
Various methods and reporter molecules are available for monitoring gene activity and protein distribution within cells. These include the formation of fusion proteins with coding sequences for reporter molecules (markers) such as beta-galactosidase, luciferase, green fluorescent protein (GFP) and red fluorescent protein (RFP). Particularly useful reporter is GFP from the bioluminescent jellyfish Aequorea victoria, which is frequently used as a fluorescent marker, and is described in U.S. Pat. No. 5,491,084. However, the known reporter molecules have a variety of limitations, including short wavelength of the fluorescence emission and small separation between excitation and emission wavelength maxima. The discovery of novel reporter molecules for monitoring gene activity, protein synthesis, and protein distribution within cells, can provide very useful tools for biotechnology applications. The present invention addresses these and related needs.
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USPTO Backgrounds