Femtosecond dynamics of a drug-protein complex: daunomycin with Apo riboflavin-binding protein

In this contribution, we report studies of the primary dynamics of the drug-protein complexes of daunomycin with apo riboflavin-binding protein. With femtosecond resolution, we observed the ultrafast charge separation between daunomycin and aromatic amino acid residues of the protein, tryptophan(s). Electron transfer occurs from tryptophan(s) to daunomycin with two reaction times, 1 ps and 6 ps, depending on the local complex structure. The formation of anionic daunomycin radical is crucial for triggering a series of chemical reactions in redox cycling. One of the subsequent reactions is the reduction of dioxygen to form active superoxide by the reduced daunomycin. This catalytic process was found to occur within 10 ps. In the absence of dioxygen, charge recombination takes a much longer time, more than 100 ps. These results, along with similar findings in DNA and nucleotides, elucidate that the ultrafast generation of reduced daunomycin radicals by photoactivation is a primary step for the observed photoenhancement of drug cytotoxicity by several orders of magnitude. We also studied the dependence of the dynamics on protein conformations at different ionic strengths and denaturant concentrations. We observe a sharp transition from the tertiary structure to the unfolding state at 2 M of denaturant concentration.     

Primary reactions of sensory rhodopsins

The first steps in the photocycles of the archaeal photoreceptor proteins sensory rhodopsin (SR) I and II from Halobacterium salinarum and SRII from Natronobacterium pharaonis have been studied by ultrafast pump/probe spectroscopy and steady-state fluorescence spectroscopy. The data for both species of the blue-light receptor SRII suggests that their primary reactions are nearly analogous with a fast decay of the excited electronic state in 300-400 fs and a transition between two red-shifted product states in 4-5 ps. Thus SRII behaves similarly to bacteriorhodopsin. In contrast for SRI at pH 6.0, which absorbs in the orange part of the spectrum, a strongly increased fluorescence quantum yield and a drastically slower and biexponential decay of the excited electronic state occurring on the picosecond time scale (5 ps and 33 ps) is observed. The results suggest that the primary reactions are controlled by the charge distribution in the vicinity of the Schiff base and demonstrate that there is no direct connection between absorption properties and reaction dynamics for the retinal protein family.     

Insights into excited-state and isomerization dynamics of bacteriorhodopsin from ultrafast transient UV absorption

A visible-pump/UV-probe transient absorption is used to characterize the ultrafast dynamics of bacteriorhodopsin with 80-fs time resolution. We identify three spectral components in the 265- to 310-nm region, related to the all-trans retinal, tryptophan (Trp)-86 and the isomerized photoproduct, allowing us to map the dynamics from reactants to products, along with the response of Trp amino acids. The signal of the photoproduct appears with a time delay of approximately 250 fs and is characterized by a steep rise ( approximately 150 fs), followed by additional rise and decay components, with time scales characteristic of the J intermediate. The delayed onset and the steep rise point to an impulsive formation of a transition state on the way to isomerization. We argue that this impulsive formation results from a splitting of a wave packet of torsional modes on the potential surface at the branching between the all-trans and the cis forms. Parallel to these dynamics, the signal caused by Trp response rises in approximately 200 fs, because of the translocation of charge along the conjugate chain, and possible mechanisms are presented, which trigger isomerization.     

