Diese Arbeit ermöglicht ein detailliertes Verständnis der Schwingungsdynamik und Kupplungen in einem Basenpaar-Modellsystem und in künstlichen DNA-Oligomeren bei verschiedenen Hydratationsgraden. Durch die Verwendung von nichtlinearer ultraschneller IR Pump-Probe Spektroskopie sind die Schwingungsbewegungen hydratisierter DNA und die schnellsten Veränderungen in den DNA-Wasser-Wechselwirkungen und Hydrationsgeometrien direkt zugänglich. 2-pyridone/2-hydroxypyridine ist ein Modellsystem für die gekoppelte intermolekularen Wasserstoffbrücken, deren Struktur der von DNA-Basenpaaren ähnelt. In Dichlormethan existiert das Molekül als ein zyklischer 2-Pyridon-Dimer, deren Vorkommern durch NMR-und 2D-FTIR Spektroskopie verifiziert wurde. Die beobachteten kohärente Oszillationen aufgrund niederfrequenter Wellenpaketbewegungen der Dimere können für die Dynamik und räumliche Geometrie der Basenpaare in den DNA-Molekül relevant sein. Transiente Schwingungsspektren eines poly[d(A-T)]:poly[d(A-T)] Film erlauben die Zuordnung von verschiedenen NH-Streckbanden zu einer bestimmten Schwingung der Nukleinbasen und ermöglichen deren Abgrenzung zu den Beiträgen von OH-Streckschwingungen des umgebenden Wassers. Bei einem niedrigen Hydratisierungsgrad verändern die restlichen, an die Phosphatgruppen gebundenen Wassermoleküle, ihre Ausrichtung auf ultraschnellen Zeitskalen nicht. Im Fall vollständig hydratisierter DNA ist die Dynamik der Wasserhülle dem Verhalten des reinen Wassers ähnlicher und man beobachtet spektrale Diffusion der OH-Streckschwingung im Subpikosekundenbereich sowie einen Zerfall der Schwingungsanisotropie durch Molekülrotation und/oder Energietransfer. Die Wassermoleküle der Phosphat-Hydratationshülle dienen als effiziente Wärmesenke für Überschussenergie aus der DNA, wobei die Energietransferzeiten im fs-bereich liegen. Im Gegensatz dazu erfolgt Energietransport innerhalb der DNA auf einer langsameren Zeitskala von 20 ps. / This thesis provides a detailed understanding of vibrational dynamics and couplings in a base pair model system and artificial DNA oligomers at different levels of hydration. By using nonlinear ultrafast infrared pump-probe spectroscopy, the basic vibrational motions of hydrated DNA and the fastest changes in the DNA–water interactions and hydration geometries are directly accessed. 2-pyridone/2-hydroxypyridine is used as a model molecule for coupled intermolecular hydrogen bonds with a structure resembling a DNA base pair. In dichloromethane the molecule predominantly exists as a cyclic 2-pyridone dimer as determined using a combined NMR and 2D FTIR approach. The observed coherent oscillations due to low-frequency hydrogen bond wavepacket motions of the dimers are expected to be relevant for the dynamics and spatial geometry of base pairs in DNA molecule. Transient vibrational spectra of a poly[d(A-T)]:poly[d(A-T)] film enabled the assignment of different NH stretching bands to particular nucleobase vibrations, also discerning them from the OH stretching contributions of the surrounding water. At a low hydration level, residual water molecules, bound to the phosphate groups, do not alter their orientation on ultrafast time scales. In the case of fully hydrated DNA, the dynamics of the water shell are closer to those of bulk liquid water with a sub-picosecond spectral diffusion and a loss of vibrational anisotropy as a result of molecular rotation and/or energy transfer. The water shell around the phosphates serves as a efficient heat sink accepting excess energy from DNA in a femtosecond time domain, whereas the energy transfer within DNA occurs on the time scale of 20 ps.

