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Controlling the activity of proteins with azobenzene photoswitches is a potent tool for manipulating their biological function. With the help of light, one can change e.g. binding affinities, control allostery or temper with complex biological processes. Additionally, due to their intrinsically fast photoisomerisation, azobenzene photoswitches can serve as triggers to initiate out-of-equilibrium processes. Such switching of the activity, therefore, initiates a cascade of conformational events, which can only be accessed with time-resolved methods. In this Review, we will show how combining the potency of azobenzene photoswitching with transient spectroscopic techniques helps to disclose the order of events and provide an experimental observation of biomolecular interactions in real-time. This will ultimately help us to understand how proteins accommodate, adapt and readjust their structure to answer an incoming signal and it will complete our knowledge of the dynamical character of proteins.

Allosteric regulation is an innate control in most metabolic and signalling cascades that enables living organisms to adapt to the changing environment by tuning the affinity and regulating the activity of target proteins. For a microscopic understanding of this process, a protein system has been designed in such a way that allosteric communication between the binding and allosteric site can be observed in both directions. To that end, an azobenzene-derived photoswitch has been linked to the α3-helix of the PDZ3 domain, arguably the smallest allosteric protein with a clearly identifiable binding and allosteric site. Photo-induced trans-to-cis isomerisation of the photoswitch increases the binding affinity of a small peptide ligand to the protein up to 120-fold, depending on temperature. At the same time, ligand binding speeds up the thermal cis-to-trans back-isomerisation rate of the photoswitch. Based on the energetics of the four states of the system (cis vs trans and ligand-bound vs free), the concept of an allosteric force is introduced, which can be used to drive chemical reactions., Almost all allosteric systems are large multi-domain proteins which complicates the investigation of the mechanism in atomistic detail. Here authors designed a small allosteric protein system using the photocontrollable PDZ3 domain, that allows controlling allostery between the binding and allosteric site in both directions.

Phytochromes are ubiquitous photoreceptor proteins that undergo a significant refolding of secondary structure in response to initial photoisomerization of the chromophoric group. This process is important for the signal transduction through the protein and thus its regulatory function in different organisms. Here, we employ two-dimensional infrared absorption (2D-IR) spectroscopy, an ultrafast spectroscopic technique that is sensitive to vibrational couplings, to study the photoreaction of bacterial phytochrome Agp1. By calculating difference spectra with respect to the photoactivation, we are able to isolate sharp difference cross-peaks that report on local changes in vibrational couplings between different sites of the chromophore and the protein. These results indicate inter alia that a dipole coupling between the chromophore and the so-called tongue region plays a role in stabilizing the protein in the light-activated state.

Hydridocarbonyl complexes, a class of industrially relevant catalysts, contain both the M-H and M-CO moieties. Here, using two-dimensional infrared spectroscopy, we examine the coupling of the typically weak M-H stretching mode and the intense M(C≡O) mode. By studying a series of Ir(I)- and Ir(III)-based hydridocarbonyl complexes, we show that the arrangement of the H and CO ligands in a

Resonant and off-resonant mid-infrared pump–probe spectroscopy is used to measure the vibrational dynamics of CO adsorbed to thin (0.2 nm, 2 nm, and 10 nm) heterogeneous Pt layers in an aqueous solution. The transient signals observed with resonant pumping are dominated by vibrational relaxation of the CO internal stretch vibration with a lifetime of T1 ∼ 3 ps in all cases. Off-resonant pumping suppresses that contribution to the signal and singles out a signal, which is attributed to heating of the metal layer as well as transient desorption of the CO molecules. Due to the small photon energy (0.2 eV) used as pump pulses, the mechanism of desorption must be thermal, in which case the desorption yield depends exclusively on the fluence of absorbed light and not its wavelength. The thin Pt layers facilitate CO desorption, despite a relatively low pump pulse fluence, as they concentrate the absorbed energy in a small volume.

This work explores the solid-liquid interface of a rhenium-tricarbonyl complex embedded in a layer of zirconium oxide deposited by atomic layer deposition (ALD). Time-resolved and steady state infrared spectroscopy were applied to reveal the correlations between the thickness of the ALD layer and the spectroscopic response of the system. We observed a transition of the molecular environment from flexible to rigid, as well as limitations to ligand exchange and excited state quenching on the embedded complexes, when the ALD layer is roughly of the same height as the molecules.

