Kinetic study of de novo chromophore maturation of fluorescent proteins

Anal Biochem. 2011 Jul 15;414(2):173-8. doi: 10.1016/j.ab.2011.03.036. Epub 2011 Apr 1.

Abstract

Green fluorescent protein (GFP) has a chromophore that forms autocatalytically within the folded protein. Although many studies have focused on the precise mechanism of chromophore maturation, little is known about the kinetics of de novo chromophore maturation. Here we present a simple and efficient method for examining the de novo kinetics. GFP with an immature chromophore was synthesized in a reconstituted cell-free protein synthesis system under anaerobic conditions. Chromophore maturation was initiated by rapid dilution in an air-saturated maturation buffer, and the time course of fluorescence development was monitored. Comparison of the de novo maturation rates in various GFP variants revealed that some folding mutations near the chromophore promoted rapid chromophore maturation and that the accumulation of mutations could reduce the maturation rate. Our method will contribute to the design of rapidly maturing fluorescent proteins with improved characteristics for real-time monitoring of cellular events.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / biosynthesis
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Fluorescent Dyes / chemistry*
  • Fluorescent Dyes / metabolism
  • Green Fluorescent Proteins / biosynthesis
  • Green Fluorescent Proteins / chemistry*
  • Green Fluorescent Proteins / genetics
  • Kinetics
  • Luminescent Proteins / biosynthesis
  • Luminescent Proteins / chemistry
  • Luminescent Proteins / genetics
  • Mutagenesis, Site-Directed
  • Protein Folding

Substances

  • Bacterial Proteins
  • Fluorescent Dyes
  • Luminescent Proteins
  • yellow fluorescent protein, Bacteria
  • Green Fluorescent Proteins