Time-resolved infrared spectroscopy probes protein dynamics over timescales spanning more than ten orders of magnitude, yet the molecular motions underlying the observed kinetic signatures have remained elusive. Here we combine transient infrared spectroscopy with nonequilibrium molecular dynamics simulations to establish a direct connection between experimental relaxation times and local structural motions. Studying single-domain allosteric proteins, we find that inter-residue contact distances provide the structural representation that most faithfully reproduces the experimental dynamics. Correlation analysis identifies localized networks of coordinated contacts that mediate communication between secondary-structure elements. The characteristic timescales of these contact networks quantitatively match the experimentally observed relaxation processes, enabling each kinetic step to be assigned to a specific molecular motion. Applied to allosteric signal propagation in PDZ3 and photoinduced ligand unbinding in PDZ2, this framework provides an atomistic picture of hierarchical protein relaxation and establishes a general framework for connecting transient infrared spectroscopy with the molecular mechanisms of protein dynamics.

Light-oxygen-voltage (LOV) domain proteins represent a versatile class of photoreceptors capable of regulating a wide range of light-dependent biological functions. While a lot of studies have focused on the photochemistry of LOV domains, the mechanisms of signal generation and propagation in multidomain LOV proteins remain incompletely understood. Here, we investigated two multidomain proteins, using time-resolved infrared spectroscopy. The measurements resolve the entire photocycle dynamics from picoseconds to hours and uncover distinct patterns of local and global structural responses. The two multidomain proteins under study, YF1 and PAL, exhibit nearly identical dynamics during excitation and intersystem crossing on the nanosecond timescale, reflecting conserved local interactions between the chromophore and its highly conserved binding pocket. Multiscale simulations attribute minor spectral differences in this regime to a phenylalanine residue located near the chromophore present only in one of the two LOV domains. The similarities, however, end at the microsecond timescale, where adduct formation already involves global structural adaptations. By experimentally isolating the response of the histidine kinase effector domain in the synthetic photoreceptor YF1, we show that major structural adaptions of the effector domain occur concurrently with cysteine-adduct formation and that the Jα-helix putatively mediates unidirectional communication between domains. In PAL, light-induced opening of the RNA binding site during the adduct formation is additionally followed by a subsequent rearrangement in the distal PAS domain after 3 s. This highlights the pivotal yet distinct roles of the Jα-helix in signal transmission, which depend on the domain topology. Ultimately, our study not only deepens the current understanding of signal transduction in full-length LOV proteins, but also contributes to the fundamental framework for the future application of LOV domains in optogenetic engineering.

The activation mechanism of Mn-based molecular catalysts is reported in a three-component system (photosensitizer, electron donor, catalyst), investigated by time-resolved infrared spectroscopy. In total four complexes were studied that are derived from Mn(2,2'-bipyridine)(CO)3Br by varying substituents on the ligand, which impose steric constraints or modulate electronic properties. Thereby, ligand effects on catalyst activation pathways are systematically assessed. A unified feature across all systems is that the intermediate after one-electron reduction and subsequent Br- dissociation, i.e., Mn0(L)(CO)3, possesses a more positive reduction potential than the parent complex, leading to its rapid second reduction. This step outcompetes dimerization of Mn0 radicals, which instead proceeds through a symproportionation between the two-electron reduced species and the parent complex. However, when dimer formation is sterically hindered, two Mn0 species arise instead. The final process is the re-oxidation of the reduced intermediates by either hydrogen evolution or regeneration of the electron donor. Although CO2 conversion was not the focus of this work, the elucidated pathways clarify how competing re-oxidation channels can limit reduction efficiency or alter product selectivity. These mechanistic insights provide a foundation for rational strategies to control the selectivity and amplify the desired catalytic reactions.

In ferroelastic materials, spontaneous symmetry breaking leads to the formation of twin domains. Although the bulk crystal typically remains centrosymmetric, inversion symmetry can be locally broken at the domain walls, potentially changing phonon selection rules and enabling local anharmonic phonon coupling. Here we report direct evidence of such anharmonic coupling in ferroelastic LaAlO_3 using two-dimensional Raman-terahertz spectroscopy. We attribute the cross-peaks observed in the two-dimensional spectra to both mechanical and electrical anharmonicity between the A_1g Raman-active phonon and the E_g phonon, which acquires finite infrared activity through local inversion symmetry breaking at ferroelastic domain walls. These findings provide new insight into the complex lattice dynamics of ferroelastic materials and highlight the potential of two-dimensional Raman-terahertz spectroscopy to uncover subtle symmetry breaking through the detection of intrinsically weak anharmonic signals.