Over the last decades, photoreceptive proteins were extensively studied with biophysical methods to gain a fundamental understanding of their working mechanisms and further guide the development of optogenetic tools from them. Time-resolved infrared (IR) spectroscopy is one of the key methods to access their functional non-equilibrium processes with high temporal resolution, but has the major drawback that experimental data is usually highly complex. Linking the spectral response to specific molecular events is a major obstacle. Here, we investigate a cyanobacteriochrome (CBCR) photoreceptor with a combined approach of transient absorption spectroscopy in the Visible and IR spectral regions. We obtain kinetic information in both spectral regions by analysis with two different fitting methods, global multiexponential fitting and lifetime analysis. We investigate the ground state dynamics that follow photoexcitation in both directions of the bi-stable photocycle (Pr* and Pg*) in the nanosecond and microsecond time regime. We find two ground state intermediates associated with the decay of Pr* and four with Pg* and report the macroscopic time constants of their interconversions. One of these processes is assigned to a structural change in the protein backbone. At later delay times, the spectroscopic responses are stretched out in time, an effect that can be better described by the lifetime analysis than the global fit. We ascribe it to the intrinsic ruggedness of the free energy landscape of proteins., Comment: regular article 12 pages 7 figures and 2 tables

With the help of a Markov State Model (MSM), two-state behaviour is resolved for two computer models of water in a temperature range from 255 K to room temperature (295 K). The method is first validated for ST2 water, for which the so far strongest evidence for a liquid-liquid phase transition exists. In that case, the results from the MSM can be cross-checked against the radial distribution function g5(r) of the 5th-closest water molecule around a given reference water molecule. The latter is a commonly used local order parameter, which exhibits a bimodal distribution just above the liquid-liquid critical point that represents the low-density form of the liquid (LDL) and the high density liquid. The correlation times and correlation lengths of the corresponding spatial domains are calculated and it is shown that they are connected via a simple diffusion model. Once the approach is established, TIP4P/2005 will be considered, which is the much more realistic representation of real water. The MSM can resolve two-state behavior also in that case, albeit with significantly smaller correlation times and lengths. The population of LDL-like water increases with decreasing temperature, thereby explaining the density maximum at 4 °C along the lines of the two-state model of water.

In a recent paper, the experimental 2D-Raman-THz response of liquid water at ambient conditions has been presented [J. Savolainen, S. Ahmed, and P. Hamm, Proc. Natl. Acad. Sci. U. S. A. 110, 20402 (2013)]. Here, all-atom molecular dynamics simulations are performed with the goal to reproduce the experimental results. To that end, the molecular response functions are calculated in a first step, and are then convoluted with the laser pulses in order to enable a direct comparison with the experimental results. The molecular dynamics simulation are performed with several different water models: TIP4P/2005, SWM4-NDP, and TL4P. As polarizability is essential to describe the 2D-Raman-THz response, the TIP4P/2005 water molecules are amended with either an isotropic or a anisotropic polarizability a posteriori after the molecular dynamics simulation. In contrast, SWM4-NDP and TL4P are intrinsically polarizable, and hence the 2D-Raman-THz response can be calculated in a self-consistent way, using the same force field as during the molecular dynamics simulation. It is found that the 2D-Raman-THz response depends extremely sensitively on details of the water model, and in particular on details of the description of polarizability. Despite the limited time resolution of the experiment, it could easily distinguish between various water models. Albeit not perfect, the overall best agreement with the experimental data is obtained for the TL4P water model.

Halogenated groups are relevant in pharmaceutical applications and potentially useful spectroscopic probes for infrared spectroscopy. In this work, the structural dynamics and infrared spectroscopy of $para$-fluorophenol (F-PhOH) and phenol (PhOH) is investigated in the gas phase and in water using a combination of experiment and molecular dynamics (MD) simulations. The gas phase and solvent dynamics around F-PhOH and PhOH is characterized from atomistic simulations using empirical energy functions with point charges or multipoles for the electrostatics, Machine-Learning (ML) based parametrization and with full $\textit{ab initio}$ (QM) and mixed Quantum Mechanical/Molecular Mechanics (QM/MM) simulations with a particular focus on the CF- and OH-stretch region. The CF-stretch band is heavily mixed with other modes whereas the OH-stretch in solution displays a characteristic high-frequency peak around 3600 cm$^{-1}$ most likely associated with the -OH group of PhOH and F-PhOH together with a characteristic progression below 3000 cm$^{-1}$ due to coupling with water modes which is also reproduced by several of the simulations. Solvent and radial distribution functions indicate that the CF-site is largely hydrophobic except for simulations using point charges which renders them unsuited for correctly describing hydration and dynamics around fluorinated sites., Main Manuscript: 41 pages and 6 figures, SI: 10 pages and 10 figures

The photo-triggered unbinding of the intrinsically disordered S-peptide from the RNase S complex is studied with the help of transient IR spectroscopy, covering a wide range of time scales from 100 ps to 10 ms. To that end, an azobenzene moiety has been linked to the S-peptide in a way that its helicity is disrupted by light, thereby initiating its complete unbinding. The full sequence of events is observed, starting from unfolding of the helical structure of the S-peptide on a 20 ns timescale while still being in the binding pocket of the S-protein, S-peptide unbinding after 300 microseconds, and the structural response of the S-protein after 3 ms. With regard to the S-peptide dynamics, the binding mechanism can be classified as an induced fit, while the structural response of the S-protein is better described as conformational selection.

