Title: Study of protein structure and dynamics by means of optical spectroscopy Author: Tomáš Pazderka Institute: Institute of Physics of Charles University Supervisor: RNDr. Vladimír Kopecký, Ph.D., Institute of Physics of Charles University Abstract: The aim of this thesis is to improve understanding of protein structure and dynamics and extend experimental setup and data processing for such stud- ies. We focus on the extension of experimental feasability of vibrational optical activity (VOA). We have demonstrated a usability of intensity calibration in the field of Raman optical activity. Advantages for measurements on multiple instru- ments and/or using different configurations have been shown. A new instrumental setup has been developed for microsampling measurements of vibrational circular dichroism spectra with a spatial resolution of 1 mm. Using this technique, spatial inhomogeneities in a sample of protein fibrils have been observed. Model com- pounds for amide nonplanarity have been investigated utilizing several methods of optical spectroscopy and key spectral features for determination of amide non- planarity and the absolute configuration have been identified. A comprehensive set of Raman spectra of proteinogenic amino acids has been measured. Sample concentration dependencies and consequent...

The hydration and dynamics of the biomolecules appear to be vital for their proper biological functioning. In the presented thesis, various fluorescence techniques were developed and applied to access these properties and their changes upon the mutual interactions of the biomolecules. Initially, the solvent relaxation method based on recording time-dependent fluorescence shift (TDFS) was used to map DNA interactions with proteins and lipids by the newly synthesised fluorene dye covalently bound to the DNA. Secondly, copper-transporting ATPase was probed by Badan attached to the copper-binding cysteine-proline-cysteine motif. The variations in hydration were found to be crucial for the proper ATPase function. Third, a detailed study on quenching of Badan/Prodan fluorescence by tryptophan revealed the limitations of the TDFS method for protein studies, which is essential finding for further applications of TDFS. Fourth application involves investigations of heavy atom effects on the excited state relaxation processes by up-conversion approach in iodinated metallocorroles, which are promising dyes for biological imaging. The obtained findings shall help in further tuning of the optical properties of the corroles desired for the variety of applications. Finally, fluorescence correlation spectroscopy...

The objective of this preliminary study was to determine the potential of diffuse correlation spectroscopy (DCS) for assessment of coagulation depth. Coagulation of tissue can occur due to a number of different reasons such as thermal or electrical burns or radiofrequency ablation. DCS is a non-invasive optical technique which can be used to determine the optical and dynamic properties of tissue by fitting a theoretical model of photon propagation in multiply scattering tissue to experimental data obtained from measurements. The DCS measurements were performed on two-layered phantom models that represent healthy tissue with high flow properties with a layer of coagulated tissue with low flow properties on top. Three different phantom models were prepared using gelatin-Intralipid gels, PDMS, and nylon as an upper layer, and an Intralipid solution was used for the bottom layer for all three phantoms. DCS measurements were performed on all three phantom models with varying thicknesses of the upper layers, and varying source-detector separations. The acquired data from the DCS measurement were analyzed in MATLAB in order to obtain the electric field temporal autocorrelation function. A theoretical model describing photon propagation in a two-layered medium was fitted to the obtained data in order to extract the desired parameters. The results showed that the thickness of the gelatin-Intralipid gels could be extracted within a 0.5 mm certainty and the thickness of the PDMS phantoms could also be extracted within approximately 0.7 mm. For the nylon phantoms, the results obtained were not good because the fitting was not successful and the thickness was not extracted appropriately. There is potential in DCS for assessment of burn wound depth but further research and development has to be done in the field in order to obtain more accurate and reliable results.

The progress of miniaturisation towards the nanoscopic scale in science and technology has also influenced the biosciences. This is particularly important, since proteins, as the smallest functional units of life, exhibit a spectacular wealth of functionalities, enabling them to fulfil complex tasks in cells and organisms. For this reason, they are often termed molecular or cellular “machines”. To be able to investigate and better understand these fascinating molecules in their native environment, new analytical methods must be developed, with appropriately high sensitivity and spatial and temporal resolution. We describe one very promising technique based on fluorescence spectroscopy, which allows a quantitative analysis of protein- protein interactions in the live cell. / Die zunehmende Miniaturisierung bis hin zum nanoskopischen Maßstab in vielen technischen Disziplinen hat auch die Lebenswissenschaften ergriffen. Dies ist insofern von großer Bedeutung, als die Proteine als kleinste funktionale Einheiten des Lebens trotz ihrer winzigen Abmessungen eine faszinierende Komplexität aufweisen, die es ihnen erlauben, hoch differenzierte und spezialisierte Aufgaben in der Zelle und im Organismus zu übernehmen. Aus diesem Grund werden sie in der modernen Biologie auch als molekulare oder zelluläre „Maschinen“ bezeichnet. Um diese kleinen Wunderwerke zu studieren und ihre Funktionsweise in ihrer natürlichen Umgebung zu analysieren, bedarf es innovativer Technologien, die es erlauben, mit maximaler räumlicher und zeitlicher Auflösung auch einzelne Moleküle in der lebenden Zelle sichtbar zu machen und zu verfolgen. Im Folgenden wird eine von uns entwickelte fluoreszenzspektroskopische Methode vorgestellt, mit deren Hilfe die komplizierten Interaktionen zwischen Proteinen in der lebenden Zelle aufgeklärt werden können.