We report on the activation pathway of a series of CO2 reduction catalysts, trans(Cl)-[Ru(X,X'-dimethyl-2,2'-bipyridine)(CO)2Cl2], with a focus on trans(Cl)-[Ru(6,6'-dimethyl-2,2'-bipyridine)(CO)2Cl2]), in the presence of the reductive quencher 1-benzyl-1,4-dihydronicotinamide and the photosensitizer Ru(bpy)3Cl2. Most mechanistic studies of these types of catalytic systems use spectroelectrochemistry in the IR, where the vibrational frequencies of the carbonyl vibrations report on the electron density on the metal center. However, spectroelectrochemistry may miss short-lived intermediates, while at the same time the spectra can be dominated by accumulating side-products, which may play only a minor role in the reaction cycle. Transient IR spectroscopy on all relevant time scales, from picoseconds to hundreds of milliseconds, can bridge this gap, revealing a surprisingly complex reaction pathway (in combination with NMR spectroscopy as well as DFT calculations). That is, electron transfer from the reduced photosensitizer is followed by a loss of a first chloride ligand, a replacement of the second chloride ligand by a solvent molecule, and a ligand rearrangement that releases the strain between the equatorial carbonyl ligands and the methyl group on the bpy ligand in this catalyst. These reaction steps happen on a tens of nanoseconds to tens of microseconds time scale. In the case of trans(Cl)-[Ru(6,6'-dimethyl-2,2'-bipyridine)(CO)2Cl2]), the complex is then reduced a second time from the oxidized 1-benzyl-1,4-dihydronicotinamide on a significantly slower 10-100 ms time scale, protonated and the solvent ligand is exchanged back to a chloride. The final product hence is a hydride, RuII(6,6'-dmbpy)(CO)2ClH, which is stable on a minute-to-hour time scale. In case of trans(Cl)-[Ru(5,5'-dmbpy)(CO)2Cl2]), dimerization of the reduced species is possible, which eventually leads to the formation of cis(Cl)-[Ru(5,5'-dmbpy)(CO)2Cl2]. The work illustrates the power of transient IR spectroscopy to elucidate complex reaction pathways of such catalytic systems, and provides solid cornerstones for their kinetic control.

The nonequilibrium relaxation of a series of, in part, very different photoactive proteins is compared, ranging over up to eleven decades in time. The series comprises various PDZ domains and MCL 1/peptide complexes with artificial azobenzene photoswitches, as well as two different cyanobacteriochromes (Slr-g3 and TePixJ). In either case, an embedded chromophore photoisomerizes after electronic excitation on an ultrafast femtosecond to picosecond timescale, initially perturbing the structure of the protein directly around the chromophore. This local perturbation propagates over the protein in a cascade of events, which spread over a wide range of timescales from picoseconds to seconds. In a very universal manner for all protein systems, a series of kinetic steps can be identified using lifetime analysis with a roughly equidistant spacing of about one per decade on a logarithmic scale. First, the inherent resolution to disentangle exponential relaxation processes is carefully evaluated. Concluding that this is not limiting, various models are discussed that may cause such a universal relaxation response. Diffusion on a rugged free energy landscape along a one- or low-dimensional progress variable may explain that behavior, where the quasi-randomness of the kinetic matrix thins out eigenstates that contribute to transport. The separation of kinetic steps is a measure of the typical barrier heights, which, by comparison to the universal patterns observed experimentally, is found to be in the range of kBT. Such barrier heights give a protein the flexibility to quickly structurally rearrange, yet provide some level of stability, which is relevant, for example, in the context of allosteric communication.

A versatile and easy-to-implement concept is presented, which allows one to time-synchronize in essence any amplified Ti:Sa laser system to a high-repetition rate Yb-laser system. The oscillator round trip frequency of one of the oscillators needs to be tunable by a few kHz only. That is, the repetition rates of the two laser oscillators do not have to be identical, rather synchronization is achieved only when the low-repetition rate Ti:Sa laser system produces a pump pulse. Piezoactuators for cavity length adjustment in the required range are a built-in option for modern high repetition rate laser systems, in which case no modification of the Ti:Sa laser system is required. Utilizing this synchronization, single-UV/VIS-pump–multiple IR-probe experiments become possible, covering a very wide range of timescales from 1 ps to 1 s with one-and-the-same instrument and with a time resolution of about 1 ps. Two example experiments are presented to demonstrate the capability of the setup.

