MSc (Chemistry) / Department of Chemistry / The computational study of biologically active molecules is particularly important for drug development because it provides crucial information about the properties of a molecule, which determine its biological activities. The current work considers the results of a computational study of myristinin A and a structurally-related molecule (2-(4-hydroxyphenyl)-4-[2,4,6-trihydroxy-3-(9tetradecenoyl)phenyl]-3,4-dihydro-2H-benzopyran-7-ol, here denoted as DBPO). The two compounds pertain to the class of acylphloroglucinols. They were firstly isolated from Horsfieldia amygdaline, and they exhibit a variety of biological activities, including potent anti-inflammatory activity, potent DNA-damaging activity and DNA-polymerase ß inhibition. Their molecular structures differ only by the acyl chain. Both molecules have a bulky substituent meta to the acyl group consisting of a ring system (2-(4-hydroxyphenyl)-3,4-dihydro-2H-chromen-7-ol). The DBPO molecule can exist as cis and trans isomers in relation to the double bond present in the R chain, and both isomers are here investigated individually. The OHs ortho to the acyl group can form an intramolecular hydrogen bond (referred to as the first IHB) with the sp2 O atom of the acyl group. The phenol OHs neighbouring the substituent ring system can form O–Hπ interaction with the aromatic rings of the substituent, if suitable oriented. This study focuses on the identification of the stable conformers of these molecules (considering all the possible geometries obtainable by rotations about relevant single bonds), and of the factors stabilising the conformers. Full-optimisation calculations were performed in vacuo and also in three conveniently selected solvents. The results show that the dominant stabilising factors are the first IHB and the O–Hπ interactions. Other factors which have significant influence on conformational preferences are the orientation of the ring systems of the substituent, the orientation of the OHs on substituent, the mutual orientation of the OHs of the phloroglucinol moiety and also the orientation of the acyl chain. The results in solution are consistent with the findings of other acylphloroglucinols, for instance, the narrowing of the energy gaps and the increase of the dipole moment with the increase of solvent polarity. / NRF

Title: SERS spectroscopy of model biomolecules for SERS biosensing Author: Martin Šubr Department: Institute of Physics of Charles University Supervisor: doc. RNDr. Marek Procházka, Dr., Institute of Physics of Charles University Abstract: The main requirement for surface enhanced Raman scattering (SERS)-based biomolecular sensing is high sensitivity and spectral reproducibility. For this purpose, ordered silver and gold nanostructures fabricated by magnetron sputtering and lithography methods at cooperating institutes were tested in this work. Reproducible SERS spectra of employed model biomolecules (amino acids, lysozyme and albumin) were obtained on ordered silver surfaces at concentrations 10-4 M - 10-6 M and as low as ≈ 10-7 M in the case of porphyrins. SERS spectra of certain biomolecules were also compared to spectra measured on silver colloid. The limit of detection provided by hydroxylamine-reduced silver colloid, using KCl as an aggregating agent, is substantially lower (on the order of 10-8 M for cysteine), but with lower spectral reproducibility. The main drawback of SERS spectra measured on silver surfaces was the occurrence of spurious bands resulting from the preparation procedure. In the case of sputter-deposited silver surfaces, it was found that keeping the substrates several hours in...

Title: Multifunctional biomolecular assemblies for parallelized analysis of biomolecular interactions Author: Markéta Bocková Department / Institute: Institute of Physics, Charles University Supervisor of the doctoral thesis: Prof. Jiří Homola, Ph.D., DSc., Institute of Photonics and Electronics, The Czech Academy of Sciences Abstract: Surface plasmon resonance (SPR) biosensors represent the most advanced optical method for the direct, real-time monitoring of biomolecular interactions without the need for labelling. This doctoral thesis aims to advance the SPR biosensor method and to expand its utility in the investigation of biomolecular interactions. This encompasses activities on two major fronts of SPR biosensor research - immobilization methods and biosensing methodologies. Methods for the immobilization of biomolecules were researched with the aim of enabling the immobilization of a broad range of biomolecules on the SPR biosensor surface in a spatially controlled manner. The development of novel biosensing methodologies was pursued in order to address the current limitations of SPR biosensors associated with non-specific adsorption and limited analyte transport, and thus to improve the accuracy and robustness of SPR biosensor measurements. Finally, advances in the development of immobilization...