Ultrafast hydration dynamics in protein unfolding: human serum albumin

We report studies of unfolding and ultrafast hydration dynamics of the protein human serum albumin. Unique in this study is our ability to examine different domains of the same protein and the intermediate on the way to the unfolded state. With femtosecond resolution and site-selective labeling, we isolate the dynamics of domains I and II of the native protein, domain I of the intermediate at 2 M guanidine hydrochloride, and the unfolded state at 6 M of the denaturant. For studies of unfolding, we used the fluorophores, acrylodan (covalently bound to Cys-34 in domain I) and the intrinsic tryptophan (domain II), whereas for hydration dynamics, we probed acrylodan and prodan; the latter is bound to domain II. From the time-dependent spectra and the correlation functions, we obtained the time scale of dynamically ordered water: 57 ps for the more stable domain I and 32 ps for the less stable domain II, in contrast to approximately 0.8 ps for labile, bulk-type water. This trend suggests an increased hydrophilic residues-water interaction of domain I, contrary to some packing models. In the intermediate state, which is characterized by essentially intact domain I and unfolded domain II, the dynamics of ordered water around domain I is nearly the same (61 ps) as that of native state (57 ps), whereas that in the unfolded protein is much shorter (13 ps). We discuss the role of this fluidity in the correlation between stability and function of the protein.     

Ultrafast surface hydration dynamics and expression of protein functionality: alpha -Chymotrypsin

We report studies of hydration dynamics at the surface of the enzyme protein bovine pancreatic alpha-chymotrypsin. The probe is the well known 1-anilinonaphthalene-8-sulfonate, which binds selectively in the native state of the protein, not the molten globule, as shown by x-ray crystallography. With femtosecond time resolution, we examined the hydration dynamics at two pHs, when the protein is physiologically in the inactive state (pH 3.6) or the active state (pH 6.7); the global structure and the binding site remain the same. The hydration correlation function, C(t), whose decay is governed by the rotational and translational motions of water molecules at the site, shows the behavior observed in this laboratory for other proteins, Subtilisin Carlsberg and Monellin, using the intrinsic amino acid tryptophan as a probe for surface hydration. However, the time scales and amplitudes vary drastically at the two pHs. For the inactive protein state, C(t) decays with an ultrafast component, close to bulk-type behavior, but 50% of the C(t) decays at a much slower rate, tau = 43 ps. In contrast, for the active state, the ultrafast component becomes dominant (90%) and the slow component changes to a faster decay, tau = 28 ps. These results indicate that in the active state water molecules in the hydration layer around the site have a high degree of mobility, whereas in the inactive state the water is more rigidly structured. For the substrate-enzyme complex, the function and dynamics at the probe site are correlated, and the relevance to the enzymatic action is clear.     

Femtosecond linear dichroism of DNA-intercalating chromophores: solvation and charge separation dynamics of [Ru(phen)2dppz]2+ systems

The DNA-intercalating chromophore [Ru(phen)(2)dppz](2+) has unique photophysical properties, the most striking of which is the "light-switch" characteristic when binding to DNA. As a dimer, it acts as a molecular staple for DNA, exhibiting a remarkable double-intercalating topology. Herein, we report femtosecond dynamics of the monomeric and the covalently linked dimeric chromophores, both free in aqueous solution and complexed with DNA. Transient absorption and linear dichroism show the electronic relaxation to the lowest metal-to-ligand charge-transfer (CT) state, and subpicosecond kinetics have been observed for this chromophore for what is, to our knowledge, the first time. We observe two distinct relaxation processes in aqueous solution with time constants of 700 fs and 4 ps. Interestingly, these two time constants are very similar to those observed for the reorientational modes of bulk water. The 700-fs process involves a major dichroism change. We relate these observations to the change in charge distribution and to the time scales involved in solvation of the CT state. Slower processes, with lifetimes of approximately 7 and 37 ps, were observed for both monomer and dimer when bound to DNA. Such a difference can be ascribed to the change of the structural and electronic relaxation experienced in the DNA intercalation pocket. Finally, the recombination lifetime of the final metal-to-ligand CT state to the ground state, which is a key in the light-switch process, is found in aqueous solution to be sensitive to structural modification, ranging from 260 ps for [Ru(phen)(2)dppz](2+) and 360 ps for the monomer chromophore derivative to 2.0 ns for the dimer. This large change reflects the direct role of solvation in the light-switch process.     