First steps toward a molecular dynamics (MD) implementation in a cluster of field-programmable gate arrays (FPGAs) are presented, reaching a simulation speed of a few microseconds/day. The nodes in this cluster are programmed into a mid-ranged FPGA (Artix 7 XC7A200T), interconnected as a 3D torus by fast optical links. The implemented MD algorithm is highly parallelized and highly pipelined internally. The FPGA cluster is freely scalable in terms of size, i.e., a larger MD system requires more nodes, however, without compromising simulation speed. The performance in terms of energy stability and simulation speed is analyzed. At present, the focus lies on the fast networking, while only minimal MD functionality has been implemented so far, i.e., Lennard-Jones interactions and a thermostat, which were needed to demonstrate the feasibility of the FPGA cluster to run multi-microsecond simulations. To that end, the nucleation of a super-cooled Lennard-Jones liquid is investigated by unbiased MD simulations, which is a difficult MD problem since a high nucleation barrier has to be overcome. Finally, the pathways toward a full MD implementation are outlined. The current implementation will be made available as an open-source development project.

Light-sensitive proteins allow organisms to perceive and respond to their environment, and have diversified over billions of years. Among these, Light–Oxygen–Voltage (LOV) domains are widely distributed photosensors that control diverse physiological processes. Despite their broad biological roles and increasing use in optogenetics, the functional diversity of natural LOV domains and the evolutionary constraints shaping their dynamics remain poorly resolved. A key unresolved problem is how evolution modulates the timescales and efficiencies of LOV photocycles and how this kinetic flexibility relates to biological function. Here we systematically map the photodynamics of 21 natural LOV domains – including 18 previously uncharacterized variants – and one de novo photosensor generated by artificial intelligence-guided protein design. We uncover an exceptional kinetic diversity spanning picoseconds to days and identify distinct functional classes within the LOV family. These patterns holistically reveal that billion years of evolutionary adaptation led to branching photocycle kinetics, matching physiological requirements. Moreover, by extending the natural catalog of LOV photosensors with a de novo designed LOV variant, we demonstrate how computational protein design can access new biophysical niches. This work expands the optogenetic toolkit and offers a framework to dissect and harness the evolutionary design principles of light-responsive proteins. ### Competing Interest Statement The authors have declared no competing interest. * FMN : flavin mononucleotide ISC : intersystem crossing LOV : Light-Oxygen-Voltage. Swiss National Science Foundation, CRSII5_213507

An investigation of the low-frequency (i.e., less than 5 THz), inter-molecular dynamics of three imidazolium-based ionic liquids-1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([C4mim][NTf2]), 1-butyl-3-methylimidazolium dicyanamide ([C4mim][DCA]), and 1-ethyl-3-methylimidazolium dicyanamide ([C2mim][DCA])-is presented using two-dimensional (2D) Raman-THz spectroscopy combined with molecular dynamics (MD) simulations. By observing an echo in the 2D Raman-THz response, the experimental results indicate that the substitution of a small [DCA]- anion with a larger [NTf2]- one leads to a substantial increase in the structural inhomogeneity of the low-frequency modes of the system. These findings are corroborated by MD simulations, comparing the experimentally observed echo decay times to those of a computed velocity echo. The comparison suggests that the echo decay time reflects the instantaneous amount of structural order related to the charge alternation network, which is enhanced for the ionic liquid with the larger anion. (c) 2025 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license(https://creativecommons.org/licenses/by/4.0/).https://doi.org/10.1063/5.0246152