Many human diseases including cancer have been associated with altered cellular metabolism and a changed oxygen consumption in cells. Fluorophores are sensitive to their local environment due to their long life times in transient dark states. A recent study successfully utilized this sensitivity to image differences in oxygen concentrations in cells using transient state (TRAST) microscopy together with fluorescent labels [1]. A natural continuation of this study is to investigate the possibilities of using this method with natural fluorophores already present in cells and thereby avoid articial labeling. Flavin adenine dinucleotide (FAD) is an auto fluorescent coenzyme that is naturally present in cells and involved in cellular metabolism. This project is an exploratory pilot study for cellular measurements with the aim to investigate if FAD can be used to probe oxygen concentrations in aqueous solution using transient state monitoring and fluorescence correlation spectroscopy (FCS). This thesis includes the results from FCS and TRAST experiments on FAD in aqueous solutions with different oxygen concentrations as well as different ascorbic acid concentrations. The performed experiments showed that FAD monitored with TRAST is sensitive to differences in oxygen concentrations for the aqueous solutions used in this study.

The thesis describes time dependent fluorescence shift method and fluorescence correlation spectroscopy method (FCS) with its extensions FLCS, Z-scan FCS and dual-focus FCS applied on specific problems in DNA and lipid research. Compaction mechanism of a DNA molecule smaller than a resolution of a confocal microscope was elucidated. The process was revealed to be "all or non" for a polycation spermine as a condenser in contrast with the gradual compaction caused by a cationic surfactant. Biophysical properties of a phospholipid bilayer influenced by presence of oxidized phospholipids with truncated sn-2 chain were explored. The dynamics of hydrated functional groups in the headgroup region was proved to get faster while the hydration of the headgroup region increased. These effects are in relation with the reorientation of the short sn-2 chains observed in molecular dynamics simulations. Presence of oxidized species may also influence the lateral diffusion of the lipids - a slight increase of the diffusion coefficient was observed. Decrease of hydration and mobility in the headgroup region was found as an influence of heavy water on the phospholipid membrane. These finding are in line with molecular dynamics simulations which show longer lifetimes of hydrogen bonds between water and lipid molecules in...

No description available.

Powerful spectroscopic techniques increasingly involve nonlinear processes that arise due to the convolution of more than one electric field - input laser pulse. Analyzing the output of optical processes like these demands the utilization of deterministic improvement tools. Three-color coherent Raman scattering represents a complex non-degenerate four wave mixing process that includes contributions from both resonant and non-resonant interaction of the three input fields to generate a signal. In order to quantify these contributions, effective differentiation of the non- resonant (background) from the resonant (coherent signal) is required. These contributions can be differentiated based on how the molecular vibrational modes are being excited by the input pulses. The work described here demonstrates the ability of second-order correlation spectroscopy, applied along with an all-Gaussian theoretical model to analyze three color coherent Raman scattering processes. It is shown to discriminate between resonant versus non-resonant four wave mixing processes successfully. A robust, femtosecond/picosecond coherent Raman spectroscope is used to observe how the resonant signal builds up in a finite amount of time for different specimens and how it is can be controlled by input laser pulse shaping. A closed-form solution obtained via an all-Gaussian approach provides confirmatory theoretical proof of the experimental results obtained. This technique is used to study hydrogen bonding, which is a vital molecular interaction for bio-molecular systems and yet lacks a profound understanding of its ways of forming complexes. Furthermore, a novel second-order one-dimensional correlation function is introduced that replicates the results of the diagonal sum of the traditional synchronous two- dimensional correlation function, thus reducing a two-dimensional analysis to one-dimension. Along with the first demonstration of these analyses for coherent Raman scattering, a generalized approach is described, which opens up research opportunities to investigate these optical processes' dependence on multiple controlling parameters.

No description available.

