Towards the Understanding of Fluorescence Quenching Mechanisms : Molecular Dynamics Simulations of Dye-Quencher Interactions in Biomolecular Systems

The presence of antibodies directed against p53 in human blood serum is a specific and independent marker of cancer. The present thesis is a simulative study of two fluorescent dyes (rhodamine 6G and MR121) used in the development of fluorescence-based immunoassays for the detection of p53 antibodies. The "selective" fluorescence quenching property of tryptophan residues present in dye-conjugated peptide chains enables monitoring of conformational dynamics and antibody binding events by means of fluorescence spectroscopy. The molecular mechanisms of the quenching of fluorescent dyes are mostly unknown. Here MD simulations are used in combination with existing results from ensemble fluorescence experiments in order to obtain predictive theoretical insight into dye/quencher interactions. A new automated refinement method was developed for deriving reliable molecular mechanics force field parameters for small- to medium-sized molecules using reference data from high level ab initio quantum chemical calculations. Using this method CHARMM force field parameters for the two dyes were derived. The parameters were then used to perform MD simulations on two simplified, but realistic dye quencher systems: MR121/TRP and R6G/TRP. Results of these simulations have given important insight on the bimolecular interactions between the dyes and the quencher. A quantitative interpretation of the quenching mechanism based on it’s dependence on dye/quencher distance and orientation has emerged. These results were then applied to the interpretation of simulation data of an epitope from the tumor suppressor protein p53 which was labelled at the N-terminus first with MR121 and then with R6G. In all phases of the thesis care was taken to confront and/or combine the theoretical results with available experimental data

Impact of crystal structure of silica nanoparticles on Rhodamine 6G adsorption : a molecular dynamics study

Understanding the mechanism of adsorption of Rhodamine 6G (R6G) to various crystal structures of silica nanoparticles (SNPs) is important to elucidate the impact of the dye size when measuring the size of the dye-SNP complex via the time-resolved fluorescence anisotropy method. In this work, molecular dynamics (MD) simulations were used to get an insight into the R6G adsorption process, which cannot be observed using experimental methods. It was found that at low pH α-Cristobalite structured SNPs have a strong affinity to R6G, however at high pH more surface silanol groups undergo ionization when compared with α-Quartz, preventing the adsorption. Therefore, α-Quartz structured SNPs are more suitable for R6G adsorption at high pH, than α-Cristobalite ones. Furthermore, it was found that stable adsorption can occur only when the R6G xanthene core is oriented flat with respect to the SNP surface, indicating that the dye size does not contribute significantly to the measured size of the dye-SNP complex. The requirement of correct dipole moment orientation indicates that only one R6G molecule can adsorb on any size SNP and the R6G layer formation on SNP is not possible. Moreover, the dimerization process of R6G and its competition with the adsorption has been explored. It has been shown that the highest stable R6G aggregate is a dimer, in this form R6G does not adsorb to the SNPs. Finally, using Steered Molecular Dynamics (SMD) with constant velocity pulling, the binding energies of R6G dimers and R6G complexes with both α-Quartz and α-Cristobalite SNPs of 40 Å diameter were estimated. These confirm that R6G adsorption is most stable on 40 Å α-Quartz at pH7, although dimerization is equally possible

Orientation of Fluorescent Lipid Analog BODIPY-PC to Probe Lipid Membrane Properties: Insights from Molecular Dynamics Simulations

Single-molecule fluorescence measurements have been used to characterize membrane properties, and recently showed a linear evolution of the fluorescent lipid analog BODIPY-PC towards small tilt angles in Langmuir-Blodgett monolayers as the lateral surface pressure is increased. In this work, we have performed comparative molecular dynamics (MD) simulations of BODIPY-PC in DPPC (dipalmitoylphosphatidylcholine) monolayers and bilayers at three surface pressures (3, 10, and 40 mN/m) to explore 1) the microscopic correspondence between monolayer and bilayer structures, 2) the fluorophore’s position within the membrane, and 3) the microscopic driving forces governing the fluorophore’s tilting. The MD simulations reveal very close agreement between the monolayer and bilayer systems in terms of the fluorophore’s orientation and lipid chain order, suggesting that monolayer experiments can be used to approximate bilayer systems. The simulations capture the trend of reduced tilt angle of the fluorophore with increasing surface pressure as seen in the experimental results, and provide detailed insights into fluorophore location and orientation, not obtainable in the experiments. The simulations also reveal that the enthalpic contribution is dominant at 40 mN/m resulting in smaller tilt angles of the fluorophore, and the entropy contribution is dominant at lower pressures resulting in larger tilt angles

