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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 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.

Authors:
 [1];  [2];  [3];  [4];  [5];  [2];  [6];  [1]; ;
  1. Lucigen Corp., Middleton, WI (United States)
  2. Rice Univ., Houston, TX (United States)
  3. Lucigen Corp., Middleton, WI (United States); Boehringer Ingelheim, Ridgefield, CT (United States)
  4. Lucigen Corp., Middleton, WI (United States); R&D Systems, Minneapolis, MN (United States)
  5. Univ. of Wisconsin, Madison, WI (United States)
  6. Rice Univ., Houston, TX (United States); Univ. of Wisconsim, Madison, WI (United States)
Publication Date:
Research Org.:
Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
OTHER U.S. STATES
OSTI Identifier:
1178845
Resource Type:
Accepted Manuscript
Journal Name:
PLoS ONE
Additional Journal Information:
Journal Volume: 10; Journal Issue: 4; Journal ID: ISSN 1932-6203
Publisher:
Public Library of Science
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; crystal structure; enzyme structure; magnesium; yellow fluorescent protein; flavin; permutation; staphylococcus aureus; crystals

Citation Formats

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., and Michnick, Stephen W. LucY: A versatile new fluorescent reporter protein. United States: N. p., 2015. Web. doi:10.1371/journal.pone.0124272.
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. LucY: A versatile new fluorescent reporter protein. United States. https://doi.org/10.1371/journal.pone.0124272
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., and Michnick, Stephen W. Thu . "LucY: A versatile new fluorescent reporter protein". United States. https://doi.org/10.1371/journal.pone.0124272. https://www.osti.gov/servlets/purl/1178845.
@article{osti_1178845,
title = {LucY: A versatile new fluorescent reporter protein},
author = {Auldridge, Michele E. and Cao, Hongnan and Sen, Saurabh and Franz, Laura P. and Bingman, Craig A. and Yennamalli, Ragothaman M. and Phillips, Jr., George N. and Mead, David and Steinmetz, Eric J. and Michnick, Stephen W.},
abstractNote = {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.},
doi = {10.1371/journal.pone.0124272},
journal = {PLoS ONE},
number = 4,
volume = 10,
place = {United States},
year = {Thu Apr 23 00:00:00 EDT 2015},
month = {Thu Apr 23 00:00:00 EDT 2015}
}

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