Dynamic determination of the functional state in photolyase and the implication for cryptochrome

The flavin adenine dinucleotide cofactor has an unusual bent configuration in photolyase and cryptochrome, and such a folded structure may have a functional role in initial photochemistry. Using femtosecond spectroscopy, we report here our systematic characterization of cyclic intramolecular electron transfer (ET) dynamics between the flavin and adenine moieties of flavin adenine dinucleotide in four redox forms of the oxidized, neutral, and anionic semiquinone, and anionic hydroquinone states. By comparing wild-type and mutant enzymes, we have determined that the excited neutral oxidized and semiquinone states absorb an electron from the adenine moiety in 19 and 135 ps, whereas the excited anionic semiquinone and hydroquinone states donate an electron to the adenine moiety in 12 ps and 2 ns, respectively. All back ET dynamics occur ultrafast within 100 ps. These four ET dynamics dictate that only the anionic hydroquinone flavin can be the functional state in photolyase due to the slower ET dynamics (2 ns) with the adenine moiety and a faster ET dynamics (250 ps) with the substrate, whereas the intervening adenine moiety mediates electron tunneling for repair of damaged DNA. Assuming ET as the universal mechanism for photolyase and cryptochrome, these results imply anionic flavin as the more attractive form of the cofactor in the active state in cryptochrome to induce charge relocation to cause an electrostatic variation in the active site and then lead to a local conformation change to initiate signaling.     

Dissection of complex protein dynamics in human thioredoxin

We report our direct study of complex protein dynamics in human thioredoxin by dissecting into elementary processes and determining their relevant time scales. By combining site-directed mutagenesis with femtosecond spectroscopy, we have distinguished four partly time-overlapped dynamical processes at the active site of thioredoxin. Using intrinsic tryptophan as a molecular probe and from mutation studies, we ascertained the negligible contribution to solvation by protein sidechains and observed that the hydration dynamics at the active site occur in 0.47-0.67 and 10.8-13.2 ps. With reduced and oxidized states, we determined the electron-transfer quenching dynamics between excited tryptophan and a nearby disulfide bond in 10-17.5 ps for three mutants. A robust dynamical process in 95-114 ps, present in both redox states and all mutants regardless of neighboring charged, polar, and hydrophobic residues around the probe, is attributed to the charge transfer reaction with its adjacent peptide bond. Site-directed mutations also revealed the electronic quenching dynamics by an aspartate residue at a hydrogen bond distance in 275-615 ps. The local rotational dynamics determined by the measurement of anisotropy changes with time unraveled a relatively rigid local configuration but implies that the protein fluctuates on the time scale of longer than nanoseconds. These results elucidate the temporal evolution of hydrating water motions, electron-transfer reactions, and local protein fluctuations at the active site, and show continuously synergistic dynamics of biological function over wide time scales.     

Femtosecond dynamics of dative bonding: concepts of reversible and dissociative electron transfer reactions

With fs time, speed, and angular resolution of the elementary steps in electron transfer reactions, we report direct observation of reversible and dissociative processes for dative bonding involving covalent and ionic characters. For bimolecular reactions of various donors and acceptors we find strong correlation between the structure and the dynamics. The dynamics from the transition state to final products involve two elementary processes, with different reaction times, speed, and angular distributions. For example, for the R2S.I2 (R = C2H5) system, it is shown that after charge separation, the reversible electron transfer occurs in less than 150 fs (fastest trajectory) and is followed by the rupture of the I-I bond with the release of the first I-atom in 510 fs. However, the second process of the remaining and trapped I-atom takes 1.15 ps with its speed (500 m/s) being much smaller than the first one (1,030 m/s). The S-I-I average angle is 130 degrees. These findings, on this and the other systems reported here, elucidate the mechanism and address some concepts of nonconcertedness, caging, and restricted energy redistribution.     

