Self-assembly Controls Self-cleavage of HHR from ASBVd (−): a Combined SANS and Modeling Study

International audienceIn the Avocado Sunblotch Viroid (ASBVd: 249-nt) from the Avsunviroidae family, a symmetric rolling-circle replication operates through an autocatalytic mechanism mediated by hammerhead ribozymes (HHR) embedded in both polarity strands. The concatenated multimeric ASBVd (+) and ASBVd (−) RNAs thus generated are processed by cleavage to unit-length where ASBVd (−) self-cleaves with more efficiency. Absolute scale small angle neutron scattering (SANS) revealed a temperature-dependent dimer association in both ASBVd (−) and its derived 79-nt HHR (−). A joint thermodynamic analysis of SANS and catalytic data indicates the rate-determining step corresponds to the dimer/monomer transition. 2D and 3D models of monomeric and dimeric HHR (−) suggest that the inter-molecular contacts stabilizing the dimer (between HI and HII domains) compete with the intra-molecular ones stabilizing the active conformation of the full-length HHR required for an efficient self-cleavage. Similar competing intra- and inter-molecular contacts are proposed in ASBVd (−) though with a remoter region from an extension of the HI domain

R.E.D. Server: a web service for deriving RESP and ESP charges and building force field libraries for new molecules and molecular fragments

R.E.D. Server is a unique, open web service, designed to derive non-polarizable RESP and ESP charges and to build force field libraries for new molecules/molecular fragments. It provides to computational biologists the means to derive rigorously molecular electrostatic potential-based charges embedded in force field libraries that are ready to be used in force field development, charge validation and molecular dynamics simulations. R.E.D. Server interfaces quantum mechanics programs, the RESP program and the latest version of the R.E.D. tools. A two step approach has been developed. The first one consists of preparing P2N file(s) to rigorously define key elements such as atom names, topology and chemical equivalencing needed when building a force field library. Then, P2N files are used to derive RESP or ESP charges embedded in force field libraries in the Tripos mol2 format. In complex cases an entire set of force field libraries or force field topology database is generated. Other features developed in R.E.D. Server include help services, a demonstration, tutorials, frequently asked questions, Jmol-based tools useful to construct PDB input files and parse R.E.D. Server outputs as well as a graphical queuing system allowing any user to check the status of R.E.D. Server jobs

RNA Structural Dynamics As Captured by Molecular Simulations: A Comprehensive Overview

With both catalytic and genetic functions, ribonucleic acid (RNA) is perhaps the most pluripotent chemical species in molecular biology, and its functions are intimately linked to its structure and dynamics. Computer simulations, and in particular atomistic molecular dynamics (MD), allow structural dynamics of biomolecular systems to be investigated with unprecedented temporal and spatial resolution. We here provide a comprehensive overview of the fast-developing field of MD simulations of RNA molecules. We begin with an in-depth, evaluatory coverage of the most fundamental methodological challenges that set the basis for the future development of the field, in particular, the current developments and inherent physical limitations of the atomistic force fields and the recent advances in a broad spectrum of enhanced sampling methods. We also survey the closely related field of coarse-grained modeling of RNA systems. After dealing with the methodological aspects, we provide an exhaustive overview of the available RNA simulation literature, ranging from studies of the smallest RNA oligonucleotides to investigations of the entire ribosome. Our review encompasses tetranucleotides, tetraloops, a number of small RNA motifs, A-helix RNA, kissing-loop complexes, the TAR RNA element, the decoding center and other important regions of the ribosome, as well as assorted others systems. Extended sections are devoted to RNA-ion interactions, ribozymes, riboswitches, and protein/RNA complexes. Our overview is written for as broad of an audience as possible, aiming to provide a much-needed interdisciplinary bridge between computation and experiment, together with a perspective on the future of the field

Structural Characterization of Ribonucleic Acids and Their Complexes by Negative-ion Mode Mass Spectrometry

