Two-Photon Polarization Dependent Spectroscopy in Chirality: A Novel Experimental-Theoretical Approach to Study Optically Active Systems

Many phenomena, including life itself and its biochemical foundations are fundamentally rooted in chirality. Combinatorial methodologies for catalyst discovery and optimization remain an invaluable tool for gaining access to enantiomerically pure compounds in the development of pharmaceuticals, agrochemicals, and flavors. Some exotic metamaterials exhibiting negative refractive index at optical frequencies are based on chiral structures. Chiroptical activity is commonly quantified in terms of circular dichroism (CD) and optical rotatory dispersion (ORD). However, the linear nature of these effects limits their application in the far and near-UV region in highly absorbing and scattering biological systems. In order to surmount this barrier, in recent years we made important advancements on a novel non linear, low-scatter, long-wavelength CD approach called two-photon absorption circular dichroism (TPACD). Herein we present a descriptive analysis of the optics principles behind the experimental measurement of TPACD, i.e., the double L-scan technique, and its significance using pulsed lasers. We also make an instructive examination and discuss the reliability of our theoretical-computational approach, which uses modern analytical response theory, within a Time-Dependent Density Functional Theory (TD-DFT) approach. In order to illustrate the potential of this novel spectroscopic tool, we first present the experimental and theoretical results obtained in C(2)-symmetric, axially chiral R-(+)-1,1\u27-bi(2-naphthol), R- BINOL, a molecule studied at the beginning of our investigation in this field. Next, we reveal some preliminary results obtained for (R)-3,3\u27-diphenyl- 2,2\u27-bi-1-naphthol, R-VANOL, and (R)-2,2\u27-diphenyl-3,3\u27-( 4-biphenanthrol), R-VAPOL. This family of optically active compounds has been proven to be a suitable model for the structure-property relationship study of TPACD, because its members are highly conjugated yet photo-stable, and easily derivatized at the 5- and 6-positions. With the publication of these outcomes we hope to motivate more members of the scientist community to engage in state-of-the-art TPACD spectroscopy

The Design of Heteromeric and Metal-binding Alpha-Helical Barrels

Introduction: The field of protein design has drastically evolved over the past four years. Both the protein folding problem, which involves predicting the 3D arrangement of atoms from a given sequence of amino acids, and its inverse, have been technically solved after 50 years. However, the black box nature of the tools developed to address these problems limits our comprehension of protein folding and dynamics. Harnessing this knowledge could revolutionise sectors such as drug design, disease diagnosis, energy transfer, and material science. This work focuses on the rational design of a protein scaffold called coiled coils, positioning them as a model for advancing our control and understanding of proteins.Results: In this thesis, we navigate the uncharted territory of coiled coils with reduced symmetry. We generate novel A3B3 hexameric α-helical barrels with both parallel and antiparallel helix orientations, expanding understanding of coiled-coil assemblies and introducing new scaffolds. Utilising these assemblies, we create covalently attached bipyridyl functional groups situated within the barrel cores, capable of chelating iron and ruthenium ions. Additionally, we develop intrinsically disordered peptide sequences that assemble only upon the introduction of specific metal ions. This can be applied for both metal sensing, as well as metal mediated sensing of other ligands.Conclusions: This research advances the field of protein design through the generation of novel α-helical barrels and the development of coiled-coil assemblies with innovative functionalities. Our work has allowed for new potential applications in bio-sensing and catalysis and has further demonstrated the broad versatility of coiled-coil scaffolds.Implications: This study illuminates the potential of coiled coils in the understanding of protein structure-function relationships. It introduces metal-sensitive peptide sequences for bio-sensing and photocatalysis within α-helical barrels, potentially paving the way for advancements in applications for de novo designed proteins

Engineering a genetic circuit for Turing patterns in E. coli with a Synthetic Biology approach