Femtosecond dynamics of rubredoxin: Tryptophan solvation and resonance energy transfer in the protein

We report here studies of tryptophan (Trp) solvation dynamics in water and in the Pyrococcus furiosus rubredoxin protein, including the native and its apo and denatured forms. We also report results on energy transfer from Trp to the iron-sulfur [Fe-S] cluster. Trp fluorescence decay with the onset of solvation dynamics of the chromophore in water was observed with femtosecond resolution ( approximately 160 fs; 65% component), but the emission extended to the picosecond range (1.1 ps; 35% component). In contrast, the decay is much slower in the native rubredoxin; the Trp fluorescence decay extends to 10 ps and longer, reflecting the local rigidity imposed by residues and by the surface water layer. The dynamics of resonance energy transfer from the two Trps to the [Fe-S] cluster in the protein was observed to follow a temporal behavior characterized by a single exponential (15-20 ps) decay. This unusual observation in a protein indicates that the resonance transfer is to an acceptor of a well-defined orientation and separation. From studies of the mutant protein, we show that the two Trp residues have similar energy-transfer rates. The critical distance for transfer (R(0)) was determined, by using the known x-ray data, to be 19.5 A for Trp-36 and 25.2 A for Trp-3, respectively. The orientation factor (kappa(2)) was deduced to be 0.13 for Trp-36, clearly indicating that molecular orientation of chromophores in the protein cannot be isotropic with kappa(2) value of 2/3. These studies of solvation and energy-transfer dynamics, and of the rotational anisotropy, of the wild-type protein, the (W3Y, I23V, L32I) mutant, and the fmetPfRd variant at various pH values reveal a dynamically rigid protein structure, which is probably related to the hyperthermophilicity of the protein.     

Ultrafast Transient Spectroscopy of Polymer/Fullerene Blends for Organic Photovoltaic Applications

We measured the picoseconds (ps) transient dynamics of photoexcitations in blends of regio-regular poly(3-hexyl-thiophene) (RR-P3HT) (donors-D) and fullerene (PCBM) (acceptor-A) in an unprecedented broad spectral range of 0.25 to 2.5 eV. In D-A blends with maximum domain separation, such as RR-P3HT/PCBM, with (1.2:1) weight ratio having solar cell power conversion efficiency of ~4%, we found that although the intrachain excitons in the polymer domains decay within ~10 ps, no charge polarons are generated at their expense up to ~1 ns. Instead, there is a build-up of charge-transfer (CT) excitons at the D-A interfaces having the same kinetics as the exciton decay. The CT excitons dissociate into separate polarons in the D and A domains at a later time (>1 ns). This “two-step” charge photogeneration process may be typical in organic bulk heterojunction cells. We also report the effect of adding spin 1/2 radicals, Galvinoxyl on the ultrafast photoexcitation dynamics in annealed films of RR-P3HT/PCBM blend. The addition of Galvinoxyl radicals to the blend reduces the geminate recombination rate of photogenerated CT excitons. In addition, the photoexcitation dynamics in a new D-A blend of RR-P3HT/Indene C60 trisadduct (ICTA) has been studied and compared with the dynamics in RR-P3HT/PCBM.     

Femtochemistry of orange II in solution and in chemical and biological nanocavities

In this work, we report on studies of the nature of the dynamics and hydrophobic binding in cyclodextrins and human serum albumin protein complexes with orange II. With femtosecond time resolution, we examined the proton-transfer and trans-cis isomerization reactions of the ligand in these nanocavities and in pure solvents. Because of confinement at the ground state, the orientational motion in the formed phototautomer is restricted, leading to a rich dynamics. Therefore, the emission lifetimes span a large window of tens to hundreds of picoseconds in the cavities. Possible H-bond interactions between the guest and cyclodextrin do not affect the caged dynamics. For the protein-ligand complexes, slow diffusional motion ( approximately 630 ps) observed in the anisotropy decay indicates that the binding structure is not completely rigid, and the embedded guest is not frozen with the hydrophobic pocket. The ultrafast isomerization and decays are explained in terms of coupling motions between N-N and C-N stretching modes of the formed tautomer. We discuss the role of confinement on the trans-cis isomerization with the cavities and its relationships to frequency and time domains of nanostructure emission.     