Fluorescence correlation spectroscopy (FCS) analyzes the fluctuations in the fluorescence intensity, which is emitted from a tiny excition volume, to obtain information about the concentration, the mobility, and the molecular interactions of labeled molecules. The more advanced fluorescence cross-correlation spectroscopy (FCCS) increases the precision in the determination of fl ow velocities and binding constants compared to standard FCS. The miniaturization in biomedical and chemical engineering has been developing rapidly, propelled by the vision of a fully functional laboratory on a single chip and its use in human therapeutics, for example, as implanted drug delivery system. A key requirement to fulfill this vision is the ability to handle small fl uid volumes. Handling liquids using the electrohydrodynamical principle circumvents many of the disadvantages of other systems. The complex flow pattern in the active region of such a pump could not be resolved by common tracking techniques. In this thesis, two-focus FCCS (2f-FCCS) was used to map the flow pro file inside a micropump. The high precision of 2f-FCCS in the determination of fl ow measurements even with small fluorescent particles allowed the measurement of the flow velocities induced by electrohydrodynamic forces acting on the solvent, while excluding the effects of dielectrophoretic forces acting on larger particles. Analysis of the fl ow data indicates a fl ow pattern that consists of two vortices of different size and opposite direction of rotation. The flow pattern derived by 2f-FCCS explains the observed complex particle trajectories in the force field and the accumulation of particles in well-de fined regions above the microelectrode array. In the second part of this thesis, the mechanism of RNA interference (RNAi) was studied by dual-color FCCS in vivo. RNAi is an evolutionary conserved gene silencing mechanism, which uses short double-stranded RNA molecules, called short interfering RNAs (siRNAs), as effector molecules. Due to its speci city and simplicity, RNAi yields a great potential for a widespread therapeutic use. To broaden the therapeutic applications, the in vivo stability of siRNAs has to be improved by chemical modi cations, but some of these modi fications inhibit the gene silencing mechanism. The presented FCCS assays are very well suited to investigate the individual assembly steps of RNAi machinery with very high specifi city and sensitivity in real time and to study the cleavage activity of the activated RNAi machinery. A direct correlation between activity of the RNAi machinery and the results from the FCCS measurements could be shown. The in fluence of several chemical modi cations on the assembly and activity of the RNAi machinery was investigated with these assays. / Fluoreszenz-Korrelations-Spektroskopie (FCS) analysiert die Fluktuationen im Fluoreszenzsignal eines kleinen angeregten Volumens, um Informationen über die Konzentration, die Bewegung und die Interaktionen der markierten Moleküle zu erhalten. Die Fluoreszenz-Kreuzkorrelations-Spektroskopie (FCCS) erhöht die Genauigkeit bei der Messung von Fließgeschwindigkeiten und Bindungskonstanten im Vergleich zur Standard-FCS. Die Miniaturisierung der Biomedizin und Chemie hat sich rapide entwickelt, angetrieben von der Vision eines kompletten Labors auf einem Chip und dem Einsatz dieses in der medizinischen Therapie, zum Beispiel als implantierter Medikamentenspender. Ein Schlüsselelement zur Erfüllung dieser Vision ist der Transport von kleinsten Flüssigkeitsmengen in diesen miniaturisierten Systemen. Der Transport von Flüssigkeiten mittels des elektrohydrodynamischen Prinzips umgeht viele Nachteile von anderen Systemen, allerdings zeigt eine solche Pumpe ein kompliziertes Strömungsbild in der aktiven Region, welches sich mit herkömmlichen Methoden wie Teilchenverfolgung nicht vermessen ließ. Hier wurde Zwei-Fokus-FCCS (2f-FCCS) genutzt, um das Strömungsbild in der Pumpe zu vermessen. Die hohe Genauigkeit der 2f-FCCS bei der Bestimmung von Fließgeschwindigkeiten auch mit kleinen fluoreszierenden Teilchen ermöglichte die Messung der Fließgeschwindigkeiten, aufgrund der auf das Lösungsmittel wirkenden elektrohydrodynamischen Kräfte, unter Ausschluss der auf größere Teilchen wirkenden dielektrophoretischen Kräfte. Die Analyse der Daten ergab, dass das Strömungsbild aus zwei entgegengesetzt rotierenden unterschiedlich großen Wirbeln besteht. Dieses Strömungsbild erklärt die komplizierten Teilchenbewegungsbahnen und die Anreicherung der Teilchen in klar abgegrenzten Bereichen über den Mikroelektroden. Im zweiten Teil dieser Arbeit wurde der RNAi-Mechanismus in lebenden Zellen mittels Zwei-Farben-FCCS untersucht. RNA Interferenz (RNAi) ist ein evolutionär erhaltener Geninaktivierungsmechanismus, der kurze doppelsträngige RNA Moleküle, so genannte kurze interferierende RNAs (siRNAs), als Effektormoleküle nutzt. Die Spezifi tät und Einfachheit der RNAi hat ihr ein weites Feld in der medikamentösen Therapie geöffnet. Zur Erweiterung dieses Feldes ist es nötig die Stabilität der siRNAs im Körper mittels chemischer Modi fikationen zu erhöhen. Einige dieser Modifikationen hemmen aber den RNAi-Mechanismus. Die hier vorgestellten FCCS Experimente sind sehr gut geeignet, um die einzelnen Schritte des Zusammenbaus der RNAi Maschinerie mit hoher Empfi ndlichkeit und Spezi fität in Echtzeit zu untersuchen und die Aktivität der RNAi Maschinerie zu studieren. Es konnte ein Zusammenhang zwischen der Aktivität der RNAi Maschinerie und den Ergebnissen der FCCS Messungen hergestellt werden. Der Einfluss von verschiedenen chemischen Modikationen auf den Zusammenbau und die Aktivität der RNAi Maschinerie wurde mit diesen neuartigen Methoden untersucht.