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Time Resolved THz Calorimetry explores Molecular Recognition Processes

Periodic Reporting for period 4 - THZCALORIMETRY (Time Resolved THz Calorimetry explores Molecular Recognition Processes)

Reporting period: 2021-04-01 to 2022-03-31

Terahertz (THz)-Calorimetry is the science of measuring low frequency modes for deriving the solvation entropy/enthalpy changes under non-equilibrium conditions in real time. The scientific vision was to develop a 1:1 correlation between spectroscopic changes and calorimetric quantities under thereby introduce time-resolved THz-Calorimetry with envisioned time resolutions of up to picoseconds.

THz spectroscopy is a sensitive tool to probe changes in the intermolecular interaction between water molecules, which are of major importance to rationalize and predict the outcome of a reaction. While the individual changes might be small, the large number of solvent molecules involved turns this part into a major driving force for reactions in an aqueous surrounding. We were able to demonstrate for the first time that the spectroscopic signatures of hydration can be directly correlated to enthalpic and entropic changes of the solvent, which determine the reaction path and reaction rates. THz spectroscopy now allows to map and monitor local entropy/enthalpy changes in inhomogeneous samples even during reactions. This will have a major impact on applications in drug design, biomedical research and electrocatalysis.

The objectives of the proposal were implement an experimental setup that is able to monitor precisely low frequency spectra (up to 10 THz), and introduce THz-calorimetry by exploring whether and how we can correlate these spectra with thermodynamic properties, such as entropy, enthalpy and heat capacity. As outlined in our publications we were able to define a local, entropy, enthalpy and pH value, based upon THz spectroscopic studies. Thereby we were able to map and understand hydrophic hydration and explain the details of reactions during the electrochemical double layer formation in for electrocatalytic processes as well as the formation of protein aggregates (fibril formation) in local solvation hotspots.
The fundamental idea to be exploited in the ERC Advance grant was the question whether the low frequency modes of a solvated molecule can be correlated in a quantitative predictive way with thermodynamic properties. Alcohol chains served as a prototype system. In successive papers, we could demonstrate that in the THz frequency range, we are able to assign fingerprints of the different types of hydration water. Based on this breakthrough, we were able to present a completely new approach to link the thermodynamic model of the Lum-Chandler-Weeks theory, which was a pure "Gedankenexperiment", directly to spectroscopic observables: The THz signatures in the frequency range of the intermolecular stretch and the librational mode.

The free energy of alcohol hydration is the sum of a free energy cost of forming and wrapping a cavity around the solute and an enthalpic gain due to the hydrogen bonds (H-bonds) formed between the alcohol OH group and bound water molecules around it. While a band around 160 cm-1 can be used to quantify the number of H-bonds involved in the formation of the cavity, the shift of the librational band is a means to quantify the H-bond formation between water and the solute. While the first results in an entropic loss, the later in an enthalpic gain. This model is shown to be more general: The THz bands allow to quantify the two contributions. We were able to map hydration enthalpy and entropy and pointed out the role of the solvent in a number of biological and catalytic processes. This will allow to quantify entropic cost and enthalpic gain not only in equilibrium but also in non-equilibrium processes. In order to reach time resolutions between ns and ps we have developed a rapid scanning technique into THz time-domain spectrometers using an oscillating frictionless delay line, especially suited for nonlinear THz experiments. The sensitivity of the THz detection could be increased by two orders of magnitude compared to previous work. This set-up now allows time resolved THz calorimetry measurements with ps time resolution.

The application of THz calorimetry was shown to allow water mapping even beyond biological recognition processes (target of the original proposal): We proposed a framework that allows to rewrite the free energy cost of hydrophobic hydration at the interface as the free energy cost in the bulk plus a size-dependent correction term. This additional contribution could be probed by THz experiments and was carried out at the synchrotron source SOLEIL.

In a collaboration with our medical department, we mapped water in liquid-liquid phase-separated FUS protein using ATR (attenuated total reflection) spectroscopy in the THz range in combination with laser-scanning microscopy, which is sensitive to protein aggregation. THz calorimetry was able to characterize the water network inside the FUS liquid-liquid phase droplets, providing novel insight into the local solvation hotspots, which are thought to trigger the formation of protein aggregates as a precursor for neurotoxic fibrils.
We are convinced that THz calorimetry will open new horizons and will have a broader impact as a cutting-edge experimental tool. We succeeded in establishing a direct connection between thermodynamic quantities – hydration entropy and enthalpy – and spectroscopic observables, i.e. well-defined fingerprints in the THz spectral range. The two spectroscopic fingerprints of the hydration bond network are the "cavity wrap" and "H-bound" hydration water populations, which are makers for hydrophobic and hydrophilic solvation at the surface of biomolecules respectively. It was remarkably and unexpected, that these two hydration water bands a) can be detected, quantified and b) are sufficient to deduce the solvation entropy, enthalpy, etc. and thus monitor these "online" in reactions.

We were able to quantify solvation entropy and enthalpy from the amplitude of the THz bands, and to dissect the hydrophilic and hydrophobic contributions by individually evaluating the amplitude of the wrap and bound THz-fingerprints. Such decomposition goes beyond the state of the art, since it cannot be achieved by standard calorimetry approaches, and represented a long-standing challenged for both theory and experiments.

Our novel THz-calorimetry methodology allowed to experimentally test fundamental theories of hydrophobic solvation, such as the well-established Lum-Chandler-Weeks theory. This was another important achievement, since a connection of this theory to experimental observables was lacking so far and our experimental methodology will help the development and validation of hydrophobic theories.

More importantly, we are now able to use THz fingerprints to map hydrophobic and hydrophilic solvation spots around biomolecules, even during biological processes, e.g. as demonstrated for biomolecular recognition processes, in the DNA ion atmosphere, and liquid-liquid phase separation. This represents a breakthrough in biology, because it paved the way toward a rational control of biological processes based on real time THz spectroscopy screening.
Mapping solvation free energy changes during biological processes with time-resolved THz calorimetry
The entropic cost and enthalpic gain can be quantified by their THz spectroscopic fingerprints