Microfluidics-based selection of red-fluorescent proteins with decreased rates of photobleaching

Integr Biol (Camb). 2015 Feb;7(2):263-73. doi: 10.1039/c4ib00251b.

Abstract

Fluorescent proteins offer exceptional labeling specificity in living cells and organisms. Unfortunately, their photophysical properties remain far from ideal for long-term imaging of low-abundance cellular constituents, in large part because of their poor photostability. Despite widespread engineering efforts, improving the photostability of fluorescent proteins remains challenging due to lack of appropriate high-throughput selection methods. Here, we use molecular dynamics guided mutagenesis in conjunction with a recently developed microfluidic-based platform, which sorts cells based on their fluorescence photostability, to identify red fluorescent proteins with decreased photobleaching from a HeLa cell-based library. The identified mutant, named Kriek, has 2.5- and 4-fold higher photostability than its progenitor, mCherry, under widefield and confocal illumination, respectively. Furthermore, the results provide insight into mechanisms for enhancing photostability and their connections with other photophysical processes, thereby providing direction for ongoing development of fluorescent proteins with improved single-molecule and low-copy imaging capabilities.

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

  • Directed Molecular Evolution
  • Fluorescent Dyes / chemistry
  • Fluorescent Dyes / radiation effects
  • HeLa Cells
  • Humans
  • Lab-On-A-Chip Devices
  • Luminescent Proteins / chemistry*
  • Luminescent Proteins / genetics
  • Luminescent Proteins / radiation effects
  • Microfluidics / methods*
  • Molecular Dynamics Simulation
  • Mutagenesis
  • Photobleaching*
  • Protein Stability / radiation effects

Substances

  • Fluorescent Dyes
  • Luminescent Proteins
  • red fluorescent protein