Search

Phototriggers are useful molecular tools to initiate reactions in enzymes by light for the purpose of photoenzymatic design and mechanistic investigations. Here, we incorporated the non-natural amino acid 5-cyanotryptophan (W5CN) in a polypeptide scaffold and resolved the photochemical reaction of the W5CN–W motif using femtosecond transient UV/Vis and mid-IR spectroscopy. We identified a marker band of ∼2037 cm−1 from the CN stretch of the electron transfer intermediate W5CN·− in the transient IR measurement and found UV/Vis spectroscopic evidence for the W·+ radical at 580 nm. Through kinetic analysis, we characterized that the charge separation between the excited W5CN and W occurs in 253 ps, with a charge-recombination lifetime of 862 ps. Our study highlights the potential use of the W5CN–W pair as an ultrafast phototrigger to initiate reactions in enzymes that are not light-sensitive, making downstream reactions accessible to femtosecond spectroscopic detection.

Short-range protein electron transfer (ET) is crucially important in light-induced biological processes such as in photoenzymes and photoreceptors and often occurs on time scales similar to those of environment fluctuations, leading to a coupled dynamic process. Herein, we use semiquinone

Short-range protein electron transfer (ET) is ubiquitous in biology and is often observed in photosyntheses, photoreceptors and photoenzymes. These ET processes occur on an ultrafast timescale from femtoseconds to picoseconds at a short donor-acceptor distance within 10 A, and thus couple with local environmental fluctuations. Here, we use oxidized Anabaena flavodoxin as a model system and have systematically studied the photoinduced redox cycle of the wild type and seven mutant proteins by femtosecond spectroscopy. We observed a series of ultrafast dynamics from the initial charge separation in 100-200 fs, subsequent charge recombination in 1-2 ps and final vibrational cooling process of the products in 3-6 ps. We further characterized the active-site solvation and observed the relaxations in 1-200 ps, indicating a nonergodic ET dynamics. With our new ET model, we uncovered a minor outer (solvent) reorganization energy and a large inner (donor and acceptor) reorganization energy, suggesting a frozen active site in initial ultrafast ET while the back ET couples with the environment relaxations. The vibronically coupled back ET dynamics was first reported in D. vulgaris flavodoxin and here is observed in Anabaena flavodoxin again, completely due to the faster ET dynamics than the cooling relaxations. We also compared the two flavodoxin structures, revealing a stronger coupling with the donor tyrosine in Anabaena. All ultrafast ET dynamics result from the large donor-acceptor couplings and the minor activation barriers due to the reaction free energies close to the inner reorganization energies. These observations should be general to many short-distance redox reactions in flavoproteins.

Transient amyloid intermediates are likely to be cytotoxic and play an essential role in amyloid-associated neurodegenerative diseases. Characterization of their structural and dynamic evolution is the key to elucidating the molecular mechanism of amyloid formation. Here, combining circular dichroism (CD), exciton couplet theory, and Fourier transform infrared spectroscopy with site-specific tryptophan (Trp) and its noncanonical derivative 5-cyano-tryptochan (Trp5CN), we developed a method to monitor strand-to-strand tertiary and sheet-to-sheet quaternary interactions in the aggregation cascades of an amyloidogenic fragment from protein SOD128-38 (with the sequence KVKVWGSIKGL). We found that the exciton couplet generated from the Bb band of Trp can be used as a probe for side chain interactions. Its sensitivity can be further improved by four times with the incorporation of Trp5CN. We further observed a red-shift of ∼2 cm-1 and a broadening of ∼2 cm-1 in the IR band generated from the CN stretch during the aggregation, which we attributed to the transition from a corkscrew-like structure to a cross-linked intermediate phase. We show here that the integration of optical methods with unique aromatic side chain-related probes is able to elucidate amyloid intermolecular interactions and even capture elusive transient intermediates on and off the amyloid assembling pathway.

