Computational design of a red fluorophore ligase for site-specific protein labeling in living cells
Name
Liu-2014-Computational design.pdf
Size
1.64 MB
Format
Adobe PDF
Checksum (MD5)
b52d551ed3af04b843e3e565aad3d816
Author(s)
Baker, David
Ting, Alice Y.
Liu, Daniel S.
Nivon, Lucas G.
Richter, Florian
Goldman, Peter J.
Yao, Jennifer Z.
Phipps, William S.
Ye, Anne Z.
Deerinck, Thomas J.
Date Issued
October 2014
Journal
Proceedings of the National Academy of Sciences of the United States of America
Publisher
National Academy of Sciences (U.S.)
Citation
Liu, D. S., L. G. Nivon, F. Richter, P. J. Goldman, T. J. Deerinck, J. Z. Yao, D. Richardson, et al. “Computational Design of a Red Fluorophore Ligase for Site-Specific Protein Labeling in Living Cells.” Proceedings of the National Academy of Sciences 111, no. 43 (October 13, 2014): E4551–E4559.
Version
Final published version
Abstract
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.
MIT Department
Massachusetts Institute of Technology. Department of Biology
Massachusetts Institute of Technology. Department of Chemistry
Terms of Use
Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use.
Persistent DSpace Link
DOI of Published Version
http://dx.doi.org/10.1073/pnas.1404736111