Von generellem Interesse für potentielle Anwendungsmöglichkeiten sind Materialien mit speziellen elektrischen bzw. nichtlinearen optischen Eigenschaften. Im Rahmen dieser Arbeit wurden neue dipolare Verbindungen synthetisiert und charakterisiert, welche eine zentrale Organosilanspacereinheit (-SiMex-,-(SiMe2)6-), ein terminales Metallkomplexfragment (potentieller Donor, FcN-) sowie eine terminale organische Akzeptoreinheit (-PhF, -PhBr, -PhCHO) enthalten und gleichzeitig eine große Variationsbreite gewünschter Eigenschaften aufweisen. Aus Photo-EMK-Messungen, UV/VIS-, NMR-Spektroskopie, Mößbauer- und cyclovoltammetrischen Untersuchungen kann auf eine im Festkörper auftretende temperaturabhängig variierende intermolekulare Kopplung zwischen dem Donor und dem organischen Akzeptor geschlossen werden. Photo-EMK- und Einkristallröntgenstrukturanalysen der Salze (Hydrochloride, Pikrate) offenbaren starke intermolekulare Wechselwirkungen (C-H···π, D-C-H···A) mit entscheidendem Einfluß auf Festkörpereigenschaften (supramolekulare MO's).

We report a Raman technique applicable for the in situ analysis of the development of hydrogen bonds in the liquid water‐rich phase just before the onset of gas hydrate formation. Herewith, the phase transition as well as the working principle of hydrate formation inhibitors and promoters can be analyzed.

Von generellem Interesse für potentielle Anwendungsmöglichkeiten sind Materialien mit speziellen elektrischen bzw. nichtlinearen optischen Eigenschaften. Im Rahmen dieser Arbeit wurden neue dipolare Verbindungen synthetisiert und charakterisiert, welche eine zentrale Organosilanspacereinheit (-SiMex-,-(SiMe2)6-), ein terminales Metallkomplexfragment (potentieller Donor, FcN-) sowie eine terminale organische Akzeptoreinheit (-PhF, -PhBr, -PhCHO) enthalten und gleichzeitig eine große Variationsbreite gewünschter Eigenschaften aufweisen. Aus Photo-EMK-Messungen, UV/VIS-, NMR-Spektroskopie, Mößbauer- und cyclovoltammetrischen Untersuchungen kann auf eine im Festkörper auftretende temperaturabhängig variierende intermolekulare Kopplung zwischen dem Donor und dem organischen Akzeptor geschlossen werden. Photo-EMK- und Einkristallröntgenstrukturanalysen der Salze (Hydrochloride, Pikrate) offenbaren starke intermolekulare Wechselwirkungen (C-H···π, D-C-H···A) mit entscheidendem Einfluß auf Festkörpereigenschaften (supramolekulare MO's).