The GroE molecular chaperone system is a critical protein machine that assists the folding of substrate proteins in its cavity. Water in the cavity is suspected to play a role in substrate protein folding, but the mechanism is currently unknown. Herein, we report measurements of water dynamics in the equatorial and apical domains of the GroEL cavity in the apo and football states, using site-specific tryptophanyl mutagenesis as an intrinsic optical probe with femtosecond resolution combined with molecular dynamics simulations. We observed clearly different water dynamics in the two domains with a slowdown of the cavity water from the apical to equatorial region in the football state. The results suggest that the GroEL cavity provides a unique water environment that may facilitate substrate protein folding.

Fatty acid photodecarboxylase is a newly discovered flavin photoenzyme that converts a carboxylic acid into a hydrocarbon and a carbon dioxide molecule through decarboxylation. The enzymatic reactions are poorly understood. In this study, we carefully characterized its dynamic evolution with femtosecond spectroscopy. We observed initial electron transfer from the substrate to the flavin cofactor in 347 ps with a stretched dynamic behavior and subsequently captured the critical carbonyloxy radical. The dominant process following this step was decarboxylation in 5.8 ns to form an alkyl radical and a carbon dioxide molecule. We further identified the absorption bands of two carbonyloxy and alkyl radical intermediates. The overall enzymatic quantum efficiency determined by our obtained timescales is 0.81, consistent with the steady-state value. The results are essential to the elucidation of the enzyme mechanism and catalytic photocycle, providing a molecular basis for potential design of flavin-based artificial photoenzymes.

Proton-coupled electron transfer (PCET) is key to the activation of the blue light using flavin (BLUF) domain photoreceptors. Here, to elucidate the photocycle of the central FMN-Gln-Tyr motif in the BLUF domain of OaPAC, we eliminated the intrinsic interfering W90 in the mutant design. We integrated the stretched exponential function into the target analysis to account for the dynamic heterogeneity arising from the active-site solvation relaxation and the flexible H-bonding network as shown in the molecular dynamics simulation results, facilitating a simplified expression of the kinetics model. We find that, in both the functional wild-type (WT) and the nonfunctional Q48E and Q48A, forward PCET happens in the range of 105 ps to 344 ps, with a kinetic isotope effect (KIE) measured to be ∼1.8 to 2.4, suggesting that the nature of the forward PCET is concerted. Remarkably, only WT proceeds with an ultrafast reverse PCET process (31 ps, KIE = 4.0), characterized by an inverted kinetics of the intermediate FMNH˙. Our results reveal that the reverse PCET is driven by proton transfer via an intervening imidic Gln.

Cyanobacteriochromes are photoreceptors in cyanobacteria that exhibit a wide spectral coverage and unique photophysical properties from the photoinduced isomerization of a linear tetrapyrrole chromophore. Here, we integrate femtosecond-resolved fluorescence and transient-absorption methods and unambiguously showed the significant solvation dynamics occurring at the active site from a few to hundreds of picoseconds. These motions of local water molecules and polar side chains are continuously convoluted with the isomerization reaction, leading to a nonequilibrium processes with continuous active-site motions. By mutations of critical residues at the active site, the modified local structures become looser, resulting in faster solvation relaxations and isomerization reaction. The observation of solvation dynamics is significant and critical to the correct interpretation of often-observed multiphasic dynamic behaviors, and thus the previously invoked ground-state heterogeneity may not be relevant to the excited-state isomerization reaction.

Water dynamics on the protein surface mediate both protein structure and function. However, many questions remain about the role of the protein hydration layers in protein fluctuations and how the dynamics of these layers relate to specific protein properties. The fish eye lens protein γM7-crystallin (γM7) is found in vivo at extremely high concentrations nearing the packing limit, corresponding to only a few water layers between adjacent proteins. In this study, we conducted a site-specific probing of hydration water motions and side-chain dynamics at nine selected sites around the surface of γM7 using a tryptophan scan with femtosecond spectroscopy and NMR nuclear spin relaxation (NSR). We observed correlated fluctuations between hydration water and protein side chains on the time scales of a few picoseconds and hundreds of picoseconds, corresponding to local reorientations and network restructuring, respectively. These motions are heterogeneous over the protein surface and relate to the various steric and chemical properties of the local protein environment. Overall, we found that γM7 has relatively slower water dynamics within the hydration shell than a similar β-sheet protein, which may contribute to the high packing limit of this unique protein.

