Photoisomerization and Proton Transfer in the Forward and Reverse Photoswitching of the Fast-Switching M159T Mutant of the Dronpa Fluorescent Protein

Abstract

The fast-switching M159T mutant of the reversibly photoswitchable fluorescent protein Dronpa has an enhanced yield for the on-to-off reaction. The forward and reverse photoreactions proceed via cis-trans and trans-cis photoisomerization, yet protonation and deprotonation of the hydroxyphenyl oxygen of the chromophore is responsible for the majority of the resulting spectroscopic contrast. Ultrafast visible-pump, infrared-probe spectroscopy was used to detect the picosecond, nanosecond, as well as metastable millisecond intermediates. Additionally, static FTIR difference measurements of the Dronpa-M159T mutant correspond very closely to those of the wild type Dronpa, identifying the p-hydroxybenzylidene-imidazolinone chromophore in the cis anion and trans neutral forms in the bright "on" and dark "off" states, respectively. Green excitation of the on state is followed by dominant radiative decay with characteristic time constants of 1.9 ps, 185 ps, and 1.1 ns, and additionally reveals spectral changes belonging to the species decaying with a 1.1 ns time constant, associated with both protein and chromophore modes. A 1 ms measurement of the on state identifies bleach features that correspond to those seen in the static off-minus-on Fourier transform infrared (FTIR) difference spectrum, indicating that thermal protonation of the hydroxyphenyl oxygen proceeds within this time window. Blue excitation of the off state directly resolves the formation of the primary photoproduct with 0.6 and 14 ps time constants, which is stable on the nanosecond time scale. Assignment of the primary photoproduct to the cis neutral chromophore in the electronic ground state is supported by the frequency positions expected relative to those for the nonplanar distorted geometry for the off state. A 1 ms measurement of the off state corresponds closely with the on-minus-off FTIR difference spectrum, indicating thermal deprotonation and rearrangement of the Arg66 side chain to be complete.

Room temperature crystal structure of the fast switching M159T mutant of the fluorescent protein dronpa

“…Experimentally it was observed from time resolved UV–vis spectroscopy measurements that in dilute solution, the relative on‐to‐off photoswitching rate constant with continuous 473 nm illumination increased 65‐fold relative to the unmodified Dronpa samples, at pH 7.8 and 3 × 10 −6 M concentration, favoring monomeric forms . We further noted distinctly reduced on‐off phototransformation kinetics with increased protein concentration for both the wild type and mutant samples, in approximately the same concentration ranges.…”

“…The small but reproducible cavity volume differences found in this study may therefore be in line with the significant rate enhancement. By comparing the isomerization rates in the monomer and the oligomeric states, we indeed found that the reactions occurs more efficiently in the monomer …”

“…report a 1143‐fold increase of the rate of the on‐off reaction at room temperature for the M159T mutant, implying an absolute quantum yield of 0.37, 11 whereas a more recent study of monomeric species in vitro recently reported a ∼65‐fold increase . The off‐to‐on reaction was found to have increased twofold, suggesting a quantum yield of 0.72 relative to the 0.36 value estimated for the wild type whereas a more recent study of monomeric species in vitro recently reported no significant difference . Stiel et al .…”

Long‐Range Electrostatics‐Induced Two‐Proton Transfer Captured by Neutron Crystallography in an Enzyme Catalytic Site

“…In the pH 4.3 structure,A sp25 is protonated on the outer Od2o xygen, whereas the OH group of darunavir hydrogen bonds with Asp25'.T herefore,anet two-proton transfer has occurred between the catalytic Asp residues and the OH group of the drug. This transfer was triggered by changes in the electrostatic properties of the protein resulting from the protonation of the four surface residues.T he electrostatic changes occur relatively far away (11)(12)(13)(14) from the catalytic site (Supporting Information, Figure S4). Thec atalytic site is not accessible to water when an inhibitor is bound, therefore we can deduce the proton transfer pathway.D1istransferred to the darunavir oxygen, while D2 shifts to Asp25.…”

“…[8][9][10][11] Similarly, excited-state proton transfer has been studied in green fluorescent protein (GFP), mapping the pathway of the proton hopping from the fluorophore OH to a glutamate side chain through a water molecule and a serine residue. [12][13][14] However, in these examples the proton is transferred back to the OH group once the photoacids return to the ground state and regain their initial acidities.…”