Chemistry: molecular biology and microbiology – Miscellaneous
Reexamination Certificate
1997-09-12
2002-01-29
Graser, Jennifer E. (Department: 1645)
Chemistry: molecular biology and microbiology
Miscellaneous
C435S004000, C435S007100, C435S007200, C435S007800, C435S963000, C436S518000, C436S519000, C436S524000, C436S527000, C436S536000, C530S402000, C530S391300
Reexamination Certificate
active
06342389
ABSTRACT:
BACKGROUND OF THE INVENTION
Phycobilisomes are complexes of phycobiliproteins and colorless polypeptides which function as the major light harvesting antennae in blue-green and red algae (Gantt, (1975) “Phycobilisomes: light harvesting pigment complexes,”
BioScience
25:781-788). Naturally-occurring phycobilisomes from different organisms share a number of common properties, including: (1) extremely high “complex molecular weights” (5-20×10
6
daltons) i.e., the weight of one mole of a phycobilisome complex comprised of multiple molecules; (2) multiple absorption maxima in the visible range of the electromagnetic spectrum; (3) high molar absorptivities (e
max
>10
7
M
−1
·cm
−1
); (4) efficient (>90%) directional vibrational energy transfer among constituent phycobiliproteins, commonly from one or more sensitizing species to a terminal acceptor capable of fluorescence; (5) large Stokes shifts relative to isolated phycobiliproteins; (6) high quantum yields of constituent phycobiliproteins; (7) high solubility in aqueous buffers; (8) allophycocyanin-containing core structures; and (9) precisely defined phycobiliprotein and linker polypeptide composition and supramolecular organization.
Morphologically, phycobilisomes are complex assemblies of oligomeric phycobiliprotein discs arranged in ordered stacks referred to as “rods”. In general, several arm-like rods radiate out from a core assembly, also comprised of rods. Phycobilisomes from different organisms are morphologically and stoichiometrically diverse, having different numbers and types of constituent phycobiliproteins and rods. In general, peripheral rods are comprised of phycoerythrocyanin, phycoerythrin, and/or phycocyanin and associated linker proteins, and the core is comprised of allophycocyanin and associated linker proteins. The colorless polypeptides are involved in the assembly and positioning of the phycobiliproteins within the phycobilisomes for proper stability and energy transfer. The major criterion for the functional integrity of these complexes is the demonstration that they exhibit highly efficient transfer of energy between component phycobiliproteins, for example, in
Porphyridium cruentum
phycobilisomes from phycoerythrin (PE) to phycocyanin (PC) and finally to allophycocyanin (APC).
Supramolecular complexes comprising phycobilisomes are well-known in the art, as evidenced by the substantial body of literature on preparative methods (e.g., Gantt, E. 1986, “Phycobilisomes. In: Photosynthesis III: Photosynthetic Membranes and Light Harvesting Systems” (L. A. Staehelin and C. J, Arntzen, eds.), pp.260-268, Springer-Verlag, NY; Grossman, A. R. et al. 1993, “The phycobilisome, a light-harvesting complex responsive to environmental conditions,”
Microbiological Reviews
57:725-749; Hiller, et al., 1982, “Isolation of intact detergent-free phycobilisomes by trypsin.”
FEBS Lett.
156:180-184), rod and core subassemblies (e.g., Lundell, et al. 1983a, “Molecular architecture of a light-harvesting antenna: core substructure in Synechococcus 6301 phycobilisomes: two new allophycocyanin and allophycocyanin B complexes,”
J. Biol. Chem.,
258:902-908; Lundell, et al., 1983b, “Molecular architecture of a light-harvesting antenna: quaternary interactions in the Synechococcus 6301 phycobilisome core as revealed by partial tryptic digestion and circular dichroism studies,”
J. Biol. Chem.,
258:8708-8713; Lundell, et al., 1983c, “Molecular architecture of a light-harvesting antenna: structure of the 18S core-rod subassembly of the Synechococcus 6301 phycobilisome,”
J. Biol. Chem.,
258:894-901; Glazer, A. N. 1985a, “Light harvesting by phycobilisomes,”
Annual Rev. Biophys. and Biophys. Chem.,
14:47-77), phycobilisome-photosystem complexes (e.g., Diner, B. A. 1979, “Energy transfer from phycobilisomes to photosystem II reaction centers in wild type Cyanidium caldarium,”
Plant Physiol.,
63:30-34; Gantt E, et al. (1988), “Photosystem II-phycobilisome complex preparations,”
Meth. Enzymol.