Ensemble-Based Coarse-Grained Molecular Dynamics Simulations of Multifunctional DNA Nanopores

Transmembrane pores are highly specialised nano-devices, with intrinsic specificity and gate-keeping properties that can be exploited in the field of nanobiotechnology. Recently, DNA-origami inspired transmembrane pores with tailorable surface chemistry and programmable dimensions have been rationally designed in an effort to overcome the limitations of protein-based membrane pores such as their fixed lumen size and limited structural repertoire. Ongoing experimental research into the potential applications of triethylene glycol-cholesterol DNA nanopores (DNPs) has been fruitful, with a particular emphasis on drug delivery and biosensing. In this thesis, I describe an ensemble-based coarse-grained MD protocol devised to probe the interactions between bilayer lipids and DNPs, and to determine the effect of membrane encapsulation and salt concentration on the dynamics, structure and conductance of these nanopores. Furthermore, I aim to elucidate the mechanisms by which DNPs mediate translocation of small molecules across lipid bilayers, and the energetics associated with these mechanisms with constant-velocity steered MD and umbrella sampling simulations. I have found that the DNP has no distinct lumen in bulk solution, where it adopts a bloated, amorphous structure with strained and constricted termini regardless of the salt conditions, with significant kinking and fraying of helices. However, salt conditions have a profound effect on the structure of a DNP as it spans a planar lipid bilayer, where it assumes a barrel-like structure with a defined lumen. Sites of constriction in the lumen of the membrane-spanning DNP present a significant barrier to translocation of fluorophores bearing dense negative charges

Fluorescence Spectroscopy in Structural Studies of Plant Cell Walls

Plant cell walls represent the most abundant, renewable and biodegradable composite on Earth. Its highly complex structure consists mainly of three organic compounds: cellulose, hemicelluloses, and lignin. Cell walls have wide applications in different industries, especially for biofuels and biomaterials. Fluorescence spectroscopy is the method allowing investigation of cell wall structure thought monitoring of lignin autoflorescence and thus interactions of lignin with the other cell wall constituents. Deconvolution of fluorescence spectra reveals the number and location of spectral component peaks by calculation of the approximation of the probability density (APD) of component positions. A characteristic of complex CW fluorescence is that the emission spectrum contains multiple log–normal components originating from different fluorophores, shorter wavelengths corresponding to phenolic structures and longer wavelengths to conjugated structures in lignin. Fluorescence spectroscopy has been used for fast screening of the cell wall properties from plants of different origin (hardwood, softwood and herbaceous plant), that may be important for selection of plants for possible applications. Fluorescence spectroscopy may be applicable in the investigation of the effect of stress on the cell wall. Lignin fluorescence emission spectra, peak intensities and shifts in the positions of the long-wavelength spectral components may be indicators of changes in cell wall structure during the stress. There is an increasing application of quantum dots (QDs) in plant science, as fluorescent markers. The isolated cell wall is an appropriate object for study of the interactions with nanoparticles. The results of different physico-chemical techniques including fluorescence spectroscopy combined with spectral deconvolution, show that in the cell walls, CdSe QDs predominantly bind to cellulose, via OH groups, and to lignin, via the conjugated C=C/C–C chains. Variability of bond types in lignin is related to the involvement of this polymer in plant response to various types of stress, by introducing local structural modifications in the cell wall. Different lignin model compounds have been used in order to reveal spectroscopic properties of lignin. Lignin model polymers were synthesized from three monomers, coniferyl alcohol, ferulic acid and p-coumaric acid mixed in various ratios, simulating lignin synthesis in the real cell walls. Further, by using fluorescence spectroscopy and appropriate mathematical methods, it is possible to get deeper insight into the structural characteristics of the molecule. Future investigations will be based on synthetic cell walls and on variation in a portion of all three main components: cellulose, hemicelluloses and lignin, also having in mind results of fine structural modifications in lignin model compounds