Light-sensitive proteins allow organisms to perceive and respond to their environment, and have diversified over billions of years. Among these, Light-Oxygen-Voltage (LOV) domains are widespread photosensors that control diverse physiological processes and are increasingly used in optogenetics. Yet, the evolutionary constraints that shaped their protein dynamics and thereby their functional diversity remain poorly resolved. Here we systematically characterize the dynamics of 21 natural LOV core domains, significantly extending the spectroscopically resolved catalog through the addition of 18 previously unstudied variants. Using time-resolved spectroscopy, we uncover an exceptional kinetic diversity spanning from picoseconds to days and identify distinct functional clusters within the LOV family. These clusters reflect evolutionary branching, including a divergence of ≈ 1.0 billion years between investigatedLOV variants from plants and ≈ 0.4 billion years of separation within one of these functional clusters. Individual variants with extreme photocycles emerge as promising anchor points for optogenetic applications, ranging from highly efficient adduct formation to ultrafast recovery. Beyond natural diversity, we introduce a LOV domain generated by artificial intelligence-guided protein design. Despite being sequentially remote from its maternal template, this variant retains core photocycle function while exhibiting unique biophysical properties, thereby occupying a new region on the biophysical landscape. Our work emphasizes how billions of years of evolution defined LOV protein dynamics, and how protein design can expand this repertoire, engineering next-generation optogenetic tools.

Protein dynamics form a critical bridge between protein structure and function, yet the impact of evolution on ultrafast processes inside proteins remains enigmatic. This study delves deep into nanosecond-scale protein dynamics of a structurally and functionally conserved protein across species separated by almost a billion years, investigating ten homologs in complex with their ligand. By inducing a photo-triggered destabilization of the ligand inside the binding pocket, we resolved distinct kinetic footprints for each homolog via transient infrared spectroscopy . Strikingly, we found a cascade of rearrangements within the protein complex which manifest in three discrete time points of dynamic activity, conserved over hundreds of millions of years within a narrow window. Among these processes, one displays a subtle temporal shift correlating with evolutionary divergence, suggesting reduced selective pressure in the past. Our study not only uncovers the impact of evolution on molecular processes in a specific case, but has also the potential to initiate a novel field of scientific inquiry within molecular paleontology, where species are compared and classified based on the rapid pace of protein dynamic processes; a field which connects the shortest conceivable time scale in living matter (10^-9 s) with the largest ones (10^16 s).

An optically transparent thin-layer electrochemical cell with stopped-flow sample transport has been developed for optical-pump infrared-probe transient absorption spectroscopy of prereduced or preoxidized molecules. Time-resolved IR-spectra of Re(bpy)(CO)3X (X = Cl, Br) complexes in different oxidation states are presented as a proof-of-principle application for this combined electrochemical and spectroscopic tool. The excited-state lifetimes and IR-spectroscopic signatures of various oxidation states of the molecule, including follow-up reaction intermediates, are disentangled by kinetic sorting, using lifetime density analysis. The method can be applied to assign and differentiate molecular intermediates in photo- and electrochemical reactions, adding new analytic coordinates to classical FTIR- and UV-vis-spectroelectrochemistry.

Through comprehensive data analysis, we demonstrate that a χ(2)-induced artifact, arising from imperfect balancing in the conventional electro-optic sampling detection scheme, contributes significantly to the measured signal in 2D Raman-THz spectroscopy of non-centrosymmetric materials. The artifact is a product of two 1D responses, overwhelming the desired 2D response. We confirm that by analyzing the 2D Raman-THz response of an x-cut beta barium borate crystal. We furthermore show that this artifact can be effectively suppressed by implementing a special detection scheme. We successfully isolate the desired third-order 2D Raman-THz response, revealing a distinct cross-peak feature, whose frequency position suggests the coupling between two crystal phonons.

Parametrizing energy functions for ionic systems can be challenging. Here, the total energy function for an eutectic system consisting of water, SCN-, K+ and acetamide is improved vis-a-vis experimentally measured properties. Given the importance of electrostatic interactions, two different types of models are considered: the first (model M0) uses atom-centered multipole whereas the other two (models M1 and M2) are based on fluctuating minimal distributed charges (fMDCM) that respond to geometrical changes of SCN-. The Lennard-Jones parameters of the anion are adjusted to best reproduce experimentally known hydration free energies and densities, which are matched to within a few percent for the final models irrespective of the electrostatic model. Molecular dynamics simulations of the eutectic mixtures with varying water content (between 0 and 100%) yield radial distribution functions and frequency correlation functions for the CN-stretch vibration. Comparison with experiments indicates that models based on fMDCM are considerably more consistent than those using multipoles. Computed viscosities from models M1 and M2 are within 30% of measured values and their change with increasing water content is consistent with experiments. This is not the case for model M0.

