Evolution and characterization of a new reversibly photoswitching chromogenic protein, Dathail

Abstract

We report the engineering of a new reversibly switching chromogenic protein, Dathail. Dathail was evolved from the extremely thermostable fluorescent proteins thermal green protein (TGP) and eCGP123 using directed evolution and ratiometric sorting. Dathail has two spectrally distinct chromogenic states with low quantum yields, corresponding to absorbance in a ground state with a maximum at 389nm, and a photo-induced metastable state with a maximum at 497nm. In contrast to all previously described photoswitchable proteins, both spectral states of Dathail are non-fluorescent. The photo-induced chromogenic state of Dathail has a lifetime of ~50min at 293K and pH7.5 as measured by UV-Vis spectrophotometry, returning to the ground state through thermal relaxation. X-ray crystallography provided structural insights supporting a change in conformation and coordination in the chromophore pocket as being responsible for Dathail's photoswitching. Neutron crystallography, carried out for the first time on a protein from the green fluorescent protein family, showed a distribution of hydrogen atoms revealing protonation of the chromophore 4-hydroxybenzyl group in the ground state. The neutron structure also supports the hypothesis that the photo-induced proton transfer from the chromophore occurs through water-mediated proton relay into the bulk solvent. Beyond its spectroscopic curiosity, Dathail has several characteristics that are improvements for applications, including low background fluorescence, large spectral separation, rapid switching time, and the ability to switch many times. Therefore, Dathail is likely to be extremely useful in the quickly developing fields of imaging and biosensors, including photochromic Förster resonance energy transfer, high-resolution microscopy, and live tracking within the cell.

Room-temperature photo-induced martensitic transformation in a protein crystal

“…Recently, FPs that possess fluorescent properties controllable by irradiation with UV or visible light have been discovered (Ando et al, 2004;Andresen et al, 2008;Adam et al, 2008;Langan et al, 2016). Three classes of such photoswitchable FPs have emerged: (1) photo-activatable FPs, which irreversibly convert to a fluorescent state upon irradiation; (2) photo-convertible FPs, in which the emitting wavelength can be altered upon irradiation; and (3) reversibly switchable FPs (RSFPs), which can be switched repeatedly between a non-fluorescent 'off' state and a fluorescent 'on' state by irradiating the protein with light of two distinctive wavelengths.…”

Status of the neutron time-of-flight single-crystal diffraction data-processing software STARGazer

“…However, the R merge of protein diffraction data obtained using TOF diffractometers is not good: for example, the R merge values for the overall resolution obtained using synchrotron X-rays, monochromated neutrons (BioDiff, BIX-3 or BIX-4) and TOF neutrons (iBIX or MaNDi or PCS) are approximately 5, 10 and 20%, respectively (Yokoyama et al, 2015;Chatake et al, 2004;Yonezawa et al, 2017;Fisher et al, 2012;Chen et al, 2011;Vandavasi et al, 2016;Langan et al, 2016). The following four factors can be considered to be the main reasons that the R merge of synchrotron X-ray data is lower than that of TOF neutron data.…”

Neutron scattering in the biological sciences: progress and prospects

“…An obstacle is the relatively long time that is needed for data collection, so that in the near term studies are likely to be focused on long-lived metastable states. For example, photoinduced proton-transfer complexes can have very long lifetimes in photo-switchable GFP derivatives, which can be studied with neutron diffraction at room temperature or at cryotemperatures (Langan et al, 2016). Neutron protein crystallography can also help to discover unusual structures that cannot be predicted by computational techniques without first performing the experiment.…”