Ribonucleic acids (RNAs) form complexes with deoxyribonucleic acids, proteins, other RNAs, and smaller ligands. Detailed knowledge of RNA interaction sites provides a basis for understanding functions. With limited analytical techniques available to obtain deeper understanding of RNA structure, negative ion mode mass spectrometry (MS) has the potential to significantly expand RNA primary, secondary, tertiary, and quarternary structure information. This dissertation presents novel MS methods for characterizing RNAs and their complexes. Negative-ion electron capture dissociation (niECD) involves ~3.5-6.5 eV electron irradiation to yield charge-increased intermediates that further undergo radical-driven fragmentation. The proposed niECD mechanism involves gas-phase zwitterionic structures in which nucleobases are protonated and the phosphate backbone is deprotonated. We found that electron-capture efficiency is higher for purine nucleobases compared with pyrimidines and that purine radicals are more stable, presumably because purines have higher proton affinities and can form intramolecular hydrogen bonds. niECD efficiency decreases with increased charge state due to Coulomb repulsion. We show that gas-phase proton-transfer reactions can be combined with niECD for improved performance. Electrospray ionization (ESI) of a model RNA hairpin from native-like (10 mM ammonium acetate) and methanol-containing (up to 50%) solvents resulted in identical charge state distributions, suggesting a minor methanol effect on overall conformation. Experimentally determined collision cross sections (CCSs) for the 5- and 6- charge states of this RNA are smaller (789 Å2 and 830Å2, respectively) than those predicted from the NMR structure. Replica-exchange molecular dynamics showed that these charge states adopt globular collapsed structures due to self-solvation whereas the 7- charge state showed hairpin retention. Higher charge states showed extended structures (higher CCSs). Ligand (e.g., paromomycin) binding assays at varied methanol content resulted in strongest binding at 0% methanol (64+6 nM KD). However the KD remained within one standard deviation up to 50% methanol, suggesting that the binding site is mainly unperturbed in methanol. Assays at varied pHs showed strongest binding at neutral pH. Overall, these data suggest that moderate methanol concentrations, which facilitate ESI, can be tolerated in native RNA MS. Crosslinking techniques coupled with MS provide an alternative tool for identifying RNA interaction sites. We show that collisional activation can provide full sequence coverage of the RNA moiety within non-covalent RNA-peptide complexes; however complexes are disrupted, resulting in loss of site-specific information. By contrast, niECD, in combination with infrared multiphoton dissociation provided sufficient sequence coverage while retaining non-covalent interactions. We also show that IR irradiation at 10.6 µm selectively dissociates RNA-peptide crosslinked species within a peptide mixture due to resonance absorption by phosphate groups, thus allowing identification of such species. Microfluidics is a highly efficient technology for biological analysis. Microfluidic-type approaches, including nano-ESI and nano-LC, coupled with MS provide several advantages, e.g., limited sample consumption and enhanced sensitivity. In order to disseminate microfluidic principles, we developed a 2-week (8 hour) laboratory experiment for an undergraduate analytical chemistry course. Students are introduced to soft lithography concepts by designing/characterizing their own agar-based microfluidic chips. They learn about fluid dynamics by approaching the challenge of mixing in microfluidic channels. By varying solvent viscosity and channel geometries, terms that govern the Reynolds number, students achieve mixing. The optimal chip geometry/solvent condition is used to quantify salicylic acid-/iron (III) complex by colorimetric analysis. Overall, this dissertation describes the utility of MS (and its associated tools) for the study of RNA, RNA-small molecule, and RNA-protein complexes.PHDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/143953/1/ikevin_1.pd

A Mechanistic Study Of The Enzymatic Excision Mechanism In AP Endonuclease (APE1)

