skip to main content

DOE PAGESDOE PAGES

Title: LucY: A versatile new fluorescent reporter protein

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:
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
Research Org:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org:
OTHER U.S. STATES
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
OSTI Identifier:
1178845

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., 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. 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., and Michnick, Stephen W.. 2015. "LucY: A versatile new fluorescent reporter protein". United States. doi: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 = {2015},
month = {4}
}

Works referenced in this record:

The Green Fluorescent Protein
journal, June 1998

Coot model-building tools for molecular graphics
journal, November 2004
  • Emsley, Paul; Cowtan, Kevin
  • Acta Crystallographica Section D Biological Crystallography, Vol. 60, Issue 12, p. 2126-2132
  • DOI: 10.1107/S0907444904019158

Protein production by auto-induction in high-density shaking cultures
journal, May 2005

Basic local alignment search tool
journal, October 1990
  • Altschul, Stephen F.; Gish, Warren; Miller, Webb
  • Journal of Molecular Biology, Vol. 215, Issue 3, p. 403-410
  • DOI: 10.1016/S0022-2836(05)80360-2

PHENIX: a comprehensive Python-based system for macromolecular structure solution
journal, January 2010
  • Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2, p. 213-221
  • DOI: 10.1107/S0907444909052925

Visualization of Interactions among bZIP and Rel Family Proteins in Living Cells Using Bimolecular Fluorescence Complementation
journal, April 2002