A billion years of evolution manifest in nanosecond protein dynamics.

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 time points of increased 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 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[Formula: see text] s) with the largest ones (10[Formula: see text] s).

Vibrational couplings between protein and cofactor in bacterial phytochrome Agp1 revealed by 2D-IR spectroscopy.

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.

Breaking Barriers in Ultrafast Spectroscopy and Imaging Using 100 kHz Amplified Yb-Laser Systems.

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.

Transient 2D IR Spectroscopy and Multiscale Simulations Reveal Vibrational Couplings in the Cyanobacteriochrome Slr1393-g3.

Cyanobacteriochromes are bistable photoreceptor proteins with desirable photochemical properties for biotechnological applications, such as optogenetics or fluorescence microscopy. Here, we investigate Slr1393-g3, a cyanobacteriochrome that reversibly photoswitches between a red-absorbing (Pr) and green-absorbing (Pg) form. We applied advanced IR spectroscopic methods to track the sequence of intermediates during the photocycle over many orders of magnitude in time. In the conversion from Pg to Pr, we have revealed a new intermediate with distinct spectroscopic features in the IR, which precedes Pr formation using transient IR spectroscopy. In addition, stationary and transient 2D IR experiments measured the vibrational couplings between different groups of the chromophore and the protein in these intermediate states, as well as their structural disorder. Anharmonic QM/MM calculations predict spectra in good agreement with experimental 2D IR spectra of the initial and final states of the photocycle. They facilitate the assignment of the IR spectra that serve as a basis for the interpretation of the spectroscopic results and suggest structural changes of the intermediates along the photocycle.

Versatile Femtosecond Laser Synchronization for Multiple-Timescale Transient Infrared Spectroscopy.

Several ways to electronically synchronize different types of amplified femtosecond laser systems are presented based on a single freely programmable electronics 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 allow us to rapidly probe a very wide range of timescales, from picoseconds to potentially seconds, in a single transient absorption experiment without the need to move any delay stage. Experiments become possible that address a largely unexplored aspect of many photochemical reactions, in particular in the context of photo-catalysis as well as photoactive proteins, where an initial femtosecond trigger very often initiates a long-lasting cascade of follow-up processes. The approach is very versatile and allows us to synchronize very different lasers, such as a Ti:Sa amplifier and a 100 kHz Yb-laser system. The jitter of the synchronization, and therewith the time-resolution in the transient experiment, lies in the range from 1 to 3 ps, depending on the method. For illustration, transient IR measurements of the excited state solvation and decay of a metal carbonyl complex as well as the full reaction cycle of bacteriorhodopsin are shown. The pros and cons of the various methods are discussed, with regard to the scientific question one might want to address, and also with regard to the laser systems that might be already existent in a laser lab.

Low-frequency anharmonic couplings in crystalline bromoform: Theory.

Theoretical calculations of the low-frequency anharmonic couplings of the ß-phase of crystalline bromoform are presented based on density functional theory quantum chemistry calculations. The electrical and mechanical anharmonicities between intra- and intermolecular modes are calculated, revealing that the electrical anharmonicity dominates the cross-peak intensities in the 2D Raman-THz response and crystalline, as well as liquid, bromoform. Furthermore, the experimentally observed difference in relative cross-peak intensities between the two intramolecular modes of bromoform and the intermolecular modes can be explained by the C3v-symmetry of bromoform in combination with orientational averaging. The good agreement with the experimental results provides further evidence for our interpretation that the 2D Raman-THz response of bromoform is, indeed, related to the anharmonic coupling between the intra- and intermolecular modes.

MCL-1 promiscuity and the structural resilience of its binding partners.