Amplifier-based pump-probe systems, while versatile, often suffer from complexity and low measurement speeds, especially when probing samples requiring low excitation fluences. To address these limitations, we introduce a pump-probe system that leverages a 60-MHz single-cavity dual-comb oscillator and an ultra-low-noise supercontinuum. The setup can operate in equivalent time sampling or in programmable optical delay generation modes. We employ this system to study the wavelength-dependent excited-state dynamics of the non-fullerene electron acceptor Y6, a compound of interest in solar cell development, with excitation fluences as low as 1 nJ/cm2, well below the onset of nonlinear exciton annihilation effects. Our measurements reach a shot-noise-limited sensitivity in differential transmission of 3.4e–7. The results demonstrate the system's potential to advance the field of ultrafast spectroscopy.

Several ways to electronically synchronizedifferenttypes of amplifiedfemtosecond laser systems are presented based on a single freely programmableelectronics hardware: arbitrary-detuning asynchronous optical sampling(ADASOPS), as well as actively locking two femtosecond laser oscillators,albeit not necessarily to the same round-trip frequency. They allowus to rapidly probe a very wide range of timescales, from picosecondsto potentially seconds, in a single transient absorption experimentwithout the need to move any delay stage. Experiments become possiblethat address a largely unexplored aspect of many photochemical reactions,in particular in the context of photo-catalysis as well as photoactiveproteins, where an initial femtosecond trigger very often initiatesa long-lasting cascade of follow-up processes. The approach is veryversatile and allows us to synchronize very different lasers, suchas a Ti:Sa amplifier and a 100 kHz Yb-laser system. The jitter ofthe synchronization, and therewith the time-resolution in the transientexperiment, lies in the range from 1 to 3 ps, depending on the method.For illustration, transient IR measurements of the excited state solvationand decay of a metal carbonyl complex as well as the full reactioncycle of bacteriorhodopsin are shown. The pros and cons of the variousmethods are discussed, with regard to the scientific question onemight want to address, and also with regard to the laser systems thatmight be already existent in a laser lab.

ConspectusUltrafast spectroscopy and imaging have become tools utilized by a broad range of scientists involved in materials, energy, biological, and chemical sciences. Commercialization of ultrafast spectrometers including transient absorption spectrometers, vibrational sum frequency generation spectrometers, and even multidimensional spectrometers have put these advanced spectroscopy measurements into the hands of practitioners originally outside the field of ultrafast spectroscopy. There is now a technology shift occurring in ultrafast spectroscopy, made possible by new Yb-based lasers, that is opening exciting new experiments in the chemical and physical sciences. Amplified Yb-based lasers are not only more compact and efficient than their predecessors but also, most importantly, operate at many times the repetition rate with improved noise characteristics in comparison to the previous generation of Ti:sapphire amplifier technologies. Taken together, these attributes are enabling new experiments, generating improvements to long-standing techniques, and affording the transformation of spectroscopies to microscopies. This Account aims to show that the shift to 100 kHz lasers is a transformative step in nonlinear spectroscopy and imaging, much like the dramatic expansion that occurred with the commercialization of Ti:sapphire laser systems in the 1990s. The impact of this technology will be felt across a great swath of scientific communities. We first describe the technology landscape of amplified Yb-based laser systems used in conjunction with 100 kHz spectrometers operating with shot-to-shot pulse shaping and detection. We also identify the range of different parametric conversion and supercontinuum techniques which now provide a path to making pulses of light optimal for ultrafast spectroscopy. Second, we describe specific instances from our laboratories of how the amplified Yb-based light sources and spectrometers are transformative. For multiple probe time-resolved infrared and transient 2D IR spectroscopy, the gain in temporal span and signal-to-noise enables dynamical spectroscopy measurements from femtoseconds to seconds. These gains widen the applicability of time-resolved infrared techniques across a range of topics in photochemistry, photocatalysis, and photobiology as well as lower the technical barriers to implementation in a laboratory. For 2D visible spectroscopy and microscopy with white light, as well as 2D IR imaging, the high repetition rates of these new Yb-based light sources allow one to spatially map 2D spectra while maintaining high signal-to-noise in the data. To illustrate the gains, we provide examples of imaging applications in the study of photovoltaic materials and spectroelectrochemistry.