DNA with an abasic site is a cyto-toxic intermediate in the base excision repair (BER) pathway that is handled by the enzyme Apuridinic/Apyrimidinic endonuclease (APE1) [99, 56, 168, 90]. Several kinetics and thermodynamics aspects of the mechanism by which the APE1 enzyme processes its abasic DNA substrate have been discussed in this thesis. APE1 is an endonuclease that is it cleaves the DNA backbone at a non-terminal site, here at the abasic site. To obtain eminent insight about the catalytic role of amino acid residues and magnesium ions which are representatively recognized in active sites of endonuclease enzymes, quantum mechanical calculations of reaction pathways based on various cluster models mimicking such active sites of endonuclease enzymes have been performed and subsequently discussed in section 4. In this light our results underline the importance of an enzymatic active site architecture in the catalytic reaction, given the substrate is properly positioned. As a side-effect, we were able to evaluate the semi empirical method DFTB3/3OB [132] by comparison of reaction pathways calculating in different cluster models with the same reaction pathways calculations on the DFT level of theory (B3LYP/6- 31G(d,p)). Comparison of the obtained mechanisms and barriers obtained by DFT and DFTB for the minimalistic (cluster) models may nominate DFTB with reasonable accuracy and computational cost as a potential candidate for quantum method in hybrid QM/MM (quantum mechanics/molecular mechanics) approaches for phosphodiester hydrolysis in an enzymatic environment. However in the “reductionist approach” employed to evaluate multiple plausible types of reaction mechanism, the protein’s flexibility and heterogeneous electrostatic environment of protein residues is not taken into account. To enable reaction pathway caculations of the DNA cleavage mechanism in the full APE1 enzyme a model of a reaction-competent APE1-DNA reactant complex has been built, based on available crystal structure data and mutagenesis experiments from the literature [168, 133, 99, 92]. To remedy the lack of lucid information about structural details of APE1/DNA substrate bounded to Mg2+ ion molecular dynamic simulation together with pKa calculations for important amino acid residues in the active-site of the enzyme have been carried out (see section 5). To investigate the potential effect of metal ion binding on the stabilization of the active site in the Ape1-DNA substrate complex, the number and position of the metal ion(s) have been varied and single point mutations of vital active-site residues in the substrate complex of Ape1-DNA have been simulated. Taken together, the most likely model for an Ape1-DNA substrate complex has one Mg2+-ion located at binding site D and His309 in protonated form. At the D site, the metal ion may play a catalytic role in leaving group departure. This scenario allows Tyr171 and His309 to form hydrogen-bonds to the phosphate group that may help DNA binding as well as stabilizing a pentacovalent transition state/intermediate. Asn212 acts in properly positioning the nucleophile which can then transfer a proton to Asp210. A combined QM/MM approach wherein the active site was treated quantum mechanically and the remaining enzyme classically empowered us to investigate the enzymatic reaction pathways of phosphodiester backbone cleavage by APE1 enzyme and enabled us to depict a more realistic picture of the enzyme’s functions in abasic DNA cleavage (see section 6). Opposed to the “reductionist approach”, the QM/MM approach is accurate enough and computationally efficient to gain electronic level insight into the chemical reaction while taking the protein environment explicitly into account and as such allowed the contribution of individual residues in the enzyme environment to be quantified. To find the energetically most favorable pathway for phosphate hydrolysis catalyzed by APE1 enzyme, several possible reaction mechanisms were initially explored by potential energy calculations. As dynamical effects are important in the reaction progress, free energy calculations have subsequently been performed on selected pathways (based on their potential energy profile). According to our QM/MM calculated enzymatic reaction pathways of phosphodiester backbone cleavage by the APE1 enzyme, the dissociative type of mechanism is the most favorable pathway. Herein an important role is played by a metal ligated water molecule that donates a proton to the leaving group while the cleaved sugar backbone migrates toward the Mg2+ ion. This allows a water molecule, activated by proton transfer to Asp210, to attack the phosphorous atom of abasic DNA as nucleophile. The APE1 enzyme as an indispensable key player in BER has been discussed in atomic resolution in this thesis, Our efforts promote the understanding of catalytic and dynamical features of the APE1 enzyme in the abasic DNA cleavage mechanism, including effects of pH and single-site mutations. The detailed insight thus gained may be helpful in designing inhibitors for APE1 as a potential drug target in cancer chemotherapy.Abasische DNA ist ein cytotoxisches Intermediat des Basenexizions Reparaturmechanismus (BER), welches durch das Enzym apuridinische/apyrimidinische Endonuklease (APE1) [99, 56, 168, 90] weiter prozessiert wird. In dieser These wurden verschiedene kinetische und thermodynamische Aspekte des Mechanismus, durch welchen