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

Journal Article · · Proceedings of the National Academy of Sciences of the United States of America
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  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)
+ Show Author Affiliations

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.

Research Organization:
Argonne National Laboratory (ANL), Argonne, IL (United States)
Sponsoring Organization:
USDOE Office of Science (SC)
Grant/Contract Number:
AC02-06CH11357
OSTI ID:
1163387
Journal Information:
Proceedings of the National Academy of Sciences of the United States of America, Vol. 111, Issue 43; ISSN 0027-8424
Publisher:
National Academy of Sciences, Washington, DC (United States)Copyright Statement
Country of Publication:
United States
Language:
English
Citation Metrics:
Cited by: 54 works
Citation information provided by
Web of Science

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conference June 2009
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Cited By (15)

Computational Aminoacyl-tRNA Synthetase Library Design for Photocaged Tyrosine text January 2019
Protein labeling for live cell fluorescence microscopy with a highly photostable renewable signal journal January 2017
Computational Aminoacyl-tRNA Synthetase Library Design for Photocaged Tyrosine journal May 2019
Assembly of Lipoic Acid on Its Cognate Enzymes: an Extraordinary and Essential Biosynthetic Pathway journal April 2016
Broad substrate tolerance of tubulin tyrosine ligase enables one-step site-specific enzymatic protein labeling journal January 2017
Advances in chemical labeling of proteins in living cells journal March 2015
Sequence-based prediction of permissive stretches for internal protein tagging and knockdown text January 2017
Genetic Code Expansion Enables Live-Cell and Super-Resolution Imaging of Site-Specifically Labeled Cellular Proteins journal April 2015
Versatile Interacting Peptide (VIP) Tags for Labeling Proteins with Bright Chemical Reporters journal January 2017
Sequence-based prediction of permissive stretches for internal protein tagging and knockdown text January 2017
Sequence-based prediction of permissive stretches for internal protein tagging and knockdown journal October 2017
Biosynthetic engineering of nonribosomal peptide synthetases: Engineering NRPSs journal July 2016
Engineering of a Red Fluorogenic Protein/Merocyanine Complex for Live‐Cell Imaging journal November 2019
Self-labelling enzymes as universal tags for fluorescence microscopy, super-resolution microscopy and electron microscopy journal December 2015
Fluorescent Reporters and Biosensors for Probing the Dynamic Behavior of Protein Kinases journal November 2015

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