Structural and spectroscopic characterization of photoactive yellow protein and photoswitchable fluorescent protein constructs containing heavy atoms

Abstract

Photo-induced structural rearrangements of chromophore-containing proteins are essential for various light-dependent signaling pathways and optogenetic applications. Ultrafast structural and spectroscopic methods have offered insights into these structural rearrangements across many timescales. However, questions still remain about exact mechanistic details, especially regarding photoisomerization of the chromophore within these proteins femtoseconds to picoseconds after photoexcitation. Instrumentation advancements for time-resolved crystallography and ultrafast electron diffraction provide a promising opportunity to study these reactions, but achieving enough signal-to-noise is a constant challenge. Here we present four new photoactive yellow protein constructs and one new fluorescent protein construct that contain heavy atoms either within or around the chromophore and can be expressed with high yields. Structural characterization of these constructs, most at atomic resolution, show minimal perturbation caused by the heavy atoms compared to wild-type structures. Spectroscopic studies report the effects of the heavy atom identity and location on the chromophore's photophysical properties. None of the substitutions prevent photoisomerization, although certain rates within the photocycle may be affected. Overall, these new proteins containing heavy atoms are ideal samples for state-of-the-art time-resolved crystallography and electron diffraction experiments to elucidate crucial mechanistic information of photoisomerization. File list (2) download file view on ChemRxiv MainText_ChemRxiv.pdf (2.62 MiB) download file view on ChemRxiv SI_ChemRxiv.pdf (438.80 KiB) Structural and spectroscopic characterization of photoactive yellow protein and photoswitchable fluorescent protein constructs containing heavy atoms

Serial Femtosecond Crystallography Reveals that Photoactivation in a Fluorescent Protein Proceeds via the Hula Twist Mechanism

“…The normalized absorption spectrum and structure of Cl-rsEGFP2 (c) possess very similar properties as the non-chlorinated protein: the OFF state absorbing predominantly at 400 nm and the ON state at around 480 nm, while the protein tertiary structure exhibits the β-barrel fold typical of GFP-like constructs. A 2 nm shift can be observed in the absorption profile between chlorinated and non-chlorinated constructs caused by the electron-withdrawing nature of chlorine …”

“…As with previous studies, we focus here on the OFF-to-ON reaction, which has a higher quantum yield than the ON-to-OFF reaction. , The 4-hydroxybenzylidene-1,2-dimethylimidazolinone chromophore’s phenol ring in rsEGFP2 has a C 2 point group symmetry, leading to equivalent cis products regardless of the photoisomerization pathway. We exploit the introduction of a chlorine atom substituent to break this symmetry and distinguish between the products formed via the OBF and HT pathways, as previously suggested . On the basis of IUPAC recommendations, − starting from a trans chromophore with a substituent anti to the double-bonded imidazolinone nitrogen, HT leads to a cis anti product, while the configuration formed via OBF is cis syn (Figure b).…”

A Short and Practical Overview on Light-Sensing Proteins, Optogenetics, and Fluorescent Biomolecules inside Biomorphs Used as Optical Sensors

“…In optogenetics, this protein can be used as a tunable optical switch because blue light triggers trans to cis isomerization of the chromophore and production of a light state with altered conformational dynamics. Based on this property, PYP has been used as switchable protein in optogenetics and it is usually linked to effector domains resulting in soluble, well-behaved constructs [44,45]. This feasibility to form specific constructs has been used to design structure-based photoswitchable affibody scaffolds for applications in immunochemistry, particularly in the use of antibodies using the tunable properties of the PYP-Z-domain with a light-dependent binding in vitro and in vivo [46].…”