APE1 Enzyme ihr abasisches DNA Substrat umwandeln, diskutiert. APE1 ist eine Endonuklease, welche das DNA Rückgrat an nicht-terminaler Position spaltet, in diesem Fall an der abasischen Position. Um tiefere Einsicht in die katalytische Funktion von Aminosäure-Seitenketten und Magnesiumionen, welche representativ für das aktive Zentrum von Endonukleasen stehen, zu erhalten, wurden quantummechanische Berechnungen von Reaktionswegen basierend auf verschiedenen Cluster-Modellen, die das aktive Zentrum von Endonukleasen imitieren, durchgeführt und nachfolgend im Abschnitt 4 diskutiert. In diesem Licht unterstreichen unsere Ergebnisse die Relevanz der Architektur des aktiven Zentrums für die katalytische Reaktion, vorausgesetzt das Substrat ist korrekt positioniert. Als Nebeneffekt waren wir in der Lage die semi-empirische Methode DFTB3/3OB durch den Vergleich von Reaktionswegen berechnet in verschiedenen Cluster-Modellen mit gleichen Reaktionsweg-Parametern auf DFT -Level zu evaluieren (B3LYP/6-31G(d,p)). Der Vergleich, der durch DFT und DFTB erhaltenen Mechanismen und Barrieren für die minimalistischen (Cluster) Modelle, nominiert DFTB [132] mit angemessener Genauigkeit und Rechenkosten als potentiellen Kandidaten für quantummechanische Methoden in hybrid QM/MM (Quantummechaniken/ Molekulare Mechaniken) Ansätzen für Phosphodiester-Hydrolyse in enzymatischer Umbegung. Jedoch wird im oft verwendeten „reduktionistischen Ansatz“ zur Evaluierung multipler plausibler Formen von Reaktions-Mechanismen die Fexibilität des Proteins und die heterogene elektrostatische Umgebung der Protein-Seitenketten nicht berücksichtigt. Um Reaktionsweg-Berechnungen des DNASpaltungs-Mechanismus im gesamten APE1 Enzym zu ermöglichen, wurde ein Modell eines Reaktionskompetenten APE1-DNA Edukt-Komplexes gebaut, basierend auf den in der Literatur [168, 133, 99, 92]. vorhandenen Krystallstrukturdaten und Mutagenesis-Experimenten. Um den Fehlen von Informationen über strukturelle Details von APE1/DNA-Substrat gebunden an Mg2+ Ionen entgegenzuwirken, wurden Moleküldynamik-Simulationen zusammen mit pKa Berechnungen für wichtige Aminosäure-Seitenketten im aktiven Zentrum durchgeführt (siehe Abschnitt 5). Zur Untersuchung des potentiellen Effekts der Bindung von Metallionen auf die Stabilisierung des aktiven Zentrum im APE1-DNA Substratkomplex wurde die Anzahl und Position der Metallionen variiert und Punktmutationen wesentlicher Seitenketten des Substratkomplex von APE1-DNA simuliert. Detailierte Analyse dieser Simulationen versorgte uns mit angemessenen Statistiken und Informationen über strukturelle Details des aktiven Zentrums von APE1. Zusammenfassend beiinhalted das wahrscheinlichste Modell für den APE1-DNA Substratkomplex ein Mg2+-Ion lokalisiert an der Bindungsstelle D sowie ein His309 in protonierter Form. And der D Bindungsstelle könnte das Metallion eine katalytische Rolle bei der Abspaltung der Abgangsgruppe einnehmen. Dieses Szenario erlaubt Tyr171 und His309 Wasserstoffbrücken zu den Phosphatgruppen zu formen, welche bei der DNA-Bindung sowie bei der Stabilisierung eines pentakovalenten Überganszustands/ Intermediates behilflich sein könnten. Asn212 spielt bei der korrekten Positionierung des Substrats eine Rolle, welches dann ein Proton auf Asp210 übertragen kann. Ein kombinierter QM/MM Ansatz, in welchem das aktive Zentrum quantummechanisch und das übrige Enzym klassisch betrachtet wurde, ermöglichte uns die enzymatischen Reaktionswege der Phosphodiesterrückgrat-Spaltung durch APE1 zu untersuchen und befähigte uns zu einer realistischeren Abbildung der enzymatischen Funtkionen der abasischen DNA Spaltung (siehe Abschnitt 6). Im Gegensatz zum „reduktionistischen Ansatz“ ist der QM/MM Ansatz präzise genug und rechentechnisch effizient um auf elektorischem Level Einsicht in chemische Reaktionen bei explizieter Berücksichtigung der Proteinumgebung zu erhalten und erlaubt als dieses die Quantifizierung der Beiträge individueller Seitenketten der Enzym-Umgebung. Um den engergetisch favorisierten Reaktionsweg für Phosphathydrolyse katalysiert durch APE1-Enzyme zu indentifizieren, wurden mehrere Reaktionsmechanismen initial durch Potentialenergie-Berechnungen untersucht. Aufgrund der Wichtigkeit dynamischer Effekte für den Reaktionsprozess wurden Berechnungen mit freier Energie stückweise an ausgewählten Reaktionswegen durchgeführt (basierend auf ihrem potentiellen Energieprofil). Zufolge unserer durch QM/MM berechneten enzymatischen Reaktionswege der Phosphodiester RückgratSpaltung durch APE1 Enzyme ist der dissoziative Mechanismus der favorisierte Reaktionsweg. In diesem wird eine wichtige Rolle durch ein Metalliganden gebundenes Wasser-Molekül eingenommen, welches Protonen an die Abgangsgruppe überträgt während das gespaltene Zucker-Rückgrat in Richtung des Mg2+ Ions wandert. Das erlaubt einem Wasser-Molekül, aktiviert durch durch Protonentransfer zu Asp210, als Nukleophil das Phosphoratom der abasischen DNA anzugreifen. Das APE1-Enzym wurde in dieser Thesis als ein unverzichtbarer Schlüsselspieler des BER mit atomarer Auflösung diskutiert. Unsere Bemühungen fördern das Verständnis der katalytischen und dynamischen Eigenschaften des APE1 Enzyms im abasischen DNA Spaltungs-Mechanismus unter Einbeziehung von Effekten durch pH und Punktmutationen. Der hierdurch erhaltene Einblick könnte für das Design von Inhibitoren für APE1 als potentielles Ziel für Therapeutika in der Chemotherapie hilfreich sein

