title: LucY: A Versatile New Fluorescent Reporter Protein.
abstract: We report on the discovery, isolation, and use of a novel yellow fluorescent protein. Lucigen Yellow (LucY) binds one FAD molecule within its core, thus shielding it from water and maintaining its structure so that fluorescence is 10-fold higher than freely soluble FAD. LucY displays excitation and emission spectra characteristic of FAD, with 3 excitation peaks at 276 nm, 377 nm, and 460 nm and a single emission peak at 530 nm. These excitation and emission maxima provide the large Stokes shift beneficial to fluorescence experimentation. LucY belongs to the MurB family of UDP-N-acetylenolpyruvylglucosamine reductases. The high resolution crystal structure shows that in contrast to other structurally resolved MurB enzymes, LucY does not contain a potentially quenching aromatic residue near the FAD isoalloxazine ring, which may explain its increased fluorescence over related proteins. Using E. coli as a system in which to develop LucY as a reporter, we show that it is amenable to circular permutation and use as a reporter of protein-protein interaction. Fragmentation between its distinct domains renders LucY non-fluorescent, but fluorescence can be partially restored by fusion of the fragments to interacting protein domains. Thus, LucY may find application in Protein-fragment Complementation Assays for evaluating protein-protein interactions.
authors.affiliation: Lucigen Corp., Middleton, WI, United States of America.
authors.forename: Michele E
authors.initials: ME
authors.lastname: Auldridge
authors.affiliation: Department of Biochemistry and Cell Biology, Rice University, Houston, Texas, United States of America.
authors.forename: Hongnan
authors.initials: H
authors.lastname: Cao
authors.affiliation: Lucigen Corp., Middleton, WI, United States of America.
authors.forename: Saurabh
authors.initials: S
authors.lastname: Sen
authors.affiliation: Lucigen Corp., Middleton, WI, United States of America.
authors.forename: Laura P
authors.initials: LP
authors.lastname: Franz
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, WI, United States of America.
authors.forename: Craig A
authors.initials: CA
authors.lastname: Bingman
authors.affiliation: Department of Biochemistry and Cell Biology, Rice University, Houston, Texas, United States of America.
authors.forename: Ragothaman M
authors.initials: RM
authors.lastname: Yennamalli
authors.affiliation: Department of Biochemistry and Cell Biology, Rice University, Houston, Texas, United States of America; Department of Biochemistry, University of Wisconsin, Madison, WI, United States of America.
authors.forename: George N
authors.initials: GN
authors.lastname: Phillips
authors.affiliation: Lucigen Corp., Middleton, WI, United States of America.
authors.forename: David
authors.initials: D
authors.lastname: Mead
authors.affiliation: Lucigen Corp., Middleton, WI, United States of America.
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Steinmetz
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000378:metabolism; D017930:Genes, Reporter; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011994:Recombinant Proteins; C054562:yellow fluorescent protein, Bacteria; D005182:Flavin-Adenine Dinucleotide