Circular permutation of red fluorescent proteins

PLoS One. 2011;6(5):e20505. doi: 10.1371/journal.pone.0020505. Epub 2011 May 27.

Abstract

Circular permutation of fluorescent proteins provides a substrate for the design of molecular sensors. Here we describe a systematic exploration of permutation sites for mCherry and mKate using a tandem fusion template approach. Circular permutants retaining more than 60% (mCherry) and 90% (mKate) brightness of the parent molecules are reported, as well as a quantitative evaluation of the fluorescence from neighboring mutations. Truncations of circular permutants indicated essential N- and C-terminal segments and substantial flexibility in the use of these molecules. Structural evaluation of two cp-mKate variants indicated no major conformational changes from the previously reported wild-type structure, and cis conformation of the chromophores. Four cp-mKates were identified with over 80% of native fluorescence, providing important new building blocks for sensor and complementation experiments.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Color
  • Crystallography, X-Ray
  • Luminescent Proteins / chemistry*
  • Luminescent Proteins / metabolism
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Structure, Secondary
  • Spectrometry, Fluorescence

Substances

  • Luminescent Proteins
  • red fluorescent protein

Associated data

  • PDB/3RWA
  • PDB/3RWT