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Title: Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

In this study, chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.
Authors:
 [1] ;  [2] ;  [2] ;  [1] ;  [3] ;  [1] ;  [4] ;  [1] ;  [1] ;  [3] ;  [1] ;  [2] ;  [1]
  1. Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States)
  2. Univ. of Washington, Seattle, WA (United States)
  3. Univ. of California, San Diego, La Jolla, CA (United States)
  4. Max Planck Institute for Biophysical Chemistry, Gottingen (Germany)
Publication Date:
OSTI Identifier:
1163387
Grant/Contract Number:
AC02-06CH11357
Type:
Accepted Manuscript
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 111; Journal Issue: 43; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences, Washington, DC (United States)
Research Org:
Argonne National Laboratory (ANL), Argonne, IL (United States)
Sponsoring Org:
USDOE Office of Science (SC)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES fluorescence microscopy; enzyme redesign; LplA; PRIME; chemical probe targeting