The dynamics of peptide–protein binding and unbinding of a variant of the RNase S system has been investigated. To initiate the process, a photoswitchable azobenzene moiety has been covalently linked to the S-peptide, thereby switching its binding affinity to the S-protein. Transient fluorescence quenching was measured with the help of a time-resolved fluorometer, which has been specifically designed for these experiments and is based on inexpensive light-emitting diodes and laser diodes only. One mutant shows on–off behavior with no specific binding detectable in one of the states of the photoswitch. Unbinding is faster by at least 2 orders of magnitude, compared to that of other variants of the RNase S system. We conclude that unbinding is essentially barrier-less in that case, revealing the intrinsic dynamics of the unbinding event, which occurs on a time scale of a few hundred microseconds in a strongly stretched-exponential manner.

While much is known about different allosteric regulation mechanisms, the nature of the "allosteric signal", and the timescale on which it propagates, remains elusive. The PDZ3 domain from postsynaptic density-95 protein is a small protein domain with a terminal third alpha helix -- the $\alpha$3-helix, which is known to be allosterically active. By cross-linking the allosteric helix with an azobenzene moiety, we obtained a photocontrollable PDZ3 variant. Photoswitching triggers its allosteric transition, resulting in a change in binding affnity of a peptide to the remote binding pocket. Using time-resolved infrared and UV/Vis spectroscopy, we follow the allosteric signal transduction and reconstruct the timeline in which the allosteric signal propagates through the protein within 200 ns.

Significance The degree to which water is structured is an extremely intriguing problem and a matter of ongoing debate. To that end, we studied the 2D Raman–terahertz response of liquid water in dependence of temperature and isotope substitution, considering H 2 O, D 2 O, and H 2 18 O. A very short-lived echo is observed, whose lifetime slows down with decreasing temperature. It differs for D 2 O versus H 2 O, whereas that of H 2 18 O versus H 2 O is the same. The comparison of the three isotopologues allows us to disentangle nuclear quantum effects from trivial dynamical mass effects. The former dominate the echo lifetime, hence, it is concluded that it is a measure of the heterogeneity of the hydrogen bond networks of water.

A new application of high-repetition rate, femtosecond Yb-laser/amplifier systems is introduced: transient 2D IR spectroscopy covering the time range from micro- to milliseconds. This approach intertwines the measurement of 2D IR spectra with the time separation from an actinic pump pulse and utilizes the high repetition rate of these lasers systems in two ways: by offering a high time resolution (10 µs) and by enabling the measurement of many 2D IR spectra. The well-studied photocycle of bacteriorhodopsin is used as a demonstration object in this proof-of-principle experiment.

A stop-flow sample delivery system for transient spectroscopy is presented, which is, in particular, suited for laser-based instruments (quantum-cascade lasers or amplified femtosecond lasers) with excitation pulse repetition rates in the range 10–100 Hz. Two pulsing micro-valves are mounted onto a flow cuvette designed for transient IR spectroscopy, which is integrated into a flow cycle driven by a peristaltic pump. The performance of the system is demonstrated with transient IR experiments of the trans-to-cis photoisomerization of a water-soluble azobenzene derivative. The sample stands still when the micro-valves are closed and is pushed out from the probe beam focus on a 1 ms timescale when opening the micro-valves. The setup is extremely sample efficient. It needs only small sample volumes, and at the same time, it enables excitation of a large fraction of molecules in solution.

Vibrational energy transfer (VET) between two isotopologues of [Re(dcb)(CO)3Br] immobilized on a TiO2 surface is studied with the help of 2D IR spectroscopy in dependence of surface coverage. To dilute the molecules on the surface, and thereby control the intermolecular distances, two different diluents have been used: a third isotopologue of the same molecule and 4-cyanobenzoic acid. As expected, the VET rate decreases with dilution. For a quantitative investigation of the distance dependence of the VET rate, we analyze the data based on an excitonic model. This model reveals the typical 1/r6-distance dependence for a dimer of a donor and acceptor, similar to the nuclear Overhauser effect in NMR spectroscopy or Forster resonant energy transfer in electronic spectroscopy. However, VET becomes a collective phenomenon on the surface, with the existence of a network of coupled molecules and its disappearance below a percolation threshold, dominating the concentration dependence of the VET rate.

