10 research outputs found
Fluorescence from Multiple Chromophore Hydrogen-Bonding States in the Far-Red Protein TagRFP675
Far-red fluorescent proteins are critical for in vivo imaging applications, but the relative importance of structure versus dynamics in generating large Stokes-shifted emission is unclear. The unusually red-shifted emission of TagRFP675, a derivative of mKate, has been attributed to the multiple hydrogen bonds with the chromophore N-acylimine carbonyl. We characterized TagRFP675 and point mutants designed to perturb these hydrogen bonds with spectrally resolved transient grating and time-resolved fluorescence (TRF) spectroscopies supported by molecular dynamics simulations. TRF results for TagRFP675 and the mKate/M41Qvariant show picosecond time scale red-shifts followed by nanosecond time blue-shifts. Global analysis of the TRF spectra reveals spectrally distinct emitting states that do not interconvert during the S-1 lifetime. These dynamics originate from photoexcitation of a mixed ground-state population of acylimine hydrogen bond conformers. Strategically tuning the chromophore environment in TagRFP675 might stabilize the most red-shifted conformation and result in a variant with a larger Stokes shift.1122sciescopu
Assessing freshwater use impacts in LCA: Part Iâinventory modelling and characterisation factors for the main impact pathways
Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting
Random Coil to Globular Thermal Response of a Protein (H3.1) with Three Knowledge-Based Coarse-Grained Potentials
The effect of temperature on the conformation of a histone (H3.1) is studied by a coarse-grained Monte Carlo simulation based on three knowledge-based contact potentials (MJ, BT, BFKV). Despite unique energy and mobility profiles of its residues, the histone H3.1 undergoes a systematic (possibly continuous) structural transition from a random coil to a globular conformation on reducing the temperature. The range over which such a systematic response in variation of the radius of gyration (R(g)) with the temperature (T) occurs, however, depends on the potential, i.e. ÎT(MJ) â 0.013â0.020, ÎT(BT) â 0.018â0.026, and ÎT(BFKV) â 0.006â0.013 (in reduced unit). Unlike MJ and BT potentials, results from the BFKV potential show an anomaly where the magnitude of R(g) decreases on raising the temperature in a range ÎT(A) â 0.015â0.018 before reaching its steady-state random coil configuration. Scaling of the structure factor, S(q) â q(â1/Îœ), with the wave vector, qâ=â2Ï/λ, and the wavelength, λ, reveals a systematic change in the effective dimension (D(e)âŒ1/Îœ) of the histone with all potentials (MJ, BT, BFKV): D(e)âŒ3 in the globular structure with D(e)âŒ2 for the random coil. Reproducibility of the general yet unique (monotonic) structural transition of the protein H3.1 with the temperature (in contrast to non-monotonic structural response of a similar but different protein H2AX) with three interaction sets shows that the knowledge-based contact potential is viable tool to investigate structural response of proteins. Caution should be exercise with the quantitative comparisons due to differences in transition regimes with these interactions