Unveiling the solid-like dynamics of liquids at low-frequency via nano-confinement

At frequencies higher than the inverse of the structural relaxation time $\tau$, the dynamics of liquids display several solid-like properties, including propagating collective shear waves and emergent elasticity. However, in classical bulk liquids, where $\tau$ is typically of the order of 1 ps or less, this solid-like behavior cannot be observed in the low-frequency region of the vibrational density of states (VDOS), below a few meV. In this work, we provide compelling evidence for the emergent solid-like nature of liquids at short distances through inelastic neutron scattering measurements of the low-frequency vibrational density of states (VDOS) in liquid water and glycerol confined within graphene oxide membranes. In particular, upon increasing the strength of confinement, we observe a continuous evolution from a liquid-like VDOS (linear in the frequency $\omega$) to a solid-like behavior (Debye law, $\sim\omega^2$) in the range of $1$-$4$ meV. Molecular dynamics simulations confirm these findings and reveal additional solid-like features, including propagating collective shear waves and a reduction in the self-diffusion constant. Finally, we show that the onset of solid-like dynamics is pushed towards low frequency because of the slowing-down of the relaxation processes upon confinement, and that the scale at which solidity emerges is qualitatively compatible with k-gap theory and the concept of gapped momentum states. Our results provide a convincing experimental proof of the continuity between liquids and solids, as originally advocated by Frenkel and Maxwell, and a deeper understanding of the dynamics of liquids across a wide range of length scales.Comment: v1: comments are welcom

Structural analysis of a genetically encoded fret biosensor by SAXS and MD simulations

Inspired by the modular architecture of natural signaling proteins, ligand binding proteins are equipped with two fluorescent proteins (FPs) in order to obtain Förster resonance energy transfer (FRET)-based biosensors. Here, we investigated a glucose sensor where the donor and acceptor FPs were attached to a glucose binding protein using a variety of different linker sequences. For three resulting sensor constructs the corresponding glucose induced conformational changes were measured by small angle X-ray scattering (SAXS) and compared to recently published single molecule FRET results (Höfig et al., ACS Sensors, 2018). For one construct which exhibits a high change in energy transfer and a large change of the radius of gyration upon ligand binding, we performed coarse-grained molecular dynamics simulations for the ligand-free and the ligand-bound state. Our analysis indicates that a carefully designed attachment of the donor FP is crucial for the proper transfer of the glucose induced conformational change of the glucose binding protein into a well pronounced FRET signal change as measured in this sensor construct. Since the other FP (acceptor) does not experience such a glucose induced alteration, it becomes apparent that only one of the FPs needs to have a well-adjusted attachment to the glucose binding protein

Structural and dynamical properties in protein ligand interactions

In this work structural and dynamical properties of protein ligand interactions were analysed. This was done predominately by the use of scattering techniques. In addition to scattering techniques complimentary techniques were used to supplement the information obtained. The main scattering technique used was incoherent neutron scattering, as it allows for a separation of the analysed protein and its surrounding hydration layer. Two protein ligand systems were analysed. The first was the protein streptavidin(STV) and its ligand biotin (B). In addition to this, a genetically engineered Försterresonance energy transfer (FRET) based glucose sensor was analysed with and withoutadded glucose. For the selected systems, the specific challenge was to separate the influences of the hydration layer and the protein. Similarly, it is also challenging to create models that incorporate all the separate observations to form a cohesive interpretation. In order to regard the STV protein independently from the surrounding hydration layer neutronscattering was chosen as the appropriate technique. Whereas, for the glucose sensor thestructural changes were deemed to be the most important information to obtain and therefore X-ray scattering was employed. For the STV+B interaction it was considered that molecular dynamics play a vitalrole for the biological function of proteins. Changes in the proteins conformational entropy and of the hydration layer influence the binding process for many protein ligand interactions. Here this was investigated for STV, as well as the change in the internal dynamics of STV upon biotin binding. Quasi elastic neutron scattering (QENS) was used to investigate the change of conformational entropy of the protein and its dynamics. QENS results show that the conformational entropy of STV is reduced upon biotinbinding. Thermal diffusion forced Rayleigh scattering (TDFRS) results also indicatean increased entropy of the hydration layer. This indicates that the hydration layerplays an important role in stabilising the binding of biotin to STV. Comparing the change in conformational entropy per residue to that of other biological processes showed that in the STV+B binding process it is comparable to that of the transition from anunfolded myoglobin to a molten globule structure. When compared toother protein ligand interactions per residue it was found to be an order of magnitude larger. This indicates that within STV a significant change of conformational entropy occurs uponbiotin binding, more than can be explained by the already established conformational changes. Therefore, the internal STV dynamics before and after biotin were compared. This showed that the flexibility of streptavidin is greatly reduced upon biotin bindingleading to the complex being more rigid. The second system was a genetically encoded FRET-based biosensor, which was developed as a tool to quantify metabolites in nourishing solutions of living cells. The sensors consist of a central metabolite binding protein and flanking fluorescent proteins(FP) affixed by different linker sequences. FRET measurements are sensitive either to distance or orientation changes of FP domains as response to glucose binding. Size exclusion chromatography - small-angle X-ray scattering (SEC-SAXS) measurements have been performed to investigate, if either large-scale structural changes of the FP positions or relative orientation changes occur as response to glucose binding. Based on the measured SAXS curves modelling of the FP domains was performed. It was determined that glucose binding results in large-scale structural changes of the positions of linked FP domains for one of the sensors. These structures fit with the behaviour of the sensor expected due to fluorescent measurements. This was achieved by first regarding the hydration layer and the protein sepparately, by taking advantage of the sensitivity of neutrons to the scattering cross section of different hydrogen isotopes. In addition calorimetric techniques and TDFRS were used to support the neutron data obtained for the STV systems. In the case of the glucose sensor SAXS and FRET results were considered together

