Periodic Reporting for period 4 - BIOVIB (Electric Interactions and Structural Dynamics of Hydrated Biomolecules Mapped by Ultrafast Vibrational Probes)
Berichtszeitraum: 2023-11-01 bis 2025-04-30
Biomolecules exist in a liquid environment of water molecules and ions. Water molecules possess an electric dipole moment and ions are charged, leading to strong electric interactions within the liquid and with embedded biomolecules. Their structure and biological function are, thus, strongly influenced by the fluctuating water shell and the ion distribution. In the liquid itself, electric interactions couple neighboring molecules at short distance but also large groups of thousands of molecules, the latter displaying a complex collective behavior. At the molecular level, such phenomena are barely understood; the interaction energies, the strength and spatial range of electric fields, and interplay with other interactions are mainly unknown. Insight in this fundamental problem is important for designing eco-friendly polar solvents for chemical synthesis and biomolecular systems with specific and tailored functional properties for applications in bioengineering, pharmacology, and other areas of societal relevance.
Going far beyond existing methods, this project introduces a novel scientific paradigm for a direct time-resolved mapping of molecular electric forces on sub-nanometer length scales and at the genuine terahertz (THz) fluctuation frequencies of biomolecules embedded in water. Femtosecond vibrational excitations of DNA and RNA at the interface to the water shell act as noninvasive probes of charge dynamics and local electric fields. Key objectives are quantitative measurements and simulations of dynamic electric fields acting on prototypical DNA, RNA, and membrane structures, another the analysis of ion geometries around charged biomolecules and their role in stabilizing structure. This includes a critical assessment of existing concepts for describing electrostatics at the molecular scale.
As the second major objective, the project aims at a quantitative characterization of electric interactions and collective molecular dynamics in polar liquids such as water and alcohols. Experiments focus on the THz frequency range, in which the relevant elementary excitations of the liquid occur, and employ the most advanced methods of nonlinear multidimensional THz spectroscopy.
Going far beyond existing methods, this project introduces a novel scientific paradigm for a direct time-resolved mapping of molecular electric forces on sub-nanometer length scales and at the genuine terahertz (THz) fluctuation frequencies of biomolecules embedded in water. Femtosecond vibrational excitations of DNA and RNA at the interface to the water shell act as noninvasive probes of charge dynamics and local electric fields. Key objectives are quantitative measurements and simulations of dynamic electric fields acting on prototypical DNA, RNA, and membrane structures, another the analysis of ion geometries around charged biomolecules and their role in stabilizing structure. This includes a critical assessment of existing concepts for describing electrostatics at the molecular scale.
As the second major objective, the project aims at a quantitative characterization of electric interactions and collective molecular dynamics in polar liquids such as water and alcohols. Experiments focus on the THz frequency range, in which the relevant elementary excitations of the liquid occur, and employ the most advanced methods of nonlinear multidimensional THz spectroscopy.
The work done in the project has covered four interconnected research areas, the development of time-resolved THz Stark shift spectroscopy, the interactions of DNA and RNA backbones with ions in their water shells, the characterization of RNA ionic sites, and the impact of electric forces on molecular couplings and solute-solvent interactions.
The new method of ultrafast THz Stark spectroscopy, which was demonstrated for the first time in this project, allows for mapping the momentary electric response of molecular ensembles under dynamically frozen conditions. An external picosecond electric field at THz frequencies is enhanced up to several megavolts/cm with the help of metallic antenna structures and induces transient changes of electronic or vibrational absorption, which are mapped by femtosecond optical probe pulses. From the transient absorption changes, molecular dipole moments and/or polarizabilities are inferred directly. This novel technique was successfully applied to dye molecules in polar liquid solution and a variety of photobiological systems embedded in proteins. For wild-type bacteriorhodopsin (BR), mutants of BR, and neorhodopsin, surprisingly small dipole differences were found between the ground and electronically excited state of their retinal chromophore. For BR, this behavior points to a pronounced mixing of the first and second electronically excited state and a femtosecond change of the excited state dipole moment along the reaction coordinate of retinal isomerization.
The water and ion geometries at the surface of double-stranded helical RNA and transfer RNA (tRNA) were studied by two-dimensional infrared (2D-IR) spectroscopy of phosphate stretching vibrations of the RNA backbone, which serve as noninvasive probes of local interactions. Three prototypical hydration motifs were identified, one of them including hydrated contact pairs of phosphate groups with counterions. Librational water motions lead to structural fluctuations on a time scale of 300 fs while hydrogen bond lifetimes and reordering times of ions are much longer.
The strong impact of contact ion pairs on biomolecular structure was demonstrated for transfer RNA (t-RNA) and Adenine-Tri-Phosphate (ATP). The folded cloverleaf structure of t-RNA is stabilized by magnesium ions in direct touch with the RNA backbone, thus mitigating the strong repulsive interaction between adjacent phosphate groups at kinks of the backbone. The results allow for distinguishing specific interaction geometries and for calibrating the local electrostatics. In a similar way, complexes of ATP with zinc ions were analyzed.
2D-IR spectroscopy and theory were combined for analyzing RNA electrostatics at the interface to its water shell. The results clearly show that the broadly established Poisson-Boltzmann approaches fail by far in reproducing local interaction strengths and electric fields, while microscopic ab-initio calculations and molecular dynamics simulations give a realistic picture.
