Recovery of red fluorescent protein chromophore maturation deficiency through rational design

PLoS One. 2012;7(12):e52463. doi: 10.1371/journal.pone.0052463. Epub 2012 Dec 20.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Absorption
  • Crystallography, X-Ray
  • Kinetics
  • Luminescent Proteins / chemistry
  • Luminescent Proteins / metabolism*
  • Models, Molecular
  • Mutant Proteins / metabolism
  • Mutation
  • Protein Engineering*
  • Spectrometry, Fluorescence
  • Static Electricity


  • Luminescent Proteins
  • Mutant Proteins
  • red fluorescent protein

Associated data

  • PDB/4H3L
  • PDB/4H3M
  • PDB/4H3N

Grant support

This work was supported by the Natural Sciences and Engineering Research Council of Canada (, the University of Ottawa (, and the Defense Advanced Research Projects Agency Protein Design Processes ( The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.