While allostery is of paramount importance for protein regulation, the underlying dynamical process of ligand (un)binding at one site, resulting time evolution of the protein structure, and change of the binding affinity at a remote site is not well understood. Here the ligand-induced conformational transition in a widely studied model system of allostery, the PDZ2 domain, is investigated by transient infrared spectroscopy accompanied by molecular dynamics simulations. To this end, an azobenzene derived photoswitch is linked to a peptide ligand in a way that its binding affinity to the PDZ2 domain changes upon switching, thus initiating an allosteric transition in the PDZ2 domain protein. The subsequent response of the protein, covering four decades of time ranging from $\sim$1~ns to $\sim$10~$\mu$s, can be rationalize by a remodelling of its rugged free energy landscape, with ver subtle shifts in the populations of a small number of structurally well defined states. It is proposed that structurally and dynamically driven allostery, often discussed as limiting scenarios of allosteric communication, actually go hand-in-hand, allowing the protein to adapt its free energy landscape to incoming signals., Comment: This unedited earlier version of the manuscript may be downloaded for personal use only. Any other use requires prior permission of the author and the National Academy of Sciences USA. The final manuscript was published in Proceedings of the National Academy of Sciences USA 117, 26031-26039 (2020) and can be found under https://www.pnas.org/content/117/42/26031.short

Azidohomoalanine (Aha) is an unnatural amino acid containing an infrared active azido side chain group that can, through frequency shifts of the azido stretch vibration, act as a probe of local structure. To realize the potential of such structural probes for protein science, we have developed a two-dimensional infrared spectrometer employing fast mechanical scanning and intrinsic phasing of the resulting spectra, leading to a lower sensitivity limit of ∼100 μOD level samples. Using this approach, we quantify the biomolecular recognition between a PDZ2 domain and two Aha-mutated peptides. It is shown that this method can distinguish different binding modes and that the energetics of binding can be determined.

A three-point velocity correlation function ⟨v(t1 + t2)v2(t1)v(0)⟩ is introduced for a better understanding of the recent 2D-Raman-THz spectroscopy of the intermolecular degrees of freedoms of water and aqueous salt solutions. This correlation function reveals echoes in the presence of inhomogeneous broadening, which are coined “velocity echoes.” In analogy to the well-known two-point velocity correlation function ⟨v(t)v(0)⟩, it reflects the density of states (DOS) of the system under study without having to amend them with transition dipoles and transition polarizabilities. The correlation function can be calculated from equilibrium trajectories and converges extremely quickly. After deriving the theory, the information content of the three-point velocity correlation function is first tested based on a simple harmonic oscillator model with Langevin dynamics. Subsequently, velocity echoes of TIP4P/2005 water are calculated as a function of temperature, covering ambient conditions, the supercooled regime and amorphous ice, as well as upon addition of various salts. The experimentally observed trends can be reproduced qualitatively with the help of computationally very inexpensive molecular dynamics simulations.

Hybrid two-dimensional (2D) Raman-terahertz spectroscopy with the Raman-terahertz-terahertz (RTT) pulse sequence is used to explore the ultrafast intra- and intermolecular degrees of freedom of liquid bromoform (CHBr

The noncanonical amino acid azidohomoalanine (Aha) is known to be an environment-sensitive infrared probe for the site-specific investigation of protein structure and dynamics. Here, the capability of that label is explored to detect protein-ligand interactions by incorporating it in the vicinity of the binding groove of a PDZ2 domain. Circular dichroism and isothermal titration calorimetry measurements reveal that the perturbation of the protein system by mutation is negligible, with minimal influence on protein stability and binding affinity. Two-dimensional infrared spectra exhibit small (1-3 cm-1) but clearly measurable red shifts of the Aha vibrational frequency upon binding of two different peptide ligands, while accompanying molecular dynamics simulations suggest that these red shifts are induced by polar contacts with side chains of the peptide ligands. Hence, Aha is a versatile and minimally invasive vibrational label that is not only able to report on large structural changes during, e.g., protein folding, but also on very subtle changes of the electrostatic environment upon ligand binding.

We explore the potential of various hydroquinone/quinone redox couples as electron relays in a homogenous water reduction system between a Re-based photosensitizer and a sacrificial electron donor [tris-(2-carboxyethyl)-phosphine, TCEP]. By using transient IR spectroscopy, flash photolysis as well as stopped-flow techniques covering timescales from picoseconds to 100 ms, we determine quenching rates and cage escape yields, the kinetics of the follow-up chemistry of the semiquinone, the recombination rates, as well as the re-reduction rates by TCEP. The overall quantum yield of hydrogen production is low, and we show that the limiting factors are the small cage escape yields and, more importantly, the slow regeneration rate by TCEP in comparison to the undesired charge recombination with the reduced water reduction catalyst.

Three structurally very different antifreeze proteins (AFPs) are studied, addressing the question as to what extent the hypothesized preordering-binding mechanism is still relevant in the second solvation layer of the protein and beyond. Assuming a two-state model of water, the solvation layers are analyzed with the help of molecular dynamics simulations together with a Markov state model, which investigates the local tedrahedrality of the water hydrogen-bond network around a given water molecule. It has been shown previously that this analysis can discriminate the high-entropy, high-density state of the liquid (HDL) from its more structured low-density state (LDL). All investigated proteins, regardless of whether they are an AFP or not, have a tendency to increase the amount of HDL in their second solvation layer. The ice binding site (IBS) of the antifreeze proteins counteracts that trend, with either a hole in the HDL layer or a true excess of LDL. The results correlate to a certain extent with recent experiments, which have observed ice-like vibrational (VSFG) spectra for the water atop the IBS of only a subset of antifreeze proteins. It is concluded that the preordering-binding mechanism indeed seems to play a role but is only part of the overall picture.

