LucY: A versatile new fluorescent reporter protein

Auldridge, Michele E.; Cao, Hongnan; Sen, Saurabh; Franz, Laura P.; Bingman, Craig A.; Yennamalli, Ragothaman M.; Phillips, Jr., George N.; Mead, David; Steinmetz, Eric J.; Michnick, Stephen W.

doi:10.1371/journal.pone.0124272

Title: LucY: A versatile new fluorescent reporter protein

Journal Article · Thu Apr 23 00:00:00 EDT 2015 · PLoS ONE

DOI:https://doi.org/10.1371/journal.pone.0124272· OSTI ID:1178845

Auldridge, Michele E. ^[1]; Cao, Hongnan ^[2]; Sen, Saurabh ^[3]; Franz, Laura P. ^[4]; Bingman, Craig A. ^[5]; Yennamalli, Ragothaman M. ^[2]; Phillips, Jr., George N. ^[6]; Mead, David ^[1]; Steinmetz, Eric J.; Michnick, Stephen W.

Lucigen Corp., Middleton, WI (United States)
Rice Univ., Houston, TX (United States)
Lucigen Corp., Middleton, WI (United States); Boehringer Ingelheim, Ridgefield, CT (United States)
Lucigen Corp., Middleton, WI (United States); R&D Systems, Minneapolis, MN (United States)
Univ. of Wisconsin, Madison, WI (United States)
Rice Univ., Houston, TX (United States); Univ. of Wisconsim, Madison, WI (United States)

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.

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Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Organization:: OTHER U.S. STATES

OSTI ID:: 1178845

Journal Information:: PLoS ONE, Vol. 10, Issue 4; ISSN 1932-6203

Publisher:: Public Library of ScienceCopyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 2 works

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Title: LucY: A versatile new fluorescent reporter protein

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