Engineering ESPT pathways based on structural analysis of LSSmKate red fluorescent proteins with large Stokes shift
Kiryl D Piatkevich1, Vladimir N Malashkevich, Steven C Almo
1Department of Anatomy and Structural Biology, Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
Journal of the American Chemical Society
|August 5, 2010
Summary
Engineered red fluorescent proteins (RFPs) with large Stokes shifts (LSSs) utilize excited-state proton transfer for unique spectral properties. Rational design enabled LSS fluorescence across a broad spectrum in various RFPs.
Area of Science:
- Biochemistry
- Biophysics
- Molecular Biology
Background:
- Monomeric red fluorescent proteins (RFPs) with large Stokes shifts (LSSs) offer spectral advantages for biological imaging.
- LSSmKate1 and LSSmKate2 possess unique absorbance and emission profiles due to their chromophore configurations.
Purpose of the Study:
- To elucidate the mechanism behind the large Stokes shifts in LSSmKate1 and LSSmKate2.
- To engineer novel RFPs with LSS properties by modifying existing fluorescent proteins.
Main Methods:
- X-ray crystallography to determine the structures of LSSmKate1 and LSSmKate2.
- Mutagenesis studies, isotope, and temperature dependence analyses to investigate excited-state proton transfer (ESPT).
- Site-directed mutagenesis of conventional RFPs (mNeptune, mCherry, mStrawberry, mOrange, mKO) at key positions.
Main Results:
- LSSmKate1 features a cis chromophore configuration, while LSSmKate2 has a trans configuration.
- ESPT mechanisms involving specific amino acid residues (Glu160, Ser158, Asp160) were identified as responsible for the LSS.
- Engineered RFP variants exhibited LSS fluorescence emission ranging from 560 to 640 nm.
Conclusions:
- ESPT is the key mechanism conferring large Stokes shifts in LSSmKates.
- Rational engineering of amino acid residues can introduce LSS properties into various RFPs.
- This work provides a framework for designing novel RFPs with tailored spectral characteristics for diverse applications.
Related Concept Videos
01:19Protein Dynamics in Living Cells
Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
01:37Super-resolution Fluorescence Microscopy
Super-resolution fluorescence microscopy (SRFM) provides a better resolution than conventional fluorescence microscopy by reducing the point spread function (PSF). PSF is the light intensity distribution from a point that causes it to appear blurred. Due to PSF, each fluorescing point appears bigger than its actual size, and it is the PSF interference of nearby fluorophores that causes the blurred image. Various approaches to achieving higher resolution through SRFM have recently been developed.