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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.
 [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:
OSTI Identifier:
Accepted Manuscript
Journal Name:
Additional Journal Information:
Journal Volume: 10; Journal Issue: 4; Journal ID: ISSN 1932-6203
Public Library of Science
Research Org:
Advanced Photon Source (APS), Argonne National Laboratory (ANL), Argonne, IL (US)
Sponsoring Org:
Country of Publication:
United States
59 BASIC BIOLOGICAL SCIENCES crystal structure; enzyme structure; magnesium; yellow fluorescent protein; flavin; permutation; staphylococcus aureus; crystals