Nucleic Acids at the Mineral Interface - An Origins of Life Study

This thesis details work which employs classical simulation techniques to investigate the interactions of nucleic acid molecules with various charged clay mineral environments. There is a focus on the structure and stability of nucleic acids at mineral interfaces in order to understand how geological settings aided in fostering the first biomolecules at the time of the origins of life on Earth. A comparison of three nucleic acids, DNA, RNA and PNA, shows a difference in preferential stability in bulk water over the corresponding nucleic acid in a mineral environment. The comparative study showed that the prevailing geochemistry preferentially favoured DNA over potentially competing genetic candidates, such as RNA and PNA. This gives us a unique insight into how there may have been a transition from a proto-DNA world (such as the RNA world) to the current DNA/protein world. The structure and arrangement of single-stranded RNA on both cationic and anionic charged surfaces showed marked differences. Both cationic and anionic surfaces successfully adsorb on charged RNA oligonucleotides but show significant differences in the adsorbed structure. Cationic surfaces are successful in mediating the collapse of the RNA sequence from an elongated linear polymer into one that is capable of exhibiting catalytic function. The anionic surface elongates the RNA polymer and exposes the information carrying base sequence to the aqueous region allowing fidelity in templating information and replicating sequences. Studies of single-stranded RNA were extended to model a large biologically relevant RNA ribozyme using replica exchange sampling methods. The results elucidated the structure and arrangement of the catalytic centre of the ribozyme. The results in this thesis show that mineral mediated origins of life differ considerably from an aqueous one that is more commonly associated with the origins of life

