Developing Red Fluorescent Proteins – Tools and Strategies

ORAL  · Invited

Abstract

Fluorescent proteins have become an indispensable tool for biological and biomedical research owing to their genetic encodability. Red Fluorescent Proteins (RFPs) are advantageous for live-cell imaging due to low optical attenuation and phototoxicity for excitation beyond 550 nm. We are devoted to developing novel strategies and tools to improve RFPs with multiple desirable photophysical properties. The most commonly adopted strategy to enhance molecular brightness of RFPs is to reduce the nonradiative decay pathways, which lengthens the excited state lifetime and possibly reduces photostability due to the longer time spent in the excited state. We propose to simultaneously improve molecular brightness and photostability of RFPs by increasing the radiative decay rate. We also develop microfluidic sorters capable of selections based on multiple photophysical properties. We use these instruments to accelerate and visualize the evolution of RFPs towards desirable photophysical properties for library sizes on the order of 106 mutants.

*This work was supported by the NSF Physics Frontier Center at JILA (PHY 1734006 to R. J.), NIH DP1 GM114863 and R01 GM084027 (to A. E. P.), and the University of Colorado Molecular Biophysics Training Grant (T32 G-065103 to S. M.).

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Presenters

  • Sheng-Ting Hung

    • JILA, NIST and University of Colorado, Boulder, Colorado 80309, USA

Authors

  • Sheng-Ting Hung

    • JILA, NIST and University of Colorado, Boulder, Colorado 80309, USA

  • Premashis Manna

    • JILA, NIST and University of Colorado, Boulder, Colorado 80309, USA

  • Srijit Mukherjee

    • JILA, NIST and University of Colorado, Boulder, Colorado 80309, USA, Department of Chemistry, University of Colorado, Boulder, Colorado 80309, USA

  • Pia Friis

    • JILA, NIST and University of Colorado, Boulder, Colorado 80309, USA

  • David M. Simpson

    • BioFrontiers Institute, University of Colorado, Boulder, Colorado 80309, USA

  • Maria N. Lo

    • BioFrontiers Institute, University of Colorado, Boulder, Colorado 80309, USA

  • Amy E. Palmer

    • BioFrontiers Institute, University of Colorado, Boulder, Colorado 80309, USA, Department of Biochemistry, University of Colorado, Boulder, Colorado 80309, USA

  • Ralph Jimenez

    • JILA, NIST and University of Colorado, Boulder, Colorado 80309, USA, Department of Chemistry, University of Colorado, Boulder, Colorado 80309, USA