Genetic circuits that can form spatial patterns have been a major topic of interest within Synthetic Biology. Turing patterns are self-organising spatial wave, spot or labyrinthine patterns that are formed in some reaction-diffusion circuits. The simplest Turing circuit involves a slow-diffusing activator and a fast-diffusing inhibitor, interacting to regulate their own and each other’s rates of production. An unambiguous implementation of Turing patterns with a genetic circuit is still lacking because of their exquisitely fine-tuned nature. This study aims to address this shortcoming and sets out to engineer a genetic circuit for Turing patterning in E. coli from first principles. Two genetic circuits were studied. Firstly, a phage circuit was designed according to the minimal self-activation, lateral inhibition Turing topology and involves a slow-diffusing M13 filamentous phage and a fast-diffusing 3OC6HSL quorum sensing signal. This circuit was abandoned because of the many complexities of phage biology, which were working against its successful implementation as a Turing generator. The focus was shifted to circuit ‘3954’, which was designed according to a more robust three-node topology and implemented with two small molecule diffusors; this could be done because the circuit allows for equal diffusivity of the two diffusing signals. All the components of circuit ‘3954’ were tested in reduced subcircuits and were shown to be functioning as expected. Growing bacterial colonies bearing the circuit were then visualised for pattern formation using confocal microscopy. Even though no Turing patterns were detected, the colonies consistently showed a centre-surround expression pattern of the fluorescence reporters, where GFP was expressed at the colony centre, whereas mCherry was predominantly expressed at the periphery. The obtained reaction-diffusion patterns are a good foundation for further tuning and exploration.Open Acces

Probing Ultrafast Dynamics of Bacterial Reaction Centers Using Two-Dimensional Electronic Spectroscopy

In the initial steps of photosynthesis, solar energy is converted to stable charge separated states with high efficiency. Understanding the relationship between structure and function in the photosynthetic reaction centers where these conversion steps take place could guide the development of more efficient artificial light harvesting systems. Reaction centers are complicated pigment-protein complexes with multiple spectrally overlapped absorption bands, making interpretation of spectroscopic data challenging. The sub-picosecond time scales involved in the energy transfer and charge separation processes present another challenge. Two-dimensional electronic spectroscopy (2DES) has proven to be a powerful tool for disentangling features in spectrally congested systems like reaction centers by resolving the optical response with respect to excitation and detection frequencies. 2DES also obtains the excitation frequency dependence without sacrificing time resolution, which is necessary to resolve energy transfer processes in reaction centers occurring on time scales faster than 100fs. We perform 2DES on bacterial reaction centers (BRCs) from the purple bacterium Rhodobacter capsulatus, using a degenerate optical parametric amplifier producing 12fs pulses with bandwidth spanning the broad near-IR absorption bands of the BRC. The 2D spectra are analyzed using several global analysis methods to extract the underlying energy transfer and charge separation kinetics, and we compare the results to published transient absorption studies on BRCs. Commonly used 2DES global analysis techniques proved inadequate for resolving specific branched and parallel reaction mechanisms. We developed an improved 2D kinetic fitting approach which employs a common set of basis spectra for all excitation frequencies, and uses information from the linear absorption spectrum and BRC structure to model the excitation frequency dependence of the 2D spectrum. Using the improved fitting method, we show that the entire time-dependent 2D spectrum is well-represented by a sequential reaction scheme with a single charge-separation pathway. We tested several proposed alternative reaction schemes involving branched charge separation pathways, and did not find compelling evidence from our data that favors a particular branched model. Based on this analysis, we conclude that our data supports the simpler, single pathway charge separation model.PHDPhysicsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/138758/1/aniedrin_1.pd

Atomistic Simulations of Model Amyloid Beta Aggregates, Water Networks and their Optical Properties