The fluctuating electric field in water induces a spontaneous tunneling ionization of water molecules, generating free electrons. Application of a much smaller external THz field suppresses charge recombination and transfers electrons in space. The electrons eventually localize in the liquid, forming so-called solvated electrons. This electron generation mechanism was observed for the first time and characterized in detail by two-dimensional THz spectroscopy.
Beyond single-electron dynamics, the project led to the discovery of coherent many-body excitations of the electron and its solvent cloud consisting of thousands of molecules. The femtosecond electron localization process launches coherent oscillations in the molecular cloud, which persist for a time range well beyond 100 ps. Such radial, i.e. longitudinal polaron excitations generate an additional resonance in the dielectric spectrum at a THz frequency that can be tuned via the total electron concentration. The linear and nonlinear optical properties of polarons were characterized in detail and their application as a novel epsilon-near-zero material for the THz frequency range demonstrated.
The new method of ultrafast THz Stark spectroscopy, which was demonstrated for the first time in this project, allows for mapping the momentary electric response of molecular ensembles under dynamically frozen conditions. An external picosecond electric field at THz frequencies is enhanced up to several megavolts/cm with the help of metallic antenna structures and induces transient changes of electronic or vibrational absorption, which are mapped by femtosecond optical probe pulses. From the transient absorption changes, molecular dipole moments and/or polarizabilities are inferred directly. This novel technique was successfully applied to dye molecules in polar liquid solution and a variety of photobiological systems embedded in proteins. For wild-type bacteriorhodopsin (BR), mutants of BR, and neorhodopsin, surprisingly small dipole differences were found between the ground and electronically excited state of their retinal chromophore. For BR, this behavior points to a pronounced mixing of the first and second electronically excited state and a femtosecond change of the excited state dipole moment along the reaction coordinate of retinal isomerization.
The water and ion geometries at the surface of double-stranded helical RNA and transfer RNA (tRNA) were studied by two-dimensional infrared (2D-IR) spectroscopy of phosphate stretching vibrations of the RNA backbone, which serve as noninvasive probes of local interactions. Three prototypical hydration motifs were identified, one of them including hydrated contact pairs of phosphate groups with counterions. Librational water motions lead to structural fluctuations on a time scale of 300 fs while hydrogen bond lifetimes and reordering times of ions are much longer.
The strong impact of contact ion pairs on biomolecular structure was demonstrated for transfer RNA (t-RNA) and Adenine-Tri-Phosphate (ATP). The folded cloverleaf structure of t-RNA is stabilized by magnesium ions in direct touch with the RNA backbone, thus mitigating the strong repulsive interaction between adjacent phosphate groups at kinks of the backbone. The results allow for distinguishing specific interaction geometries and for calibrating the local electrostatics. In a similar way, complexes of ATP with zinc ions were analyzed.
2D-IR spectroscopy and theory were combined for analyzing RNA electrostatics at the interface to its water shell. The results clearly show that the broadly established Poisson-Boltzmann approaches fail by far in reproducing local interaction strengths and electric fields, while microscopic ab-initio calculations and molecular dynamics simulations give a realistic picture.
The fluctuating electric field in water induces a spontaneous tunneling ionization of water molecules, generating free electrons. Application of a much smaller external THz field suppresses charge recombination and transfers electrons in space. The electrons eventually localize in the liquid, forming so-called solvated electrons. This electron generation mechanism was observed for the first time and characterized in detail by two-dimensional THz spectroscopy.
Beyond single-electron dynamics, the project led to the discovery of coherent many-body excitations of the electron and its solvent cloud consisting of thousands of molecules. The femtosecond electron localization process launches coherent oscillations in the molecular cloud, which persist for a time range well beyond 100 ps. Such radial, i.e. longitudinal polaron excitations generate an additional resonance in the dielectric spectrum at a THz frequency that can be tuned via the total electron concentration. The linear and nonlinear optical properties of polarons were characterized in detail and their application as a novel epsilon-near-zero material for the THz frequency range demonstrated.
THz Stark spectroscopy paves the way for generating accurate and specific insight into biomolecular electrostatics and avoids the severe shortcomings of conventional Stark spectroscopy. The successful application of this novel method to complex rhodopsin systems demonstrates a strong potential for future applications in biophysics and –chemistry. In both methodological and scientific respect, the results are groundbreaking and advance the research field well beyond the state of the art.
The discovery of polaronic excitations in polar liquids represents an unforeseen major breakthrough in understanding the mainly unexplored physics of collective low-frequency excitations in disordered systems like polar liquids. Before this work, insight in such fundamental behavior has existed in part for solids but not for liquids. Beyond the relevance for a basic understanding of liquid dynamics, polaron excitations hold a strong potential for applications in THz nonlinear optics, as demonstrated here by the tunable epsilon-near-zero behavior of a simple liquid alcohol.
The discovery of polaronic excitations in polar liquids represents an unforeseen major breakthrough in understanding the mainly unexplored physics of collective low-frequency excitations in disordered systems like polar liquids. Before this work, insight in such fundamental behavior has existed in part for solids but not for liquids. Beyond the relevance for a basic understanding of liquid dynamics, polaron excitations hold a strong potential for applications in THz nonlinear optics, as demonstrated here by the tunable epsilon-near-zero behavior of a simple liquid alcohol.