The allosteric protein MCL-1 and its natural inhibitors, the BH3-only proteins PUMA, BIM, and NOXA regulate apoptosis by interacting promiscuously within an entangled binding network. Little is known about the transient processes and dynamic conformational fluctuations that are the basis for the formation and stability of the MCL-1/BH3-only complex. In this study, we designed photoswitchable versions of MCL-1/PUMA and MCL-1/NOXA, and investigated the protein response after an ultrafast photo-perturbation with transient infrared spectroscopy. We observed partial α-helical unfolding in all cases, albeit on strongly varying timescales (1.6 ns for PUMA, 9.7 ns for the previously studied BIM, and 85 ns for NOXA). These differences are interpreted as a BH3-only-specific "structural resilience" to defy the perturbation while remaining in MCL-1's binding pocket. Thus, the presented insights could help to better understand the differences between PUMA, BIM, and NOXA, the promiscuity of MCL-1, in general, and the role of the proteins in the apoptotic network.

Real-time observation of ligand-induced allosteric transitions in a PDZ domain.

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 are 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 ∼1 ns to ∼µs, can be rationalized by a remodeling of its rugged free-energy landscape, with very 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.

Structure, Organization, and Heterogeneity of Water-Containing Deep Eutectic Solvents.

The spectroscopy and structural dynamics of a deep eutectic mixture (KSCN/acetamide) with varying water content is investigated from 2D IR (with the C-N stretch vibration of the SCN- anions as the reporter) and THz spectroscopy. Molecular dynamics simulations correctly describe the nontrivial dependence of both spectroscopic signatures depending on water content. For the 2D IR spectra, the MD simulations relate the steep increase in the cross-relaxation rate at high water content to the parallel alignment of packed SCN- anions. Conversely, the nonlinear increase of the THz absorption with increasing water content is mainly attributed to the formation of larger water clusters. The results demonstrate that a combination of structure-sensitive spectroscopies and molecular dynamics simulations provides molecular-level insights into the emergence of heterogeneity of such mixtures by modulating their composition.

Hydration dynamics and IR spectroscopy of 4-fluorophenol.

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 parametrizations and with full 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. The hydrophobic character of the CF-group is particularly relevant for applications in pharmaceutical chemistry with a focus on local hydration and interaction with the surrounding protein.

Low-frequency anharmonic couplings in bromoform revealed from 2D Raman-THz spectroscopy: From the liquid to the crystalline phase.

Two-dimensional (2D) Raman-THz spectroscopy in the frequency of up to 7 THz has been applied to study the crystalline ß-phase of bromoform (CHBr3). As for liquid CHBr3, cross peaks are observed, which, however, sharpen up in the crystalline sample and split into assignable sub-contributions. In the Raman dimension, the frequency positions of these cross peaks coincide with the intramolecular bending modes of the CHBr3 molecules and in the THz dimension with the IR-active lattice modes of the crystal. This work expands the applicability of this new 2D spectroscopic technique to solid samples at cryogenic temperatures. Furthermore, it provides new experimental evidence that the cross peaks, indeed, originate from the coupling between intra- and intermolecular vibrational modes.

Impact of nuclear quantum effects on the structural inhomogeneity of liquid water.

The 2D Raman-terahertz (THz) response of liquid water is studied in dependence of temperature and isotope substitution ([Formula: see text]O, [Formula: see text]O, and [Formula: see text]O). In either case, a very short-lived (i.e., between 75 and 95 fs) echo is observed that reports on the inhomogeneity of the low-frequency intermolecular modes and hence, on the heterogeneity of the hydrogen bond networks of water. The echo lifetime slows down by about 20% when cooling the liquid from room temperature to the freezing point. Furthermore, the echo lifetime of [Formula: see text]O is [Formula: see text] slower than that of [Formula: see text]O, and both can be mapped on each other by introducing an effective temperature shift of [Formula: see text] K. In contrast, the temperature-dependent echo lifetimes of [Formula: see text]O and [Formula: see text]O are the same within error. [Formula: see text]O and [Formula: see text]O have identical masses, yet [Formula: see text]O is much closer to [Formula: see text]O in terms of nuclear quantum effects. It is, therefore, concluded that the echo is a measure of the structural inhomogeneity of liquid water induced by nuclear quantum effects.

