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PLoS One. 2015 Apr 23;10(4):e0124272. doi: 10.1371/journal.pone.0124272. eCollection 2015.

LucY: A Versatile New Fluorescent Reporter Protein.

Author information

1
Lucigen Corp., Middleton, WI, United States of America.
2
Department of Biochemistry and Cell Biology, Rice University, Houston, Texas, United States of America.
3
Department of Biochemistry, University of Wisconsin, Madison, WI, United States of America.
4
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.

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.

PMID:
25906065
PMCID:
PMC4408115
DOI:
10.1371/journal.pone.0124272
[Indexed for MEDLINE]
Free PMC Article

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