Based on the amyloid hypothesis, amyloid oligomers and the fibrils that they aggregate into, have been implicated in neurodegenerative diseases. Most of these amyloid proteins live in a solvent environment. The role of solvent in modulating the structural and dynamical properties of amyloid proteins remains poorly understood. In this thesis, computer simulations are used to reveal the structural properties of the amyloid protein and the coupling between protein and water using model systems. After assessing the validity of the force fields by comparison with high-level quantum chemistry calculations, we examine further the conformational free energy landscape of an amyloid protein. Different conformations characterized in the free energy surface are driven by internal protein interactions as well as interactions between protein and water, resulting in the collective reorganization of protein and water hydrogen bond networks. We show that these proteins are surrounded by water wires that add a roughness to the free energy surface. To better understand the water hydrogen bond network and particularly the water wires around protein, we used data-science algorithms allowing for the dimensionality and free energy landscape of different water coordinates to be determined. These results confirm that using water wire coordinates encodes more information on the underlying secondary structure of the protein. Finally, ab initio calculations are used to investigate the optical properties of amyloid proteins to help rationalize recent experiments suggesting the intrinsic fluorescence in fibrils that can occur without aromatic residues

Two dimensional infrared four wave mixing spectroscopy of simple molecules, peptides and proteins

In this thesis, measurements of electron-vibration-vibration two dimensional infrared (EVV 2DIR) spectra are demonstrated for the first time from aliphatic, aromatic and amide containing compounds. Bioanalytical applications of EVV 2DIR spectroscopy are explored and the first EVV 2DIR measurements on proteins reported. Operational protocols for collecting EVV 2DIR spectra are described and it is shown for the case of benzene Fermi resonances how excitation pulse ordering can be used to isolate different EVV coherence pathways, giving signals that report on several aspects of a molecule’s electrical and mechanical anharmonicity. Experimental spectra are compared with first principles simulations and in general found to be in good agreement with one another. It is shown that inter-pulse delays can substantially clean up frequency domain spectra and for the range of compounds studied, the 2DIR spectra are significantly decongested compared with their 1D infrared and Raman counterparts. EVV 2DIR coherence lifetime measurements are reported and applied to a range of simple organic compounds. Measurements of exponential dephasing, nonexponential dephasing and quantum beating are demonstrated, with the exponential decays used to accurately measure vibrational linewidths and the quantum beats to measure line splittings

Computation of optical properties of chromophores in different environments using QM/MM methods

Die theoretische Beschreibung der Wechselwirkung zwischen Molekülen und Licht kann herausfordernd sein, insbesondere dann, wenn es sich um flexible Farbstoffe in einer komplexen und dynamischen Umgebung handelt. Obgleich quantenmechanische (QM) Methoden den angeregten Zustand eines Moleküls beschreiben können, sind sie zu rechenaufwändig, um strukturelle Fluktuationen simulieren zu können. Darüber hinaus ist die mögliche Systemgröße, die beschrieben werden kann, durch die Rechenkosten begrenzt. Aus diesem Grund kommen für die Untersuchung von Farbstoffen in Proteinumgebung semiempirische und Multiskalenansätze ins Spiel. Die semiempirische Time-Dependent Long-range Corrected Density Functional Tight Binding (TD-LC-DFTB2) Methode wurde als effiziente Alternative zu ab initio Methoden oder der Dichtefunktionaltheorie in Bezug auf Geometrien im angeregten Zustand und Anregungsenergien getestet. Sie wurde in QM/MM Simulationen angewandt, in denen sie einen angeregten Fluorophor beschrieb, dessen Umgebung von einem klassischen Kraftfeld beschrieben wurde. Diese neue Strategie für die Untersuchung von Fluoreszenz wurde sorgfältig anhand von Literaturergebnissen bewertet, indem die Ergebnisse sowohl mit experimentellen als auch mit theoretischen Studien, die auf anderen Ansätzen basieren, verglichen wurden. Es wurde herausgefunden, dass TD-LC-DFTB2 im Allgemeinen Geometrien und Anregungsenergien von ausreichender Qualität liefert, aber es wurden auch einige Schwächen entdeckt. Außerdem wurde ein optischer Glukosesensor untersucht, der aus dem Glukosebindeprotein und einem angefügten Fluorophor besteht. Mit Hilfe von klassischen Molekulardynamiksimulationen (MD Simulationen) konnten Zusammenhänge zwischen der Anwesenheit von Glukose, den Proteinkonformationen und dem Aufenthaltsort des Farbstoffs gefunden werden. Daraus ergab sich ein starker Hinweis auf die Funktionsweise des Sensors. Schließlich wurde der Energietransfer in einem Pigment-Protein-Komplex untersucht. Der Fenna-Matthews-Olson-Komplex von Photosynthese betreibenden grünen Schwefelbakterien beinhaltet mehrere Bakteriochlorophyll a -- Pigmente in seinem Proteingerüst. Diese leiten die im Chlorosome gesammelte Anregungsenergie mit erstaunlicher Effizienz zum Reaktionszentrum weiter. Es wird Vorarbeit für eine Simulation der Exzitonenpropagation durch den Komplex gezeigt. Anregungsenergien und die Kopplungen zwischen den Pigmenten, das heißt die Elemente des exzitonischen Hamiltonoperators, wurden mit TD-LC-DFTB2 für Strukturen aus klassischen MD Simulationen berechnet. Dadurch wurde ein Eindruck zu deren Entwicklung über die Zeit und den Einfluss der Proteinumgebung gewonnen. Weiterhin wurden diese Daten genutzt, um neuronale Netze zu trainieren, die Anregungsenergien und Kopplungen noch schneller als TD-LC-DFTB2 vorhersagen können