We present direct observation of ultrafast proton rocking in the central motif of a BLUF domain protein scaffold. The mutant design has taken consideration of modulating the proton-coupled electron transfer (PCET) driving forces by replacing Tyr in the original motif with Trp, in order to remove the interference of a competing electron transfer pathway. Using femtosecond pump-probe spectroscopy and detailed kinetics analysis, we resolved an electron-transfer-coupled Grotthuss-type forward and reverse proton rocking along the FMN-Gln-Trp proton relay chain. The rates of forward and reverse proton transfer are determined to be very close, namely 51 ps vs. 52 ps. The kinetic isotope effect (KIE) constants associated with the forward and reverse proton transfer are 3.9 and 5.3, respectively. The observation of ultrafast proton rocking is not only a crucial step towards revealing the nature of proton relay in the BLUF domain, but also provides a new paradigm of proton transfer in proteins for theoretical investigations.

Cryptochromes are signaling proteins activated by photoexcitation of the flavin adenine dinucleotide (FAD) cofactor. Although extensive research has been performed, the mechanism for this allosteric process is still unknown. We constructed three computational models, corresponding to different redox states of the FAD cofactor in Drosophila cryptochrome (dCRY). Analyses of the dynamics trajectories reveal that the activation process occurs in the semiquinone state FAD-●, resulting from excited-state electron transfer. The Arg381-Asp410 salt bridge acts as an allosteric switch, regulated by the change in the redox state of FAD. In turn, Asp410 forms new hydrogen bonds, connecting allosteric networks of the amino-terminal and carboxyl-terminal domains initially separated in the resting state. The expansion to a global dynamic network leads to enhanced protein fluctuations, an increase in the radius of gyration, and the expulsion of the carboxyl-terminal tail. These structural features are in accord with mutations and spectroscopic experiments.

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., The light-harvesting network from distal and peripheral to central tryptophans with transfer efficiencies determined from measured energy-transfer rates.

Short-range electron-transfer (ET) reactions in biological systems are usually ultrafast, having transfer rates comparable to or even faster than corresponding environmental fluctuations, and often display nonexponential behaviors. To understand these nonequilibrium ET dynamics, we carried out detailed theoretical analyses based on the Sumi–Marcus model. It is shown that the ET dynamics is largely determined by the relative time scales of the ET reaction and its surrounding motions. Significantly, different environmental fluctuations can produce a variety of apparent ET dynamics even with the same driving force, ΔGo, and reorganization energy, λ. We applied our analyses to an ultrafast ET process in DNA repair by (6–4) photolyase and directly obtained the inner and outer reorganization energies (λi and λo) as well as the free energy ΔGo of various mutants, providing mechanical insight into ultrafast short-range ET reactions in proteins.

The formation of transient water photolysis products is followed with ultrafast electron diffraction

Photoexcitation of FAD triggers ion-pair breakage and allosteric conformational change, leading to cryptochrome activation., Cryptochromes are signaling proteins activated by photoexcitation of the flavin adenine dinucleotide (FAD) cofactor. Although extensive research has been performed, the mechanism for this allosteric process is still unknown. We constructed three computational models, corresponding to different redox states of the FAD cofactor in Drosophila cryptochrome (dCRY). Analyses of the dynamics trajectories reveal that the activation process occurs in the semiquinone state FAD−●, resulting from excited-state electron transfer. The Arg381-Asp410 salt bridge acts as an allosteric switch, regulated by the change in the redox state of FAD. In turn, Asp410 forms new hydrogen bonds, connecting allosteric networks of the amino-terminal and carboxyl-terminal domains initially separated in the resting state. The expansion to a global dynamic network leads to enhanced protein fluctuations, an increase in the radius of gyration, and the expulsion of the carboxyl-terminal tail. These structural features are in accord with mutations and spectroscopic experiments.