The vibrational dynamics of Pt–H on a nanostructured platinum surface has been examined by ultrafast infrared spectroscopy. Three bands are observed at 1800, 2000, and 2090 cm–1, which are assigned to Pt–CO in a bridged and linear configuration and Pt–H, respectively. Lifetime analysis revealed a time constant of (0.8 ± 0.1) ps for the Pt–H mode, considerably shorter than that of Pt–CO because of its stronger coupling to the metal substrate. Two-dimensional attenuated total reflection infrared spectroscopy provided additional evidence for the assignment based on the anharmonic shift, which is large in the case of Pt–H (90 cm–1), in agreement with the density functional theory calculations. The absorption cross section of Pt–H is smaller than that of the very strong Pt–CO vibration by only a modest factor of ∼1.5–3. Because Pt–H is transiently involved in catalytic water splitting on Pt, the present spectroscopic characterization paves the way for in-operando kinetic studies of such reactions.

We present experimental and simulated 2D IR spectra of some high-pressure forms of isotope-pure D2O ice and compare the results to those of ice Ih published previously [F. Perakis and P. Hamm, Phys. Chem. Chem. Phys. 14, 6250 (2012); L. Shi et al., ibid. 18, 3772 (2016)]. Ice II, ice V, and ice XIII have been chosen for this study, since this selection covers many aspects of the polymorphism of ice. That is, ice II is a hydrogen-ordered phase of ice, in contrast to ice Ih, while ice V and ice XIII are a hydrogen-disordered/ordered couple that shares essentially the same oxygen structure and hydrogen-bonded network. For the transmission 2D IR spectroscopy, a novel method had to be developed for the preparation of ultrathin films (1-2 mu m) of high-pressure ices with good optical quality. We also simulated 2D IR spectra based on molecular dynamics simulations connected to a vibrational exciton picture. These simulations agree with the experimental results in a semi-quantitative manner for ice II, while the same approach failed for ice V and ice XIII. From the perspective of 2D IR spectroscopy, ice II appears to be more inhomogeneously broadened than ice Ih, despite its hydrogen-order, which we attribute to the fact that ice II is structurally more complex with four distinguishable hydrogen bonds that mix due to exciton coupling. Ice V and ice XIII, on the other hand, behave as expected with the hydrogen-disordered case (ice V) being more inhomogenously broadened. Furthermore, in all hydrogen-ordered forms (ice II and ice XIII), cross peaks could be identified in the anisotropic 2D IR spectrum, whose signs reveal the relative direction of the corresponding excitonic states. Published by AIP Publishing.

Nuclear quantum effects in liquid water have profound implications for several of its macroscopic properties related to the structure, dynamics, spectroscopy, and transport. Although several of water's macroscopic properties can be reproduced by classical descriptions of the nuclei using interaction potentials effectively parameterized for a narrow range of its phase diagram, a proper account of the nuclear quantum effects is required to ensure that the underlying molecular interactions are transferable across a wide temperature range covering different regions of that diagram. When performing an analysis of the hydrogen-bonded structural networks in liquid water resulting from the classical (class) and quantum (qm) descriptions of the nuclei with two interaction potentials that are at the two opposite ends of the range in describing quantum effects, namely the flexible, pair-wise additive q-TIP4P/F, and the flexible, polarizable TTM3-F, we found that the (class) and (qm) results can be superimposed over the temperature range T = 250-350 K using a surprisingly simple, linear scaling of the two temperatures according to T(qm) = α T(class) + ΔT, where α = 0.99 and ΔT = -6 K for q-TIP4P/F and α = 1.24 and ΔT = -64 K for TTM3-F. This simple relationship suggests that the structural networks resulting from the quantum and classical treatment of the nuclei with those two very different interaction potentials are essentially similar to each other over this extended temperature range once a model-dependent linear temperature scaling law is applied.

We show that it is possible to phase multidimensional infrared spectra generated by a boxcars geometry four-wave mixing spectrometer directly from the signal generated by the molecular vibration of interest, without the need for auxiliary phasing measurements. For isolated vibrations, the phase profile of the 2D response smoothly varies between fixed phase limits, allowing for a general target for phasing independent of the degree of anharmonicity exhibited between the ground and excited state. As a proof of principle, the 2D response of the similar to 2155 cm(-1) thiocyanate stretch vibration of MeSCN, a system exhibiting anharmonicity such that the 0-1 and 1-2 transitions are spectrally isolated, is successfuly phased directly from the experimental spectra. The methodology is also applied to correctly phase extremely weak signals of the unnatural amino acid azidohomoalanine following background subtraction.