Fluorescence Methods for Investigation of Living Cells and Microorganisms

Fluorescence methods play a leading role in the investigation of biological objects. They are the only non-destructive methods for investigating living cells and microorganisms in vivo. Using intrinsic and artificial fluorescence methods provides deep insight into mechanisms underlying physiological and biochemical processes. This book covers a wide range of modern methods involved in experimental biology. It illustrates the use of fluorescence microscopy and spectroscopy, confocal laser scanning microscopy, flow cytometry, delayed fluorescence, pulse-amplitude-modulation fluorometry, and fluorescent dye staining protocols. This book provides an overview of practical and theoretical aspects of fluorescence methods and their successful application in the investigation of static and dynamic processes in living cells and microorganisms

Toward Hydrogenase mimicry : subjecting the problem to three different approaches

In this work, the challenging task of modelling Hydrogenase is subjected to three different approaches. The first strategy used here is bioorganometallic. A wide range of sulfur containing ferrocene-peptide derivatives were synthesized and fully characterized. As a second approach, organic self-assembled oligoquinoline were designed and synthesized. These two kind of structures were further used a scaffold for Hydrogenase mimics. In the third approach, a theoretical computational study of the ferrocene-peptide derivatives is led. In order to investigate a wider range of ferrocene-peptide as a molecular scaffold, a molecular force field was successfully implemented and validated for CHARMM. After the deprotection of the thiol group in the bioorganometallic approach, the free SH groups were coordinated with iron-carbonyl, so as to mimic the Fe-only Hydrogenase active site. The complexes thus obtained were comprehensively characterized and their electronic and electrochemical properties were extensively studied

Toward hydrogenase mimicry : subjecting the problem to three different approaches

In this work, the challenging task of modelling Hydrogenase is subjected to three different approaches. The first strategy used here is bioorganometallic. A wide range of sulfur containing ferrocene-peptide derivatives were synthesized and fully characterized. A new class of structural mimic of Fe-only hydrogenase with ferrocene-peptide backbone was obtained. In the second approach, a theoretical computational study of the ferrocene peptide derivatives is led. In order to investigate a wider range of ferrocene-peptide as a molecular scaffold, a molecular force field was successfully implemented in CHARMM27. As a third approach, organic self-assembled oligoquinoline were studied as potential scaffold of hydrogenase mimics

Experimental Methods in Cryogenic Spectroscopy: Stark Effect Measurements in Substituted Myoglobin

Dawning from well-defined tertiary structure, the active regions of enzymatic proteins exist as specifically tailored electrostatic microenvironments capable of facilitating chemical interaction. The specific influence these charge distributions have on ligand binding dynamics, and their impact on specificity, reactivity, and biological functionality, have yet to be fully understood. A quantitative determination of these intrinsic fields would offer insight towards the mechanistic aspects of protein functionality. This work seeks to investigate the internal molecular electric fields that are present at the oxygen binding site of myoglobin. Experiments are performed at 1 K on samples located within a glassy matrix, using the high-resolution technique spectral hole-burning. The internal electric field distributions can be explored by implementing a unique mathematical treatment for analyzing the effect that externally applied electric fields have on the spectral hole profiles. Precise control of the light field, the temperature, and the externally applied electric field at the site of the sample is crucial. Experimentally, the functionality of custom cryogenic temperature confocal scanning microscope was extended to allow for collection of imaging and spectral data with the ability to modulate the polarization of the light at the sample. Operation of the instrumentation was integrated into a platform allowing for seamless execution of input commands with high temporal inter-instrument resolution for collection of data streams. For the regulated control and cycling of the sample temperature. the thermal characteristics of the research Dewar were theoretically modeled to systematically predict heat flows throughout the system. A high voltage feedthrough for delivering voltages of up to 5000 V to the sample as positioned within the Dewar was developed. The burning of spectral holes with this particular experimental setup is highly repeatable. The quantum mechanical treatment that is employed during analysis of the experimental data requires the state energies and the transition dipole moments of the porphyrin probe. The configuration interaction, as well as the coupled-cluster approaches, have been investigated for their ability to produce realistic valuations for these calculated quantities as gauged by their ability to accurately reproduce valuations for spectroscopically observable transition energies. A capacitive cell, for the determination of a material’s dielectric permittivity, necessary for defining the magnitude of the externally applied electric field at the sample, was developed and shown to successfully yield permittivity valuations for various media in accordance with those reported the literature, while offering the ability to provide measures for permittivities over the temperature range of 1-300 K