Die Struktur von DNS Molekülen und ihre Wechselwirkung mit Wasser werden seit langer Zeit heiß diskutiert. In der vorliegenden Arbeit wird nichtlineare Spektroskopie zur Untersuchung dieser Systeme angewendet. Oligomere, die aus 23 alternierenden Adenin-Thymin-Basenpaaren bestehen und eine Doppelhelix bilden, wurden mit Hilfe von 2D IR Spektroskopie für verschiedene Hydratisierungsgrade untersucht. Für DNS-Filme bei 0% relativer Feuchte (r.F.) erlauben die transienten Spektren eine Unterscheidung der NH Streckschwingung von Thymin ((NH)), der symmetrischen und asymmetrischen NH2 Streckschwingung von Adenin (s(NH2) and a(NH2)) sowie die Bestimmung der jeweiligen Linienprofile. Die Spektren zeigen eine homogene Verbreiterung für die (NHT) wohingegen die s(NH2) and a(NH2) eine ausgeprägte und zeitunabhängige inhomogene Verbreiterung zeigen, welche auf Unordnungen in der DNS-Struktur hinweisen. Außerdem kann Energietransfer von der a(NH2) zur (NH) beobachtet werden. Bei Erhöhung der r.F. hat die erhöhte Anzahl von Wassermolekülen nur einen geringen Einfluss auf die Positionen und Linienprofile der NH Streckschwingungen. Dadurch wird nahegelegt, dass die spektrale Dynamik vom DNS Molekül selbst und nicht vom umgebenen Wasser bestimmt ist. Im Gegensatz dazu zeigt die OH Streckmode der Wasserhülle um die DNS spektrale Diffusion auf einer 500 fs Zeitskala. Guanosin-Cytidin(GC)-Basenpaare wurden in Chloroformlösung untersucht, um die Wechselwirkung zwischen Basenpaaren zu verstehen. Dabei wurden die NH Schwingungen in einer local mode Darstellung betrachtet, die zwei freie NH Gruppen von G und C und drei wasserstoffverbrückte NH Gruppen beeinhaltet. Die Kopplungen und Relaxationsdynamik der NH Streckanregungen wurden mit Femtosekunden-Pump-Probe und 2D IR Experimenten studiert. Die Ergebnisse zeigen eine Verringerung der Lebensdauer mit der Bildung von Wasserstoffbrücken sowie Energietransfer zwischen zwei wasserstoffverbrückten NH Streckschwingungen. / The structure of DNA molecule and the interactions with its surrounding water is a hot topic for long time. In this thesis, we employ the nonlinear spectroscopy, including femtosecond pump-probe and two-dimensional infrared (2D IR) experiment, to study the vibrational dynamics of the systems. Double-stranded DNA short oligomers containing 23 alternating adenine-thymine base pairs were studied at different hydration levels by femtosecond 2D IR spectroscopy. For a DNA film at 0% relative humidity, the transient spectra enable a separation of the NH stretching mode of thymine from the symmetric and asymmetric NH2 stretching modes of adenine and determine the individual line shapes. For the NH stretch of thymine, the spectra demonstrate an essential homogeneous broadening, whereas for the symmetric and asymmetric NH2 stretches a pronounced and time-independent inhomogeneous broadening suggests a disorder in DNA structure. An energy transfer from the asymmetric NH2 stretch of adenine to the NH stretch of thymine is also observed. When the relative humidity increases, the increased water molecules have limited influence on the positions and line shapes of NH stretching frequencies, suggesting the spectral dynamics governed by DNA rather than water fluctuations. In contrast, the OH stretching mode of water shell around hydrated DNA undergoes a spectral diffusion on a 500 fs time scale, which is slower than the neat water. The guanosine-cytidine (GC) base pairs in chloroform solution were investigated to understand the interactions within base pairs. A local mode representationof NH stretching mode is adopted, consisting two free NH groups of G and C and three hydrogen bonded NH groups. The coupling and relaxation dynamics of the NH stretching excitations are studied by femtosecond pump-probe and 2D IR experiments. The results demonstrate a lifetime shortening upon the formation of hydrogen bonds, and an energy transfer between two hydrogen-bonded NH stretches.

We report the application of a novel optical Raman-based measurement technique for the simultaneous determination of the progress of mixing on the micro- and on the macro-scale. The introduced measurement technique is applicable to mixing systems containing one compound, which potentially can form hydrogen bonds, such as water, alcohols or amines, and does not rely on the addition of traces of indicator compounds. Here we demonstrate its applicability by analyzing the lag of micro-mixing behind macro-mixing when liquid ethanol is injected into a supercritical bulk environment mainly composed of carbon dioxide (CO2). While the degree of mixing on the macro-scale is determined from the ratio of the intensities of characteristic Raman signals of ethanol and CO2, the degree of mixing on the micro-scale is determined from the shape of the OH stretching vibration Raman signal of ethanol, which is a function of the development of hydrogen bonds.