THERMODIFFUSION AS A PROBE FOR PROTEIN HYDRATION

Protein-ligand interactions are of fundamental importance to numerous processes in living organisms. A very sensitive method to observe the reaction kinetics is Microscale thermophoresis (MST), which uses the changed thermophoretic behaviour as an indicator for complex formation [1]. This sensitivity of thermodiffusion is attributed to modifications in the hydration shell of the protein upon complex formation, which can be strong due to conformational changes of the protein. There is, however, no detailed understanding how protein conformation, hydration and thermophoretic behaviour are connected. Our aim is to elucidate that point and find out if MST could be used to obtain information about protein hydration in addition to the reaction kinetics. Several cyclodextrins and their complexes with acetylsalicylic acid were investigated as a simple model system [2]. We found that the temperature dependence of a solute’s thermodiffusion is correlated to its hydrophilicity, but the observed change upon complex formation is relatively small due to the rigidity of cyclodextrin. The second model system is the protein streptavidin and its complex with biotin [3]. Data from quasi-elastic neutron scattering (QENS) and isothermal titration calorimetry (ITC) show a reduced entropy of the complex in comparison to the free protein, which is partly compensated by an increased entropy of the hydration shell. This is in agreement with a breaking of hydrogen bonds between protein and surrounding water due to the reduced flexibility of the protein [4] and fits with the reduced hydrophilicity of the complex indicated by thermodiffusion.REFERENCES[1] M. Jerabek-Willemsen et al., J. Mol. Struct. 1077, 101-113 (2014)[2] D. Niether et al., Langmuir, 33, 8483 (2017).[3] D. Niether et al., AIP Conference Proceedings 1929, 020001 (2018).[4] S. Liese et al., ACS Nano, 11 702 (2017)

Complementary approaches to obtaining thermodynamic parameters from protein ligand systems-challenges and opportunities

Protein ligand interactions play an important role in biology. Increasingly the aim is to understandand influence protein ligand binding. The binding process is heavily influenced by its thermodynamicparameters. In order to understand how the whole system thermodynamics work it is important to characterisethe individual contribution of each of the systems components. While the change in conformational entropyof the protein can be determined using QENS complementary methods are necessary in order to characteriseall components. This paper will describe the challenges that can occur when combining the different methods,as well as how they can be overcome

Thermodiffusion as a Probe of Protein Ligand Binding

Molecular recognition via protein-ligand interactions is of fundamental importance to numerous processes in living organisms. The behaviour of biomolecules in a temperature gradient, known as thermodiffusion or thermophoresis, changes when a ligand binds. Microscale thermophoresis (MST) uses this sensitivity of the thermophoretic response to access information on binding dynamics, although the physicochemical processes are still unclear [1]. Additionally, thermophoresis is promising as a tool to gain information on the hydration layer and how it changes due to complex formation. We use infra-red thermal diffusion forced Rayleigh scattering (IR-TDFRS) in a temperature range from 10 to 50°C to investigate the thermodiffusion properties. In previous studies [2] we used cyclodextrin-aspirin as a model system for complexes and showed that the temperature dependence of the thermodiffusion behaviour is sensitive to solute-solvent interactions. Now we shift our focus to the protein streptavidin (SA) and its biotin complex. Similar to the cyclodextrins, formation of the SA-biotin complex leads to a weaker temperature sensitivity of the thermodiffusion behaviour, although the effect is more pronounced. This indicates a less hydrophilic complex. To quantify the influence of structural fluctuations and conformational motion of the protein on the entropy change of its hydration layer upon ligand binding, we combine quasi-elastic incoherent neutron scattering (QENS) and isothermal titration calorimetry (ITC) data. As the QENS measurements are only possible in heavy water, the ITC need to be performed in heavy water as well in order to gain a better understanding of the hydration layer. The aim of this work is to develop a microscopic understanding of the correlation between the strength of solute-solvent interactions and the thermophoretic behaviour.[1] M. Jerabek-Willemsen et al., J. Mol. Struct. (2014).[2] D. Niether et al., Langmuir 33(34), 8483-8492 (2017)

Thermophoresis: The Case of Streptavidin and Biotin

Thermophoretic behavior of a free protein changes upon ligand binding and gives access to information on the binding constants. The Soret effect has also been proven to be a promising tool to gain information on the hydration layer, as the temperature dependence of the thermodiffusion behavior is sensitive to solute–solvent interactions. In this work, we perform systematic thermophoretic measurements of the protein streptavidin (STV) and of the complex STV with biotin (B) using thermal diffusion forced Rayleigh scattering (TDFRS). Our experiments show that the temperature sensitivity of the Soret coefficient is reduced for the complex compared to the free protein. We discuss our data in comparison with recent quasi-elastic neutron scattering (QENS) measurements. As the QENS measurement has been performed in heavy water, we perform additional measurements in water/heavy water mixtures. Finally, we also elucidate the challenges arising from the quantiative thermophoretic study of complex multicomponent systems such as protein solutions

Supplementary Materials from Computational and spectroscopic characterisation of thianthrene

Computational and spectroscopic characterisation of thianthrene_ESM_Revise