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 276nm, 377nm, and 460nm and a single emission peak at 530nm. 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.
Highlights
Fluorescent proteins (FPs) make it possible to visualize biological processes through in vivo imaging and in vitro fluorescence labeling
Lucigen Yellow (LucY) was discovered while screening for carbohydrate active enzymes in a metagenomic library prepared from corn stalks using a fluorescent substrate
The purified MurB protein from E. coli exhibits a yellow color [8], and quenching of the fluorescence of the tightly bound flavin adenine dinucleotide (FAD) cofactor is used to assay binding of compounds to S. aureus MurB protein [9], suggesting that fluorescence of the metagenomic clone is due to expression of the murB gene
Summary
Fluorescent proteins (FPs) make it possible to visualize biological processes through in vivo imaging and in vitro fluorescence labeling. Processes such as protein expression, localization, degradation, and interaction can be observed through fusion of a protein of interest with a FP. Green Fluorescent Protein (GFP) from Aequorea victoria and its varied derivatives constitute a multi-colored toolbox ranging from blue to yellow, with red-shifted FPs (RFPs) originating mostly from the sea anemone Discosoma striata [1]. LucY Fluorescent Protein design, data collection and analysis, decision to publish, or preparation of the manuscript
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