Die Energierelaxationsdynamik und strukturelle Dynamik der DNA spielen eine entscheidende Rolle für das Verständnis der photochemischen Eigenschaften und biologischen Funktion von DNA. Die schnellsten Prozesse geschehen dabei auf Zeitskalen im Femto- bis Pikosekundenbereich und können durch zeitaufgelöste (ultraschnelle) Messungen an molekularen Schwingungsanregungen in Echtzeit beobachtet werden. In dieser Arbeit werden NH-Streck Schwingungsmoden der Nukleobasen sowie OH-Streckmoden der Hydrathülle mittels linearer infrarot (IR) Spektroskopie sowie ultraschneller Pump-Probe und zweidimensionaler IR Spektroskopie untersucht. Messungen an monomerartigen Nukleobasen, wasserstoffverbrückten Nukleobasenpaaren und kurzen DNA Fragmenten in Doppelhelixstruktur ermöglichen es, die Effekte der verschiedenen Wasserstoffbrücken und Schwingungskopplungen separat voneinander zu untersuchen. Im Zusammenspiel mit exzitonischen und quantenchemischen Rechnungen werden so weitreichende Einsichten in die spektroskopischen Eigenschaften und die Relaxationsdynamik von Schwingungsanregungen in DNA gewonnen. Es wird u.a. gezeigt, dass Wasserstoffbrücken zwischen Nukleobasen eine Lebensdauer größer 1 ps besitzen und eine beschleunigte Energierelaxation durch Fermiresonanzen mit niederfrequenten Schwingungsmoden des Fingerprintbereichs bewirken. Die DNA-Hydrathülle zeigt eine ultraschnelle strukturelle Dynamik unabhängig von der Basenpaarzusammensetzung und fungiert als effiziente Wärmesenke für hochfrequente Schwingungsanregungen. / Energy relaxation and structural dynamics in DNA play a crucial role for the understanding of the photochemical properties and biological function of DNA. The fastest of such processes occur on the femto- to picosecond time scale and can be followed in real time through time-resolved (ultrafast) measurements on molecular vibrations. In this work, NH stretching excitations of nucleobases and OH stretching modes of the hydration shell are analyzed through linear infrared (IR) spectroscopy as well as ultrafast pump-probe and two-dimensional IR spectroscopy. Measurements on monomeric nucleobases, hydrogen-bonded nucleobase pairs, and short DNA fragments in double helix structure allow one to examine the effects of the different hydrogen bonds and vibrational couplings separately from each other. The combination with excitonic and quantum chemical calculations provides profound new insights into the spectroscopic properties and relaxation dynamics of vibrational excitations in DNA. This work shows that hydrogen bonds between nucleobases have a lifetime greater than 1 ps and lead to an accelerated dissipation of energy due to Fermi resonances with vibrational modes in the fingerprint range. The hydration shell of DNA exhibits ultrafast structural dynamics independent of the base pair composition and serves as an efficient heat sink for high-energy vibrational excitations.

