Dynamics and mechanism of dimer dissociation of photoreceptor UVR8.

Photoreceptors are a class of light-sensing proteins with critical biological functions. UVR8 is the only identified UV photoreceptor in plants and its dimer dissociation upon UV sensing activates UV-protective processes. However, the dissociation mechanism is still poorly understood. Here, by integrating extensive mutations, ultrafast spectroscopy, and computational calculations, we find that the funneled excitation energy in the interfacial tryptophan (Trp) pyramid center drives a directional Trp-Trp charge separation in 80 ps and produces a critical transient Trp anion, enabling its ultrafast charge neutralization with a nearby positive arginine residue in 17 ps to destroy a key salt bridge. A domino effect is then triggered to unzip the strong interfacial interactions, which is facilitated through flooding the interface by channel and interfacial water molecules. These detailed dynamics reveal a unique molecular mechanism of UV-induced dimer monomerization.

A leap in quantum efficiency through light harvesting in photoreceptor UVR8.

Plants utilize a UV-B (280 to 315 nm) photoreceptor UVR8 (UV RESISTANCE LOCUS 8) to sense environmental UV levels and regulate gene expression to avoid harmful UV effects. Uniquely, UVR8 uses intrinsic tryptophan for UV-B perception with a homodimer structure containing 26 structural tryptophan residues. However, besides 8 tryptophans at the dimer interface to form two critical pyramid perception centers, the other 18 tryptophans' functional role is unknown. Here, using ultrafast fluorescence spectroscopy, computational methods and extensive mutations, we find that all 18 tryptophans form light-harvesting networks and funnel their excitation energy to the pyramid centers to enhance light-perception efficiency. We determine the timescales of all elementary tryptophan-to-tryptophan energy-transfer steps in picoseconds to nanoseconds, in excellent agreement with quantum computational calculations, and finally reveal a significant leap in light-perception quantum efficiency from 35% to 73%. This photoreceptor is the first system discovered so far, to be best of our knowledge, using natural amino-acid tryptophans to form networks for both light harvesting and light perception.

Effects of nonequilibrium fluctuations on ultrafast short-range electron transfer dynamics.

A variety of electron transfer (ET) reactions in biological systems occurs at short distances and is ultrafast. Many of them show behaviors that deviate from the predictions of the classic Marcus theory. Here, we show that these ultrafast ET dynamics highly depend on the coupling between environmental fluctuations and ET reactions. We introduce a dynamic factor, γ (0 ≤ γ ≤ 1), to describe such coupling, with 0 referring to the system without coupling to a "frozen" environment, and 1 referring to the system's complete coupling with the environment. Significantly, this system's coupling with the environment modifies the reaction free energy, ΔGγ, and the reorganization energy, λγ, both of which become smaller. This new model explains the recent ultrafast dynamics in flavodoxin and elucidates the fundamental mechanism of nonequilibrium ET dynamics, which is critical to uncovering the molecular nature of many biological functions.

Dynamics and mechanism of light harvesting in UV photoreceptor UVR8.

Photosynthetic pigments form light-harvesting networks to enable nearly perfect quantum efficiency in photosynthesis via excitation energy transfer. However, similar light-harvesting mechanisms have not been reported in light sensing processes in other classes of photoreceptors during light-mediated signaling. Here, based on our earlier report, we mapped out a striking energy-transfer network composed of 26 structural tryptophan residues in the plant UV-B photoreceptor UVR8. The spectra of the tryptophan chromophores are tuned by the protein environments, funneling all excitation energy to a cluster of four tryptophan residues, a pyramid center, where the excitation-induced monomerization is initiated for cell signaling. With extensive site-directed mutagenesis, various time-resolved fluorescence techniques, and combined QM/MM simulations, we determined the energy-transfer rates for all donor-acceptor pairs, revealing the time scales from tens of picoseconds to nanoseconds. The overall light harvesting quantum efficiency by the pyramid center is significantly increased to 73%, compared to a direct excitation probability of 35%. UVR8 is the only photoreceptor discovered so far using a natural amino-acid tryptophan without utilizing extrinsic chromophores to form a network to carry out both light harvesting and light perception for biological functions.

Short-Range Electron Transfer in Reduced Flavodoxin: Ultrafast Nonequilibrium Dynamics Coupled with Protein Fluctuations.

Short-range electron transfer (ET) in proteins is an ultrafast process on the similar time scales as local protein-solvent fluctuation, and thus the two dynamics are coupled. Here we use semiquinone flavodoxin and systematically characterized the photoinduced redox cycle with 11 mutations of different aromatic electron donors (tryptophan and tyrosine) and local residues to change redox properties. We observed the forward and backward ET dynamics in a few picoseconds, strongly following a stretched behavior resulting from a coupling between local environment relaxations and these ET processes. We further observed the hot vibrational-state formation through charge recombination and the subsequent cooling dynamics also in a few picoseconds. Combined with the ET studies in oxidized flavodoxin, these results coherently reveal the evolution of the ET dynamics from single to stretched exponential behaviors and thus elucidate critical time scales for the coupling. The observed hot vibration-state formation is robust and should be considered in all photoinduced back ET processes in flavoproteins.

Origin of strong synergism in weakly perturbed binary solvent system: a case study of primary alcohols and chlorinated methanes.

A strong synergistic solvation was observed for the mixtures of hydrogen bond donating and accepting solvent pairs. The nature of the interactions between two solvent pairs was investigated with different dye molecules viz. coumarin 480, coumarin 153, 4-aminophthalimide, and p-nitroaniline. Coumarin 480 in differenet alcohols-CHCl(3) (alcohols: MeOH, EtOH, BuOH) binary mixture shows a strong synergism, which is explained in the backdrop of solvent-solvent interactions. Fluorescence quenching of C480 by 1,2-phenylenediamine in the binary solvent mixture exhibited the maximum deviation in quenching constant corresponding to ~0.45 mol fraction of MeOH in MeOH-CHCl(3) binary mixture and hence suggested the maximum extent of hydrogen-bonding interactions prevailing at this proportion of mixture. The solvation behavior of MeOH-CHCl(3) mixture shows strong probe dependence with no synergism observed in p-nitroaniline, which is ascribed to its higher ground state dipole moment (8.8 D) relative to C480 (6.3 D). Interestingly, the strong synergistic signature observed through spectrophotometric measurement of C480 in alcohol-CHCl(3) binary mixture is absent when studied by fluorescence measurement. The higher excited state dipole moment of coumarin 480 (13.1 D) is considered to be the driving force for the absence of synergism in the excited state. In such strongly perturbed systems (due to high dipole moment values) the dominant phenomenon is preferential solvation. Analysis of proton NMR of MeOH-CHCl(3) binary solvent mixture indicates the existence of MeOH-CHCl(3) clusters in the stoichiometric ratio of 1:2.15. Refractive index measurement also infers the existence of hydrogen bonded network structure between MeOH and CHCl(3). A modified Bosch solvent exchange model has been used to determine the feasibility of synergistic behavior and polarity parameter of the mixed solvent structure of MeOH-CHCl(3) binary solvent mixture.