Primary charge-recombination in an artificial photosynthetic reaction center

Photoinduced primary charge-separation and charge-recombination are characterized by a combination of time-resolved optical and EPR measurements of a fullerene-porphyrin-linked triad that undergoes fast, stepwise charge-separation processes. The electronic coupling for the energy-wasting charge recombination is evaluated from the singlet-triplet electronic energy gap in the short-lived, primary charge-separated state. The electronic coupling is found to be smaller by approximately 40% than that for the primary charge-separation. This inhibition of the electronic interaction for the charge-recombination to excited triplet state largely results from a symmetry-broken electronic structure modulated by configuration interaction between 3(b1u,b3g) and 3(au, b3g) electronic states of the free-base porphyrin.     

Ultrafast electron crystallography: transient structures of molecules, surfaces, and phase transitions

The static structure of macromolecular assemblies can be mapped out with atomic-scale resolution by using electron diffraction and microscopy of crystals. For transient nonequilibrium structures, which are critical to the understanding of dynamics and mechanisms, both spatial and temporal resolutions are required; the shortest scales of length (0.1-1 nm) and time (10(-13) to 10(-12) s) represent the quantum limit, the nonstatistical regime of rates. Here, we report the development of ultrafast electron crystallography for direct determination of structures with submonolayer sensitivity. In these experiments, we use crystalline silicon as a template for different adsorbates: hydrogen, chlorine, and trifluoroiodomethane. We observe the coherent restructuring of the surface layers with subangstrom displacement of atoms after the ultrafast heat impulse. This nonequilibrium dynamics, which is monitored in steps of 2 ps (total change 相似文献   

Biological water at the protein surface: dynamical solvation probed directly with femtosecond resolution

Biological water at the interface of proteins is critical to their equilibrium structures and enzyme function and to phenomena such as molecular recognition and protein-protein interactions. To actually probe the dynamics of water structure at the surface, we must examine the protein itself, without disrupting the native structure, and the ultrafast elementary processes of hydration. Here we report direct study, with femtosecond resolution, of the dynamics of hydration at the surface of the enzyme protein Subtilisin Carlsberg, whose single Trp residue (Trp-113) was used as an intrinsic biological fluorescent probe. For the protein, we observed two well separated dynamical solvation times, 0.8 ps and 38 ps, whereas in bulk water, we obtained 180 fs and 1.1 ps. We also studied a covalently bonded probe at a separation of approximately 7 A and observed the near disappearance of the 38-ps component, with solvation being practically complete in (time constant) 1.5 ps. The degree of rigidity of the probe (anisotropy decay) and of the water environment (protein vs. micelle) was also studied. These results show that hydration at the surface is a dynamical process with two general types of trajectories, those that result from weak interactions with the selected surface site, giving rise to bulk-type solvation (approximately 1 ps), and those that have a stronger interaction, enough to define a rigid water structure, with a solvation time of 38 ps, much slower than that of the bulk. At a distance of approximately 7 A from the surface, essentially all trajectories are bulk-type. The theoretical framework for these observations is discussed.     

Femtosecond studies of protein-ligand hydrophobic binding and dynamics: human serum albumin

In this contribution, we report studies of the nature of the dynamics and hydrophobic binding in protein-ligand complexes of human serum albumin with 2-(2'-hydroxyphenyl)-4-methyloxazole. With femtosecond time resolution, we examined the orientational motion of the ligand, its intrinsic nuclear motions, and the lifetime changes in the hydrophobic phase. For comparisons, with similar but chemical nanocavities, we also studied the same ligand in micelles and cyclodextrins. The hydrophobic interactions in the binding crevice are much stronger than those observed in cyclodextrins and micelles. The confined geometry restrains the nonradiative decay and significantly lengthens the excited-state lifetime. The observed dynamics over the femtosecond-to-nanosecond time scale indicate that the binding structure is rigid and the local motions of the ligand are nearly "frozen" in the protein. Another major finding is the elucidation of the directed dynamics by the protein. Proton transfer and intramolecular twisting of 2-(2'-hydroxyphenyl)-4-methyloxazole were observed to evolve along two routes: one involves the direct stretching motion in the molecular plane (approximately 200 fs) and is not sensitive to the environment; the second, less dominant, is related to the twisting motion (approximately 3 ps) of the two heterocyclic rings and drastically slows down in the protein hydrophobic pocket.     