Intrinsic Dynamics of Protein-Peptide Unbinding.

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.

Needles in a haystack: H-bonding in an optogenetic protein observed with isotope labeling and 2D-IR spectroscopy.

Recently, re-purposing of cyanobacterial photoreceptors as optogentic actuators enabled light-regulated protein expression in different host systems. These new bi-stable optogenetic tools enable interesting new applications, but their light-driven working mechanism remains largely elusive on a molecular level. Here, we study the optogenetic cyanobacteriochrome Am1-c0023g2 with isotope labeling and two dimensional infrared (2D-IR) spectroscopy. Isotope labeling allows us to isolate two site-specific carbonyl marker modes from the overwhelming mid-IR signal of the peptide backbone vibrations. Unlike conventional difference-FTIR spectroscopy, 2D-IR is sensitive to homogeneous and inhomogeneous broadening mechanisms of these two vibrational probes in the different photostates of the protein. We analyse the 2D-IR line shapes in the context of available structural models and find that they reflect the hydrogen-bonding environment of these two marker groups.

Dielectric response of light, heavy and heavy-oxygen water: isotope effects on the hydrogen-bonding network's collective relaxation dynamics.

Isotopic substitutions largely affect the dielectric relaxation dynamics of hydrogen-bonded liquid water; yet, the role of the altered molecular masses and nuclear quantum effects has not been fully established. To disentangle these two effects we study the dielectric relaxation of light (H216O), heavy (D216O) and heavy-oxygen (H218O) water at temperatures ranging from 278 to 338 K. Upon 16O/18O exchange, we find that the relaxation time of the collective orientational relaxation mode of water increases by 4-5%, in quantitative agreement with the enhancement of viscosity. Despite the rotational character of dielectric relaxation, the increase is consistent with a translational mass factor. For H/D substitution, the slow-down of the relaxation time is more pronounced and also shows a strong temperature dependence. In addition to the classical mass factor, the enhancement of the relaxation time for D216O can be described by an apparent temperature shift of 7.2 K relative to H216O, which is higher than the 6.5 K shift reported for viscosity. As this shift accounts for altered zero-point energies, the comparison suggests that the underlying thermally populated states relevant to the activation of viscous flow and dielectric relaxation differ.

Flexible to rigid: IR spectroscopic investigation of a rhenium-tricarbonyl-complex at a buried interface.

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.

Transient 2D IR spectroscopy from micro- to milliseconds.

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.

Transient CO desorption from thin Pt films induced by mid-IR pumping.

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.

2D-Raman-THz spectroscopy with single-shot THz detection.

We present a 2D-Raman-terahertz (THz) setup with multichannel (single-shot) THz detection, utilizing two crossed echelons, in order to reduce the acquisition time of typical 2D-Raman-THz experiments from days to a few hours. This speed-up is obtained in combination with a high repetition rate (100 kHz) Yb-based femtosecond laser system and a correspondingly fast array detector. The wavelength of the Yb-laser (1030 nm) is advantageous, since it assures almost perfect phase matching in GaP for THz generation and detection and since the dispersion in the transmissive echelons is minimal. 2D-Raman-THz test measurements on liquid bromoform (CHBr3) are reported. An enhancement of a factor ∼5.8 in signal-to-noise ratio is obtained for single-shot detection when compared to conventional step-scanning measurements in the THz time domain, corresponding to a speed-up of acquisition time of ∼34.

About Control: Kinetics in Molecule- based Photochemical Water Reduction Investigated by Transient IR Spectroscopy.

This review aims to promote the role of transient IR spectroscopy to investigate molecular-based photocatalytic water reduction. Examples are discussed in which this method has been successfully applied to elucidate reaction mechanisms. Focus is given to kinetic changes and their consequences when a photochemical water reduction system, which is functional and well understood in solution, is brought onto a metal oxide surface.