Proteins have an indispensable role in the cell. They carry out a wide variety of structural, catalytic and signaling functions in all known biological systems. To perform their biological functions, proteins establish interactions with other bioorganic molecules including other proteins. Therefore, protein-protein interactions is one of the central topics in molecular biology. My thesis is devoted to three different topics in the field of protein-protein interactions. The first one focuses on solvent contribution to protein interfaces as it is an important component of protein complexes. The second topic discloses the structural and functional potential of fluorine's unique properties, which are attractive for protein design and engineering not feasible within the scope of canonical amino acids. The last part of this thesis is a study of the impact of charged amino acid residues within the hydrophobic interface of a coiled-coil system, which is one of the well-established model systems for protein-protein interactions studies. I. The majority of proteins interact in vivo in solution, thus studies of solvent impact on protein-protein interactions could be crucial for understanding many processes in the cell. However, though solvent is known to be very important for protein-protein interactions in terms of structure, dynamics and energetics, its effects are often disregarded in computational studies because a detailed solvent description requires complex and computationally demanding approaches. As a consequence, many protein residues, which establish water-mediated interactions, are neither considered in an interface definition. In the previous work carried out in our group the protein interfaces database (SCOWLP) has been developed. This database takes into account interfacial solvent and based on this classifies all interfacial protein residues of the PDB into three classes based on their interacting properties: dry (direct interaction), dual (direct and water-mediated interactions), and wet spots (residues interacting only through one water molecule). To define an interaction SCOWLP considers a donor–acceptor distance for hydrogen bonds of 3.2 Å, for salt bridges of 4 Å, and for van der Waals contacts the sum of the van der Waals radii of the interacting atoms. In previous studies of the group, statistical analysis of a non-redundant protein structure dataset showed that 40.1% of the interfacial residues participate in water-mediated interactions, and that 14.5% of the total residues in interfaces are wet spots. Moreover, wet spots have been shown to display similar characteristics to residues contacting water molecules in cores or cavities of proteins. The goals of this part of the thesis were: 1. to characterize the impact of solvent in protein-protein interactions 2. to elucidate possible effects of solvent inclusion into the correlated mutations approach for protein contacts prediction To study solvent impact on protein interfaces a molecular dynamics (MD) approach has been used. This part of the work is elaborated in section 2.1 of this thesis. We have characterized properties of water-mediated protein interactions at residue and solvent level. For this purpose, an MD analysis of 17 representative complexes from SH3 and immunoglobulin protein families has been performed. We have shown that the interfacial residues interacting through a single water molecule (wet spots) are energetically and dynamically very similar to other interfacial residues. At the same time, water molecules mediating protein interactions have been found to be significantly less mobile than surface solvent in terms of residence time. Calculated free energies indicate that these water molecules should significantly affect formation and stability of a protein-protein complex. The results obtained in this part of the work also suggest that water molecules in protein interfaces contribute to the conservation of protein interactions by allowing more sequence variability in the interacting partners, which has important implications for the use of the correlated mutations concept in protein interactions studies. This concept is based on the assumption that interacting protein residues co-evolve, so that a mutation in one of the interacting counterparts is compensated by a mutation in the other. The study presented in section 2.2 has been carried out to prove that an explicit introduction of solvent into the correlated mutations concept indeed yields qualitative improvement of existing approaches. For this, we have used the data on interfacial solvent obtained from the SCOWLP database (the whole PDB) to construct a “wet” similarity matrix. This matrix has been used for prediction of protein contacts together with a well-established “dry” matrix. We have analyzed two datasets containing 50 domains and 10 domain pairs, and have compared the results obtained by using several combinations of both “dry” and “wet” matrices. We have found that for predictions for both intra- and interdomain contacts the introduction of a combination of a “dry” and a “wet” similarity matrix improves the predictions in comparison to the “dry” one alone. Our analysis opens up the idea that the consideration of water may have an impact on the improvement of the contact predictions obtained by correlated mutations approaches. There are two principally novel aspects in this study in the context of the used correlated mutations methodology : i) the first introduction of solvent explicitly into the correlated mutations approach; ii) the use of the definition of protein-protein interfaces, which is essentially different from many other works in the field because of taking into account physico-chemical properties of amino acids and not being exclusively based on distance cut-offs. II. The second part of the thesis is focused on properties of fluorinated amino acids in protein environments. In general, non-canonical amino acids with newly designed