The photochemistry of a molecular pentad composed of a central anthraquinone (AQ) acceptor flanked by two Ru(bpy)32+ photosensitizers and two peripheral triarylamine (TAA) donors was investigated by transient IR and UV–vis spectroscopies in the presence of 0.2 M p-toluenesulfonic acid (TsOH) in deaerated acetonitrile. In ∼15% of all excited pentad molecules, AQ is converted to its hydroquinone form (AQH2) via reversible intramolecular electron transfer from the two TAA units (τ = 65 ps), followed by intermolecular proton transfer from TsOH (τ ≈ 3 ns for the first step). Although the light-driven accumulation of reduction equivalents occurs through a sequence of electron and proton transfer steps, the resulting photoproduct decays via concerted PCET (τ = 4.7 μs) with an H/D kinetic isotope effect of 1.4 ± 0.2. Moreover, the reoxidation of AQH2 seems to take place via a double electron transfer step involving both TAA+ units rather than sequential single electron transfer events. Thus, the overall charge-recombination reaction seems to involve a concerted proton-coupled two-electron oxidation of AQH2. The comparison of experimental data obtained in neat acetonitrile with data from acidic solutions suggests that the inverted driving-force effect can play a crucial role for obtaining long-lived photoproducts resulting from multiphoton, multielectron processes. Our pentad provides the first example of light-driven accumulation of reduction equivalents stabilized by PCET in artificial molecular systems without sacrificial reagents. Our study provides fundamental insight into how light-driven multielectron redox chemistry, for example the reduction of CO2 or the oxidation of H2O, can potentially be performed without sacrificial reagents.

By covalently linking an azobenzene photoswitch across the binding groove of a PDZ domain, a conformational transition, similar to the one occurring upon ligand binding to the unmodified domain, can be initiated on a picosecond timescale by a laser pulse. The protein structures have been characterized in the two photoswitch states through NMR spectroscopy and the transition between them through ultrafast IR spectroscopy and molecular dynamics simulations. The binding groove opens on a 100-ns timescale in a highly nonexponential manner, and the molecular dynamics simulations suggest that the process is governed by the rearrangement of the water network on the protein surface. We propose this rearrangement of the water network to be another possible mechanism of allostery.

Accumulation and temporary storage of redox equivalents with visible light as an energy input is of pivotal importance for artificial photosynthesis because key reactions, such as CO2 reduction or water oxidation, require the transfer of multiple redox equivalents. We report on the first purely molecular system, in which a long-lived charge-separated state (τ≈870 ns) with two electrons accumulated on a suitable acceptor unit can be observed after excitation with visible light. Importantly, no sacrificial reagents were employed.

Amyloid aggregates are highly ordered fibrillar assemblies of polypeptides involved in a number of neurodegenerative diseases. Very little is known on the pathways of self-assembly of peptides into the final amyloid fibrils, which is due in part to the difficulty of triggering the aggregation process in a controlled manner. Here we present the design and validation of a cross-linked hexapeptide that reversibly aggregates and dissociates under ultraviolet light irradiation control. First molecular dynamics simulations were carried out to identify, among hundreds of possible sequences, those with the highest propensity to form ordered (β-sheet) oligomers in the trans state of the azobenzene cross-linker, and at the same time with the highest solubility in the cis state. In the simulations, the peptides were observed to spontaneously form ordered oligomers with cross-β contacts when the cross-linker was in the trans state, whereas in the cis state they self-assemble into amorphous aggregates. For the most promising sequence emerging from the simulations (Ac-Cys-His-Gly-Gln-Cys-Lys-NH(2) cross-linked at the two cysteine residues), the photoisomerization of the azobenzene group was shown to induce reversible aggregation by time-resolved light scattering and fluorescence measurements. The amyloid-like fibrillar topology was confirmed by electron microscopy. Potential applications of minimally designed peptides with photoswitchable amyloidogenic propensity are briefly discussed.

In photocatalytic H2 formation, tertiary amines are commonly used as sacrificial electron donors, thereby limiting the pH range for studies in water and the concentration of free protons. We found that ascorbate rapidly reductively quenches the excited state of [Re(CO)3(bipy)(py)]+ (bpy = 2,2′-bipyridyl; py = pyridine). In combination with the water reduction catalyst (WRC) [Co{(DO)(DOH)pn}Br2] [(DO)(DOH)pn = N2,N2′-propanediylbis(2,3-butanedione 2-imine 3-oxime)], this system produces H2 upon irradiation with light under acidic conditions (pH range 2–6) and with significantly enhanced turnover numbers. Furthermore, we observed that, in contrast to similar systems with tertiary amines, oxidized ascorbate (dehydroascorbic acid) is slowly re-reduced during the course of photocatalysis.

