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Published December 20, 2012 | Published + Supplemental Material
Journal Article Open

Recovery of Red Fluorescent Protein Chromophore Maturation Deficiency through Rational Design


Red fluorescent proteins (RFPs) derived from organisms in the class Anthozoa have found widespread application as imaging tools in biological research. For most imaging experiments, RFPs that mature quickly to the red chromophore and produce little or no green chromophore are most useful. In this study, we used rational design to convert a yellow fluorescent mPlum mutant to a red-emitting RFP without reverting any of the mutations causing the maturation deficiency and without altering the red chromophore's covalent structure. We also created an optimized mPlum mutant (mPlum-E16P) that matures almost exclusively to the red chromophore. Analysis of the structure/function relationships in these proteins revealed two structural characteristics that are important for efficient red chromophore maturation in DsRed-derived RFPs. The first is the presence of a lysine residue at position 70 that is able to interact directly with the chromophore. The second is an absence of non-bonding interactions limiting the conformational flexibility at the peptide backbone that is oxidized during red chromophore formation. Satisfying or improving these structural features in other maturation-deficient RFPs may result in RFPs with faster and more complete maturation to the red chromophore.

Additional Information

© 2012 Moore et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Received: September 18, 2012; Accepted: November 19, 2012; Published: December 20, 2012. This work was supported by the Natural Sciences and Engineering Research Council of Canada (http://www.nserc-crsng.gc.ca), the University of Ottawa (http://www.uottawa.ca), and the Defense Advanced Research Projects Agency Protein Design Processes (http://www.darpa.mil). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank Marie L. Ary for help with the manuscript. We would like to acknowledge the Gordon and Betty Moore Foundation for support of the Molecular Observatory at Caltech, and the Department of Energy and National Institutes of Health for supporting the Stanford Synchrotron Radiation Lightsource. Author Contributions: Conceived and designed the experiments: MMM SLM RAC. Performed the experiments: MMM SKO ATP. Analyzed the data: MMM SKO ATP RAC. Wrote the paper: MMM RAC.

Attached Files

Published - journal.pone.0052463.pdf

Supplemental Material - journal.pone.0052463.s001.tif

Supplemental Material - journal.pone.0052463.s002.tif

Supplemental Material - journal.pone.0052463.s003.tif

Supplemental Material - journal.pone.0052463.s004.tif

Supplemental Material - journal.pone.0052463.s005.docx


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August 19, 2023
October 20, 2023