167, 286-290; Clement-Metral, J. D. and Gantt (1983a), “Isolation of oxygen-evolving phycobilisome-photosystem II particles from Porphyridium cruentum,”
FEBS Letters
156:185-188; Clement-Metral J D, et al. (1983b), “A photosystem II-phycobilisome preparation from the red alga Porphyridium cruentum: oxygen evolution, ultrastructure, and polypeptide resolution,”
Arch. Biochem. Biophys.
238:10-17; Kirilovsky D, et al. (1986). “Functional assembly in vitro of phycobilisomes with isolated photosystem II particles of eukaryotic chloroplasts,”
J. Biol. Chem.,
261:12317-12323), phycobilisome-membrane preparations (e.g., Clement-Metral, J. D., et al. (1971), “Fluorescence transfer in glutaraldehyde fixed particles of the red alga Porphyridium cruentrum (N),”
FEBS Letters
12:225-228), phycobilisome dissociation (e.g., Rigbi, et al. (1980), “Cyanobacterial phycobilisomes: Selective dissociation monitored by fluorescence and circular dichroism.”
Proc. Natl. Acad. Sci. USA,
77:1961-1965) and reconstitution (e.g., Gantt, et al. (1979), “Phycobilisomes from blue-green and red algae: Isolation criteria and dissociation characteristics,”
Plant Physiology
63:615-620; Kirilovsky et al. (1986), Glick, et al. (1983), “Role of the colorless polypeptides in phycobilisome reconstitution from separated pycobiliproteins,”
Plant Physiol.,
69:991-997), genetic modifications (e.g., Bryant, D. A., 1991, “Cyanobacterial phycobilisomes: progress toward complete structural and functional analysis via molecular genetics,” In L. Bogorad and I.K. Vasil (ed.),
Cell Culture and Somatic Genetics of Plants. Molecular Biology of Plastids
and
Mitochondria,
Vol. 7, pp. 257-300, Academic Press, San Diego, Calif.); Yamanaka, et al. (1978), “Cyanobacterial phycobilisomes. Characterization of the phycobilisomes of Synechococcus sp. 6302,”
J. Biol. Chem.,
253:8303-8310; Yamanaka, et al. (1980), “Molecular architecture of a light-harvesting antenna. Comparison of wild type and mutant Synechococcus 6301 phycobilisomes,”
J. Biol. Chem.,
255:11004-11010), and environmental effects (e.g., Grossman et al. (1993)), including chromatic adaptation (e.g., Bryant, et al. 1981, “Effects of chromatic illumination on cyanobacterial phycobilisomes: Evidence for the specific induction of a second pair of phycocyanin subunits in Pseudanabaena 7409 grown in red light,”
Eur. J. Biochem.
119:415-424).
Isolated phycobilisomes readily dissociate into free phycobiliproteins and a variety of phycobiliprotein complexes under all but the most favorable conditions. Low to moderate ionic strength (<0.5 M phosphate), low phycobilisome concentration (<1 mg/ml), and low and high temperatures lead to dissociation of phycobilisomes (Katoh, (1988)
Methods in Enzymology,
162:313-318; Gantt et al., (1979)). Freezing of algae is also reported to lead to destruction of phycobilisomes (Gantt et al., (1972)
Journal of Cell Biology,
54:313-324).
Isolated phycobiliproteins, the component fluorescent proteins of phycobilisomes, have been used as labels in immunoassays. See e.g., Stryer et al., U.S. Pat. No. 4,520,110 and Kronick et al. (1983)
Clinical Chemistry,
29:1582-1586. However, because of the difficulty in isolating and manipulating intact phycobilisomes, the art has not recognized that these macromolecular assemblies could be similarly utilized. Because the signal which phycobilisomes can provide is theoretically so much larger than that of isolated phycobiliproteins, there is a need in the art for methods of treating phycobilisomes so that they can be used as detectable markers for a host of assays and other applications.
SUMMARY OF THE INVENTION
One object of this invention is to provide supramolecular complexes with diverse spectral properties for use as highly detectable tracers and labels.
Another object of this invention is to provide a versatile set of highly sensitive signal-generating systems and conjugates for use in, inter alia, various assay methods.
Yet another object of this invention is to provide biotransducers comprising phycobilisomes o
Brobeck Phleger & Harrison LLP
Graser Jennifer E.
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