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., Photoreceptor UVR8 in plants senses environmental UV levels through 26 structural tryptophan residues, but the role of 18 of them was unknown. The authors show, by experiments and computations, how these form a light-harvesting network that funnels the excitation to the pyramid centers enhancing the light-perception efficiency.

Red-light bacteriophytochromes regulate many physiological functions through photoisomerization of a linear tetrapyrrole chromophore. In this work, we mapped out femtosecond-resolved fluorescence spectra of the excited Pr state and observed unique active-site relaxations on the picosecond time scale with unusual spectral tuning of rises on the blue side and decays on the red side of the emission. We also observed initial wavepacket dynamics in femtoseconds with two low-frequency modes of 38 and 181 cm-1 as well as the intermediate product formation after isomerization in hundreds of picoseconds. With critical mutations at the active site, we observed similar dynamic patterns with different times for both relaxation and isomerization, consistent with the structural and chemical changes induced by the mutations. The observed multiphasic dynamics clearly represents the active-site relaxation, not different intermediate reactions or excitation of heterogeneous ground states. The active-site relaxation must be considered in understanding overall isomerization reactions in phytochromes, and such a molecular mechanism should be general.

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., Ultrafast electron-transfer reactions are fundamental to protein functions. Here the authors show that these reaction dynamics are affected by the ruggedness of protein energy landscape, which even modifies the reaction free energy and reorganization energy.

Redox reaction, involving the gain and loss of electrons between reactants, is one type of common chemical reaction governing fundamental energy issues in nature. However, reports of vividly visualizing such key processes with simultaneous structural determination of new phases that are involved are rare. Here, by achieving simultaneous recording in both real and reciprocal space, we demonstrate in situ imaging of the redox reaction dynamics in perovskite nanocrystals. The thorough atomic-scale movies enable an in-depth understanding of the reaction-induced nucleation and growth mechanism of clusters with the aid of carbon, and a simple way of using SiN films at room temperature to fully prevent the irradiation-induced degradation in perovskites is proposed, in contrast to the costly low-temperature strategy. Real-time atomic-scale imaging in both real and reciprocal space paves the way for revealing various chemical and physical events at targeted nanoscale positions with complementary structural information.

The biological functions of photoenzymes are often triggered by photoinduced electron transfer (ET) reactions. An ultrafast backward ET (BET) reaction follows the initial photoinduced forward ET (FET), which dissipates the energy of absorbed photons and terminates the biological function in vain. Based upon our previous works, we reasoned that the dynamics of the BET is coupled with that of the FET and other local motions. In this work, the dynamics of the FET and BET is modeled as the master equation of the reduced density operator of a three-state system coupled with a classical harmonic reservoir. The coupling of the FET and BET is reflected in the time-evolution of the charge-transfer state’s population, which is generated by a source, the reaction flux for the FET, and annihilated by a sink, the reaction flux for the BET. Surprisingly, numerical simulations show that when the BET is in the Marcus normal region, the BET can be accelerated by nonequilibrium local motions and becomes faster than what is predicted by the Marcus theory. The experimental confirmation of this novel dynamics would provide qualitative evidence for nonequilibrium effects on ultrafast ET dynamics. Additionally, the effects of quantum vibrational modes on the dynamics are discussed. This work can help understand the dynamical interactions between the chain of ultrafast reactions and the complex local environmental motions, revealing the physical nature underlying biological functions.

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

We present the first exact solution to the time-independent Schrodinger equation of a model Hamiltonian consisting of a vibrational mode coupled to three electronic states. This Hamiltonian serves as a generic model for photo-induced electronic transfer reactions. The solution is non-perturbative and can be applied to ET reactions with weak and strong electronic and vibrational coupling strengths. This work suggests a new direction towards understanding the vibronic effects in ET dynamics beyond the non-adiabatic limit and Condon approximation.