In a previous paper [M. Schade, P. Hamm, Vibrational energy transport in the presence of intrasite vibrational energy redistribution, J. Chem. Phys. 131 (2009) 044511], it has been shown that on ultrashort length and time scales, the speed of vibrational energy transport along a molecular chain is limited by intra site vibrational relaxation rather than the actual inter site propagation. However, since intra site vibrational relaxation is length independent, the inter site propagation rate is expected to become rate-limiting at some length scale, where propagation approaches the bulk limit. In the present paper, we investigate the transition between both regimes. The response of different types of modes may be very different at early times, depending on how much they contribute directly to energy transport. Surprisingly though, when averaging the energy content over all vibrational modes of the various chain sites, the complexity of the intra site vibrational relaxation process is completely hidden so that energy transport on the nanoscale can be described by an effective propagation rate, that equals the bulk value, even at short times.

In a combined experimental-theoretical study, we investigated the transport of vibrational energy from the surrounding solvent into the interior of a heme protein, the sperm whale myoglobin double mutant L29W-S108L, and its dependence on temperature from 20 to 70 K. The hindered libration of a CO molecule that is not covalently bound to any part of the protein but is trapped in one of its binding pockets (the Xe4 pocket) was used as the local thermometer. Energy was deposited into the solvent by IR excitation. Experimentally, the energy transfer rate increased from (30 ps)(-1) at 20 K to (8 ps)(-1) at 70 K. This temperature trend is opposite to what is expected, assuming that the mechanism of heat transport is similar to that in glasses. In order to elucidate the mechanism and its temperature dependence, nonequilibrium molecular dynamics (MD) simulations were performed, which, however, predicted an essentially temperature-independent rate of vibrational energy flow. We tentatively conclude that the MD potentials overestimate the coupling between the protein and the CO molecule, which appears to be the rate-limiting step in the real system at low temperatures. Assuming that this coupling is anharmonic in nature, the observed temperature trend can readily be explained.

Ultrafast dynamics of molecules at solid–liquid interfaces are of outstanding importance in chemistry and physics due to their involvement in processes of heterogeneous catalysis. We present a new spectroscopic approach to resolve coherent, time-resolved, 2D vibrational spectra as well as ultrafast vibrational relaxation dynamics of molecules adsorbed on metallic thin films in contact with liquids. The setup is based on the technique of attenuated total reflectance (ATR) spectroscopy, which is used at interfaces between materials that exhibit different refractive indices. As a sample molecule, we consider carbon monoxide adsorbed in different binding configurations on different metals and resolve its femtosecond vibrational dynamics. It is presented that mid-infrared, multidimensional ATR spectroscopy allows for obtaining a surface-sensitive characterization of adsorbates’ vibrational relaxation, spectral diffusion dynamics, and sample inhomogeneity on the femtosecond time scale.

We present two-dimensional infrared (2D IR) spectra of organic monolayers immobilized on thin metallic films at the solid liquid interface. The experiments are acquired under Attenuated Total Reflectance (ATR) conditions which allow a surface-sensitive measurement of spectral diffusion, sample inhomogeneity, and vibrational relaxation of the monolayers. Terminal azide functional groups are used as local probes of the environment and structural dynamics of the samples. Specifically, we investigate the influence of different alkyl chain-lengths on the ultrafast dynamics of the monolayer, revealing a smaller initial inhomogeneity and faster spectral diffusion with increasing chain-length. Furthermore, by varying the environment (i.e., in different solvents or as bare sample), we conclude that the most significant contribution to spectral diffusion stems from intra- and intermolecular dynamics within the monolayer. The obtained results demonstrate that 2D ATR IR spectroscopy is a versatile tool for measuring interfacial dynamics of adsorbed molecules.

2D-IR spectroscopy has matured to a powerful technique to study the structure and dynamics of peptides, but its extension to larger proteins is still in its infancy, the major limitations being sensitivity and selectivity. Site-selective information requires measuring single vibrational probes at sub-millimolar concentrations where most proteins are still stable, which is a severe challenge for conventional (FT)IR spectroscopy. Besides its ultrafast time-resolution, a so far largely underappreciated potential of 2D-IR spectroscopy lies in its sensitivity gain. The present paper sets the goals and outlines strategies how to use that sensitivity gain together with properly designed vibrational labels to make IR spectroscopy a versatile tool to study a wide class of proteins.

