title: Biophysical characterization of natural and mutant fluorescent proteins cloned from zooxanthellate corals.
abstract: Two novel colored fluorescent proteins were cloned and biophysically characterized from zooxanthellate corals (Anthozoa). A cyan fluorescent protein derived from the coral Montastrea cavernosa (mcCFP) is a trimeric complex with strong blue-shifted excitation and emission maxima at 432 and 477 nm, respectively. The native complex has a fluorescence lifetime of 2.66+/-0.01 ns and an inferred absolute quantum yield of 0.385. The spectroscopic properties of a green fluorescent protein cloned from Meandrina meandrites (mmGFP) resemble the commercially available GFP derived originally from the hydrozoan Aequorea victoria (avGFP). mmGFP is a monomeric protein with an excitation maximum at 398 nm and an emission maximum at 505 nm, a fluorescence lifetime of 3.10+/-0.01 ns and an absolute quantum yield of 0.645. Sequence homology with avGFP and the red fluorescent protein (DsRed) indicates that the proteins adopt the classic beta-barrel configuration with 11 beta-strands. The three amino acid residues that comprise the chromophore are QYG for mcCFP and TYG for mmGFP, compared with SYG for avGFP. A single point mutation, Ser-110 to Asn, was introduced into mmGFP by random mutagenesis. Denaturation and refolding experiments showed that the mutant has reduced aggregation, increased solubility and more efficient refolding relative to the wild type. Time-resolved emission lifetimes and anisotropies suggest that the electronic structure of the chromophore is highly dependent on the protonation state of adjoining residues.
authors.affiliation: Environmental Biophysics and Molecular Ecology Program, Institute of Marine and Coastal Sciences, Rutgers University, 71 Dudley Road, New Brunswick, NJ 08901, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Sun
authors.forename: Edward W
authors.initials: EW
authors.lastname: Castner
authors.forename: Catherine L
authors.initials: CL
authors.lastname: Lawson
authors.forename: Paul G
authors.initials: PG
authors.lastname: Falkowski
mesh_terms: D000818:Animals; D016880:Anisotropy; D037421:Anthozoa; D001216:Asparagine / Q000737:chemistry; D055592:Biophysical Phenomena; D001703:Biophysics; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D049452:Green Fluorescent Proteins; D019791:Guanidine / Q000737:chemistry; D007700:Kinetics; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010802:Phylogeny; D017354:Point Mutation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012694:Serine / Q000737:chemistry; D013053:Spectrophotometry; D013696:Temperature; D013997:Time Factors
chemical_list: D017931:DNA Primers; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D012694:Serine; D001216:Asparagine; D019791:Guanidine