Theoretical Investigation on the Biomolecular Systems using Multiscale Modelling

Die Untersuchung von Protein-Ligand-Wechselwirkungen ist für biomolekulare Systeme von entscheidender Bedeutung und eine Herausforderung. Insbesondere haben traditionelle Laborexperimente oft Schwierigkeiten, die Mechanismen der Reaktionen zu erklären, während klassische theoretische Berechnungsmethoden Defizite im Umgang mit der System- und Zeitskala biomolekularer Systeme aufweisen. In dieser Arbeit werden sogenannte enhanced Sampling-Methoden auf der Grundlage von Molekulardynamiksimulationen (MD) und Algorithmen für künstliche neuronale Netze (ANN), die auf semi-empirischen quantenmechanischen (QM) Ansätzen beruhen, zur Untersuchung verschiedener biomolekularer Systeme eingesetzt. Im ersten Teil wurde die Wirt-Gast-Chemie von [4+4]- und [2+3]-Iminkäfigen untersucht. Bei der Untersuchung von [4+4]-Käfigen wurde der Aufnahmeprozess von unterschiedlich großen Ammoniumionen in Käfigen mit alternativen Volumina durch wohltemperierte Metadynamik (MetaD) simuliert. Es wurden drei mögliche Mechanismen vorgeschlagen, um die Gastaufnahmeprozesse zu erklären. Bei der Untersuchung von [2+3]-Käfigen wurde der Stickstoffmolekültransfer in drei verschiedenen Käfigkristallen mit Funnel-Metadynamik (FM) berechnet. Die erhaltenen freien Energieflächen deuten auf die Existenz von zwei möglichen Wegen hin, auf denen der Stickstofftransfer erfolgen kann. Im zweiten Teil wurde eine neuartige Fluoreszenzsonde auf der Basis eines Glukose bindenden Proteins untersucht. Ein detailliertes molekulares Verständnis der Veränderungen an der Glukosebindestelle aufgrund von Mutationen und deren Auswirkungen auf die Glukosebindung wurde durch MD-Simulationen erreicht. Die Energetik der Dissoziation von Protein und Glukose wurde aufgedeckt und stimmte mit den experimentellen Ergebnissen überein. Schließlich wurde eine Reihe von künstlichen neuronalen Netzen (ANNs) trainiert, um die falsche Darstellung von angeregten Zuständen durch LC-DFTB zu korrigieren, wenn Energieniveaus kreuzen. Die meisten der trainierten Maschinen sind in der Lage, die durch LC-DFTB verursachten Fehler bei der Beschreibung des angeregten Zustands zuverlässig zu korrigieren, während die für Farbstoffgeometrien in Wasser trainierte Maschine weniger genaue Ergebnisse liefert und weiteres Training erfordert