Photoswitchable red fluorescent protein with a large Stokes shift

Area of Science:

• Biochemistry
• Biophysics
• Molecular Biology

Background:

• Fluorescent proteins (FPs) are vital tools in biological research.
• Large Stokes shift (LSS) FPs offer advantages due to spectral separation.
• Photoswitchable FPs allow for dynamic imaging applications.

Purpose of the Study:

• To develop and characterize a photoswitchable red FP with a large Stokes shift.
• To investigate the molecular mechanisms underlying its properties.
• To demonstrate its utility in advanced microscopy and live-cell imaging.

Main Methods:

• Spectroscopic analysis (excitation/emission spectra, photophysics).
• Biochemical characterization in vitro and in mammalian cells.
• Crystal structure determination.
• Mass spectrometry and mutagenesis studies.
• Superresolution photoactivated localization microscopy (PALM).

Main Results:

• PSLSSmKate exhibits initial excitation/emission at 445/622 nm.
• Violet irradiation photoswitches PSLSSmKate to a red form (573/621 nm).
• Detailed characterization of spectral, photophysical, and biochemical properties.
• Proposed molecular mechanisms for LSS, pH dependence, and photoswitching.
• Successful application in PALM and live-cell protein dynamics.

Conclusions:

• PSLSSmKate is a versatile photoswitchable LSS FP.
• Its properties are well-defined and suitable for advanced imaging.
• PSLSSmKate facilitates photoactivatable imaging and tracking of intracellular objects.