Solid-state NMR (ssNMR) has matured into a versatile method to provide structural information, probe protein dynamics and detect small molecule binding and -protein interaction of a variety of biomolecular assemblies including amyloid fibrils, viral particles and membrane proteins. Membrane proteins embedded in liposomes are natural targets for ssNMR as their native states are solids. Magic angle spinning (MAS) ssNMR studies using moderate spinning frequencies provide detailed structural information and probe subtle conformational change. Development of fast magic angle spinning ssNMR enables proton-detection which increases sensitivity and facilitates protein dynamics measurements. In this dissertation, we applied moderate and fast MAS ssNMR to study potassium ion channel and protein dynamics Chapter 1 will introduce concepts and theory of solid-state NMR pulse sequences and experiments. Chapter 2 will discuss the application and perspectives of solid-state NMR to membrane protein systems. In Chapter 3, we test an allostery mechanism for inactivation using a KcsA mutant (H25R/E118A) that exhibits an open pH gate across a broad range of pH values. We present solid-state NMR measurements of this open mutant at neutral pH to probe the affinity for potassium at the selectivity filter. This result strongly supports our assertion that the open pH gate allosterically affects the potassium binding affinity of the selectivity filter. In this mutant the protonation state of a glutamate residue (E120) in the pH sensor is sensitive to potassium binding, suggesting that this mutant also has flexibility in the activation gate and is subject to transmembrane allostery. In Chapter 4, I optimize protein expression, purification and reconstitution into native environment protocols of a bacterial potassium transporter, KtrB. In chapter 5, methods and experimental details of setting up 60 and 40 kHz fast MAS ssNMR are discussed. With fast MAS ssNMR setup, multidimensional NMR experiments with higher sensitivity could be collected on a perdeuterated sample with less sample mass required. In Chapter 6, we employ fast MAS ssNMR to measure bulk and residue site-specific 15N and carbonyl 13C relaxation of microcrystalline ubiquitin. Carbonyl R1ρ relaxation profiles provide additional information on protein backbone dynamics.

The conformational dynamics of RNA are crucial to its role in numerous essential biological functions, requiring a comprehensive view of masses of individual molecular motions in order to fully understand these processes. Obtaining such a unified view, however, presents many challenges. Even the simplest RNA structures undergo rearrangements within an intricate three-dimensional network of secondary and tertiary interactions, resulting in motions that span a broad range of timescales. This complexity gives rise to a large number of experimental techniques sampling different aspects of the folding process, leaving a rather fragmented picture of RNA folding overall. In order to address the divergence and limitations of existing ensemble and single-molecule methods, this work describes the application of carbon nanotube (CNT)-based single-molecule field-effect transistors (smFET) as a platform for studying the folding and unfolding dynamics of RNA. smFET is capable of measuring individual biomolecular dynamics for long durations, while at a sufficiently high time resolution to capture the relevant timescales for RNA folding. This technique, moreover, avoids potential sources of interference or damage to the molecule as found in other available methods, and capitalizes on the detailed information that can be garnered from studying a single molecule as opposed to an ensemble. By taking advantage of the highly sensitive electronic properties and nanoscale dimensions of CNTs, and tethering a comparably sized and charged single biomolecule like RNA, it is possible to monitor the folding and unfolding of the molecule and characterize the kinetics associated with these motions. This thesis describes the optimization and application of such smFET technology to RNA stem-loops, which are extremely prevalent and thermodynamically stable elements of RNA secondary structure. Chapter 1 introduces the biological context of these molecules as well as the mechanisms of CNT-based smFET sensing. In Chapter 2, the methods used to fabricate smFET devices are described along with the experiments conducted to optimize single-molecule tethering. These methods and protocols were then applied to the studies detailed in Chapter 3, which examines the implications of the thermodynamic and kinetic analyses of RNA stem-loop folding and unfolding as investigated with smFET. Chapter 4 concludes with a brief overview of what has been accomplished and potential future directions for this platform. The work expressed here thus presents a cohesive view of RNA stem-loop folding, integrating the past results of both experimental and computational studies. With this improved smFET methodology, this technique can be applied to many other essential and increasingly complex biological systems to achieve a fuller and richer understanding of the processes that govern life.