side-chain functionalities are powerful tools that can be used to improve structural, catalytic, kinetic and thermodynamic properties of peptides and proteins, which otherwise are not feasible within the use of canonical amino acids. In this context fluorinated amino acids have increasingly gained in importance in protein chemistry because of fluorine's unique properties: high electronegativity and a small atomic size. Despite the wide use of fluorine in drug design, properties of fluorine in protein environments have not been yet extensively studied. The aims of this part of the dissertation were: 1. to analyze the basic properties of fluorinated amino acids such as electrostatic and geometric characteristics, hydrogen bonding abilities, hydration properties and conformational preferences (section 3.1) 2. to describe the behavior of fluorinated amino acids in systems emulating protein environments (section 3.2, section 3.3) First, to characterize fluorinated amino acids side chains we have used fluorinated ethane derivatives as their simplified models and applied a quantum mechanics approach. Properties such as charge distribution, dipole moments, volumes and size of the fluoromethylated groups within the model have been characterized. Hydrogen bonding properties of these groups have been compared with the groups typically presented in natural protein environments. We have shown that hydrogen and fluorine atoms within these fluoromethylated groups are weak hydrogen bond donors and acceptors. Nevertheless they should not be disregarded for applications in protein engineering. Then, we have implemented four fluorinated L-amino acids for the AMBER force field and characterized their conformational and hydration properties at the MD level. We have found that hydrophobicity of fluorinated side chains grows with the number of fluorine atoms and could be explained in terms of high electronegativity of fluorine atoms and spacial demand of fluorinated side-chains. These data on hydration agrees with the results obtained in the experimental work performed by our collaborators. We have rationally engineered systems that allow us to study fluorine properties and extract results that could be extrapolated to proteins. For this, we have emulated protein environments by introducing fluorinated amino acids into a parallel coiled-coil and enzyme-ligand chymotrypsin systems. The results on fluorination effect on coiled-coil dimerization and substrate affinities in the chymotrypsin active site obtained by MD, molecular docking and free energy calculations are in strong agreement with experimental data obtained by our collaborators. In particular, we have shown that fluorine content and position of fluorination can considerably change the polarity and steric properties of an amino acid side chain and, thus, can influence the properties that a fluorinated amino acid reveals within a native protein environment. III. Coiled-coils typically consist of two to five right-handed α-helices that wrap around each other to form a left-handed superhelix. The interface of two α-helices is usually represented by hydrophobic residues. However, the analysis of protein databases revealed that in natural occurring proteins up to 20% of these positions are populated by polar and charged residues. The impact of these residues on stability of coiled-coil system is not clear. MD simulations together with free energy calculations have been utilized to estimate favourable interaction partners for uncommon amino acids within the hydrophobic core of coiled-coils (Chapter 4). Based on these data, the best hits among binding partners for one strand of a coiled-coil bearing a charged amino acid in a central hydrophobic core position have been selected. Computational data have been in agreement with the results obtained by our collaborators, who applied phage display technology and CD spectroscopy. This combination of theoretical and experimental approaches allowed to get a deeper insight into the stability of the coiled-coil system. To conclude, this thesis widens existing concepts of protein structural biology in three areas of its current importance. We expand on the role of solvent in protein interfaces, which contributes to the knowledge of physico-chemical properties underlying protein-protein interactions. We develop a deeper insight into the understanding of the fluorine's impact upon its introduction into protein environments, which may assist in exploiting the full potential of fluorine's unique properties for applications in the field of protein engineering and drug design. Finally we investigate the mechanisms underlying coiled-coil system folding. The results presented in the thesis are of definite importance for possible applications (e.g. introduction of solvent explicitly into the scoring function) into protein folding, docking and rational design methods. The dissertation consists of four chapters: ● Chapter 1 contains an introduction to the topic of protein-protein interactions including basic concepts and an overview of the present state of research in the field. ● Chapter 2 focuses on the studies of the role of solvent in protein interfaces. ● Chapter 3 is devoted to the work on fluorinated amino acids in protein environments. ● Chapter 4 describes the study of coiled-coils folding properties. The experimental parts presented in Chapters 3 and 4 of this thesis have been performed by our collaborators at FU Berlin. Sections 2.1, 2.2, 3.1, 3.2 and Chapter 4 have been submitted/published in peer-reviewed international journals. Their organization follows a standard research article structure: Abstract, Introduction, Methodology, Results and discussion, and Conclusions. Section 3.3, though not published yet, is also organized in the same way. The literature references are summed up together at the end of the thesis to avoid redundancy within different chapters.

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