The mechanism of photocatalytic hydrogen production was studied with a three-component system consisting of fac-[Re(py)(CO)3bipy](+) (py = pyridine, bipy = 2,2'-bipyridine) as photosensitizer, [Co(TPY-OH)(OH2)](2+) (TPY-OH = 2-bis(2-pyridyl)(hydroxy)methyl-6-pyridylpyridine), a polypyridyl-based cobalt complex, as water reduction catalyst (WRC), and triethanolamine (TEOA) as sacrificial electron donor in aqueous solution. A detailed mechanistic picture is provided, which covers all processes from excited state quenching on the time scale of a few nanoseconds to hydrogen release taking place between seconds and minutes at moderately basic reaction conditions. Altogether these processes span 9 orders of magnitude in time. The following reaction sequence was found to be the dominant pathway for hydrogen generation: After reductive quenching by TEOA, the reduced photosensitizer (PS) transfers an electron to the Co(II)-WRC. Protonation of Co(I) yields Co(III)H which is reduced in the presence of excess Co(I). Co(II)H releases hydrogen after a second protonation step, which is detected time-resolved by a clark-type hydrogen electrode. Aside from these productive steps, the role of side and back reactions involving TEOA-derived species is assessed, which is particularly relevant in laser flash photolysis measurements with significantly larger transient concentrations of reactive species as compared to continuous photolysis experiments. Most notable is an equilibrium reaction involving Co(I), which is explained by a nucleophilic addition of Co(I) to the oxidation product of TEOA, an electrophilic iminium ion. Quantum chemical calculations indicate that the reaction is energetically feasible. The calculated spectra of the adduct are consistent with the spectroscopic observations.

By covalently binding a photoswitchable linker across the binding groove of the PDZ2 domain, a small conformational change can be photo-initiated that mimics the allosteric transition of the protein. The response of its binding groove is investigated with the help of ultrafast pump-probe IR spectroscopy from picoseconds to tens of microseconds. The temperature dependence of that response is compatible with diffusive dynamics on a rugged energy landscape without any prominent energy barrier. Furthermore, the dependence of the kinetics on the concentration of certain viscogens, sucrose, and glycerol, has been investigated. A pronounced viscosity dependence is observed that can be best fit by a power law, i.e., a fractional viscosity dependence. The change of kinetics when comparing sucrose with glycerol as viscogen, however, provides strong evidence that direct interactions of the viscogen molecule with the protein do play a role as well. This conclusion is supported by accompanying molecular dynamics simulations.

The integrity of a small model protein, the 36-residue villin headpiece HP36, attached to gold nanoparticles (AuNP) is examined, and its response to laser excitation of the AuNPs is investigated. To that end, it is first verified by stationary IR and CD spectroscopy, together with denaturation experiments, that the folded structure of the protein is fully preserved when attached to the AuNP surface. It is then shown by time-resolved IR spectroscopy that the protein does not unfold, even upon the highest pump fluences that lead to local temperature jumps on the order of 1000 K of the phonon system of the AuNPs, since that temperature jump persists for too short a time of a few nanoseconds only to be destructive. Judged from a blue shift of the amide I band, indicating destabilized or a few broken hydrogen bonds, the protein either swells, becomes more unstructured from the termini, or changes its degree of solvation. In any case, it recovers immediately after the excess energy dissipates into the bulk solvent. The process is entirely reversible for millions of laser shots without any indication of aggregation of the protein or the AuNPs and with only a minor fraction of broken protein-AuNP thiol bonds. The work provides important cornerstones in designing laser pulse parameters for maximal heating with protein-capped AuNPs without destroying the capping layer.

Using 3D infrared (IR) exchange spectroscopy, the ultrafast hydrogen-bond forming and breaking (i.e., complexation) kinetics of phenol to benzene in a benzene/CCl4 mixture is investigated. By introducing a third time point at which the hydrogen-bonding state of phenol is measured (in comparison with 2D IR exchange spectroscopy), the spectroscopic method can serve as a critical test of whether the spectroscopic coordinate used to observe the exchange process is a memory-free, or Markovian, coordinate. For the system under investigation, the 3D IR results suggest that this is not the case. This conclusion is reconfirmed by accompanying molecular dynamics simulations, which furthermore reveal that the non-Markovian kinetics is caused by the heterogeneous structure of the mixed solvent.

It is generally assumed that the hydrated electron occupies a quasi-spherical cavity surrounded by only a few water molecules in its equilibrated state. However, in the very moment of its generation, before water has had time to respond to the extra charge, it is expected to be significantly larger in size. According to a particle-in-a-box picture, the frequency of its absorption spectrum is a sensitive measure of the initial size of the electronic wavefunction. Here, using transient terahertz spectroscopy, we show that the excess electron initially absorbs in the far-infrared at a frequency for which accompanying ab initio molecular dynamics simulations estimate an initial delocalization length of ≈40 A. The electron subsequently shrinks due to solvation and thereby leaves the terahertz observation window very quickly, within ≈200 fs. It is generally believed that, after being generated, an excess electron in water shrinks from a strongly delocalized to a localized state in about a picosecond. Now, these early stages in the behaviour of this electron have been observed using a combination of transient THz spectroscopy and ab initio molecular dynamics simulations.