The ability to identify and confirm a compound using mass spectrometry usually involves time consuming sample preparation and method development. The open-air ion source DART (Direct Analysis in Real Time) can ionize compounds in the gas, solid, or liquid phase without chromatography or sample preparation due to the interactions of helium metastable atoms with gas molecules commonly found in air. The coupling of the DART to a time-of-flight (TOF) mass spectrometer allows the rapid determination of an analyte's elemental composition based on accurate mass measurement and isotope peak intensities. Mass spectrometric fragmentation data can aid in the structural identification of an analyte as compounds produce characteristic fragment-ions based on their structure. The TOP's ability to produce fragmentation spectra was compared to the more traditional tandem mass spectral method (MS/MS) considering the TOF lacks the ability to select pre-cursor ions. The TOF produced in-source CAD (collisionally activated dissociation) spectra comparable to MS/MS spectra for three well known pharmaceuticals acetaminophen, phenylbutazone and clenbuterol. Further structural confirmation was explored through a determination of the number of active hydrogen atoms in an analyte molecule achieved by hydrogen/deuterium (H/D) exchange by treatment with deuterium oxide (D20) in the DART sample gap. Mass spectra acquired in the presence of D20 of analytes containing active hydrogen atoms associated with hydroxyl, amino and carboxylic acid groups showed that H/D exchange was predictable and reproducible. Using accurate mass measurement and isotope peak intensities, the elemental composition of an unknown captured on filter paper was identified as dipropylene glycol (DPG) analyzed directly from the surface of the filter paper. Data from in-source CAD and H/D exchange of both the unknown and authentic standards confirmed that the unknown was DPG. The cross-correlation of accurate mass measurement and isotope peak intensities, in-source CAD and HID exchange data provided an unambiguous identification of the contaminant melamine in dog food without the need for any sample preparation. Once analytes are identified and confirmed, quantitation of the analyte is desirable. The calibration curves here are constructed using the net extracted ion-current associated with the analyte relative to the internal standard. In cough syrup, a complicated matrix, the linearity, R2, is shown to be 0.992.

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Biomolecular machines are responsible for carrying out a host of essential cellular processes. In accordance to the wide range of functions they execute, the architectures of these also vary greatly. Yet, despite this diversity in both structure and function, they have some common characteristics. They are all large macromolecular complexes that enact multiple steps during the course of their functions. They are also ’Brownian’ in nature, i.e., they rectify the thermal motions of their surroundings into work. Yet how these machines can utilise their surrounding thermal energy in a directional manner, and do so in a cycle over and over again, is still not well understood. The work I present in this thesis spans the development, evaluation and use of biophysical, in particular single-molecule, tools in the study of the functional mechanisms of biomolecular machines. In Chapter 2, I describe a mathematical framework which utilises both the framework of Bayesian inference to relate any experimental data to an ideal template irrespective of the scale, background and noise in the data. This framework may be used for the analysis of data generated by multiple experimental techniques in an accurate, fast, and human-independent manner. One such application is described in Chapter 3, where this framework is used to evaluate the extent of spatial information present in experimental data generated using cryogenic electron microscopy (cryoEM). This application will not only aid the study of biomolecular structure using cryoEM by structural biologists, but also enable biophysicists and biochemists who use structural models to interpret and design their experiments to evaluate the cryoEM data they need to use for their investigations. In Chapter 4, I describe an investigation into the use of one class of analytical models, hidden Markov models (HMMs) to accurately extract kinetic information from single-molecule experimental data, such as the data generated by single-molecule fluorescence resonance energy transfer (smFRET) experiments. Finally in Chapter 5, I describe how single-molecule experiments have led to the discovery of a mechanism by which ligands can modulate and drive the conformational dynamics of the ribosome in a manner that facilitates ribosome-catalysed protein synthesis. This mechanism has implications to our understanding of the functional mechanisms of the ribosome in particular, and of biomolecular machines in general.

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