Water-protein coupled interactions are essential to the protein structural stability, flexibility and dynamic functions. The ultimate effects of the hydration dynamics on the protein fluctuations remain substantially unexplored. Here, we investigated the dynamics of both hydration water and protein sidechains at 13 different sites around the polymerase β protein surface using a tryptophan scan with femtosecond spectroscopy. Three types of hydration-water relaxations and two types of protein sidechain motions were determined, reflecting a highly dynamic water-protein interactions fluctuating on the picosecond time scales. The hydration-water dynamics dominate the coupled interactions with higher flexibility.

Protein surface hydration is fundamental to its structure, flexibility, dynamics, and function, but it has been challenging to disentangle their ultimate relationships. Here, we report our systematic characterization of hydration dynamics around a β-barrel protein, rat liver fatty acid-binding protein (rLFABP), to reveal the effect of different protein secondary structures on hydration water. We employed a tryptophan scan to the protein surface one at a time and examined a total of 17 different sites. We observed three types of hydration water relaxation with distinct time scales, from hundreds of femtoseconds to a hundred picoseconds. We also examined the anisotropy dynamics of the corresponding tryptophan side chains and observed two distinct relaxations from tens to hundreds of picoseconds. Integrating our previous findings on α-helical proteins, we conclude the following: (1) The hydration dynamics is highly heterogeneous around the protein surface of both α-helical and β-sheet proteins. The outer-layer water of the hydration shell behave like a bulk and relaxes in hundreds of femtoseconds. The inner-layer water collectively relaxes in two time scales, reorientation motions in a few picoseconds and network restructuring in tens to a hundred picoseconds. (2) The hydration dynamics are always faster than local protein relaxations and in fact drive the protein fluctuations on the picosecond time scale. (3) The hydration dynamics in general are more retarded around β-sheet structures than α-helical motifs. A thicker hydration shell and a more rigid interfacial hydration network are observed in the β-sheet protein. Overall, these findings elucidate the intimate relationship between water-protein interactions and dynamics on the ultrafast time scale.

Protein surface hydration is critical to the protein's structural properties and biological activities. However, it is still unknown whether the hydration shell is intrinsically connected and how its fluctuations dynamically interact with protein motion. Here, by selecting five site-specific locations with distinctly different environments around the surface of apomyoglobin, we used a tryptophan scan with femtosecond fluorescence spectroscopy and simultaneously detected hydration water dynamics and tryptophan side-chain relaxations with temperature dependence. We observed two types of relaxations for both interfacial hydration water and the tryptophan side chain. The former is always faster than the latter, and both motions show direct linear correlations with temperature changes, indicating one origin of their motions and hydration water driving of side-chain fluctuations. Significantly, we found the relaxation energy barriers are uniform across the entire protein surface, all less than 20 kJ/mol, strongly suggesting highly extended cooperative water networks and the nature of global dynamic collectivity of the entire hydration shell.

Photolyase, a photomachine discovered half a century ago for repair of sun-induced DNA damage of cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs), has been characterized extensively in biochemistry (function), structure and dynamics since 1980s. The molecular mechanism and repair photocycle have been revealed at the most fundamental level. Using femtosecond spectroscopy, we have mapped out the entire dynamical evolution and determined all actual timescales of the catalytic processes. Here, we review our recent efforts in studies of the dynamics of DNA repair by photolyases. The repair of CPDs in three life kingdoms includes seven electron-transfer (ET) reactions among ten elementary steps through initial bifurcating ET pathways, a direct tunneling route and a two-step hopping path both through an intervening adenine from the cofactor to CPD, with a conserved folded structure at the active site. The repair of 6-4PPs is challenging and requires similar ET reactions and a new cyclic proton transfer with a conserved histidine residue at the active site of (6-4) photolyases. Finally, we also summarize our efforts on multiple intraprotein ET of photolyases in different redox states and such mechanistic studies are critical to the functional mechanism of homologous cryptochromes of blue-light photoreceptors.