Direct observation of vibrational coherence in bacterial reaction centers using femtosecond absorption spectroscopy.

It is shown that vibrational coherence modulates the femtosecond kinetics of stimulated emission and absorption of reaction centers of purple bacteria. In the DLL mutant of Rhodobacter capsulatus, which lacks the bacteriopheophytin electron acceptor, oscillations with periods of approximately 500 fs and possibly also of approximately 2 ps were observed, which are associated with formation of the excited state. The kinetics, which reflect primary processes in Rhodobacter sphaeroides R-26, were modulated by oscillations with a period of approximately 700 fs at 796 nm and approximately 2 ps at 930 nm. In the latter case, at 930 nm, where the stimulated emission of the excited state, P*, is probed, oscillations could only be resolved when a sufficiently narrow (10 nm) and concomitantly long pump pulse was used. This may indicate that the potential energy surface of the excited state is anharmonic or that low-frequency oscillations are masked when higher frequency modes are also coherently excited, or both. The possibility is discussed that the primary charge separation may be a coherent and adiabatic process coupled to low-frequency vibrational modes.     

Determining complete electron flow in the cofactor photoreduction of oxidized photolyase

The flavin cofactor in photoenzyme photolyase and photoreceptor cryptochrome may exist in an oxidized state and should be converted into reduced state(s) for biological functions. Such redox changes can be efficiently achieved by photoinduced electron transfer (ET) through a series of aromatic residues in the enzyme. Here, we report our complete characterization of photoreduction dynamics of photolyase with femtosecond resolution. With various site-directed mutations, we identified all possible electron donors in the enzyme and determined their ET timescales. The excited cofactor behaves as an electron sink to draw electron flow from a series of encircling aromatic molecules in three distinct layers from the active site in the center to the protein surface. The dominant electron flow follows the conserved tryptophan triad in a hopping pathway across the layers with multiple tunneling steps. These ET dynamics occur ultrafast in less than 150 ps and are strongly coupled with local protein and solvent relaxations. The reverse electron flow from the flavin is slow and in the nanosecond range to ensure high reduction efficiency. With 12 experimentally determined elementary ET steps and 6 ET reaction pairs, the enzyme exhibits a distinct reduction–potential gradient along the same aromatic residues with favorable reorganization energies to drive a highly unidirectional electron flow toward the active-site center from the protein surface.     

Excitation trapping and primary charge stabilization in Rhodopseudomonas viridis cells, measured electrically with picosecond resolution

The transmembrane primary charge separation in the photosynthetic bacterium Rhodopseudomonas viridis was monitored by electric measurements of the light-gradient type [Trissl, H. W. & Kunze, U. (1985) Biochim. Biophys. Acta 806, 136-144]. Excitation of whole cells with 30-ps laser pulses at either 532 nm or 1064 nm gave rise to a biphasic increase of the photovoltage. The fast phase, contributing about 50% of the total, rose with an exponential time constant ≤40 ps and was independent of the redox state of the quinone electron acceptor. It is assigned to the migration of the excitation energy in the antenna and its subsequent trapping by the reaction center, monitored by the ultrafast charge separation between the primary electron donor and the bacteriopheophytin intermediary acceptor. The slower phase (125 ± 50 ps) only occurred when the quinone was oxidized and disappeared when it was reduced (either chemically or photochemically). It is assigned to the forward electron transfer from the bacteriopheophytin to the quinone. The relative amplitudes of these two electrogenic steps demonstrate that the bacteriopheophytin intermediary acceptor is located halfway between the primary donor and the quinone.