The Dynamic Functions Of Bax Are Dependent On Key Structural And Regulatory Features

Bax is an essential mediator of cell fate. Since its discovery in 1985 as a protein that interacts with the anti-apoptotic protein, Bcl-2, key elements related to its function, structure and regulation remains to be determined. To this end, mitochondrial metabolism was examined in non-apoptotic Bax-deficient HCT-116 cells as well as primary hepatocytes from Bax-deficient mice. Although mitochondrial density and mitochondrial DNA content was the same in Bax-containing and Bax -deficient cells, MitoTracker staining patterns differed, suggesting the existence of Bax -dependent functional differences in mitochondrial physiology. Oxygen consumption and cellular ATP levels were reduced in Bax -deficient cells, while glycolysis was increased. These results suggest that cells lacking Bax have a deficiency in the ability to generate ATP through cellular respiration, supported by detection of reduced citrate synthase activity in Bax -deficient cells. Expression of either full length or C-terminal truncated Bax in Bax -deficient cells rescued ATP synthesis and oxygen consumption and reduced glycolytic activity, suggesting that this metabolic function of Bax was not dependent upon its C-terminal helix. Expression of BCL-2 in Bax-containing cells resulted in a subsequent loss of ATP measured, implying that, even under non-apoptotic conditions, an antagonistic interaction exists between the two proteins. Bax is composed of nine alpha-helices. While three of these helices have features of a trans-membrane region, the contribution of each domain to the apoptotic or non-apoptotic functions of Bax remains unknown. To examine this, we focused on the C-terminal alpha-9 helix, an amphipathic domain with putative membrane binding iv properties and discovered that it has an inherent membrane-binding and cytotoxic capacity. A peptide based on the last twenty amino acids (CT20p) of the alpha-9 helix was synthesized and proved a potent inducer of cell death independent of any apoptotic stimuli. The solubility of CT20p allowed it to be encapsulated in polymeric nanoparticles (NPs), and these CT20p-NPs caused the death of colon and breast cancer cells in vitro and induced tumor regression in vivo, using a murine breast cancer tumor model. CT20p caused increased mitochondrial membrane potential followed by cell death via membrane rupture, without the characteristic membrane asymmetry associated with apoptosis. Hence, while CT20p is based on Bax, its innate cytotoxic activity is unlike the parent protein and could be a powerful anti-cancer agent that bypasses drug resistance, can be encapsulated in tumor-targeted nanoparticles and has potential application in combination therapies to activate multiple death pathways in cancer cells. While previous work revealed novel aspects of the biology of Bax that were unrecognized, new structural information is needed to fully elucidate the complexity of Bax’s function. One approach is to use computational modeling to assess the solved structure of Bax and provide insight into the structural components involved in the activity of the protein. Use of molecular dynamics simulators such as GROMACS, as well as other computational tools provides a powerful means by which to test the feasibility of certain modifications in defined parameters. Such work revealed that the removal of the C-terminal alpha-9 helix of Bax, which normally resides within a hydrophobic pocket, significantly destabilized the protein, perhaps explaining how the protein transitions from soluble to membrane-bound form and maintain energy v production via aerobic respiration or, conversely, how the C-terminus helix conveys cytotoxicity. Collectively, this work reveals that Bax is more than an inducer of cell death but has complex activities yet to be determined

Molecular Modeling of Membrane Embedded Proteins

Over the past years, molecular modeling and simulation techniques have had a major impact on experimental life sciences. They are capable of providing accurate insight into microscopic mechanisms, which are usually difficult to investigate experimentally. Moreover, the integration of experimental data with molecular modeling appears to be a promising strategy to better understand complex biological processes. In this thesis, molecular dynamics simulation has been used in combination with experimental data to investigate two transmembrane proteins: (i) the bacterial chemoreceptor PhoQ and (ii) the Amyloid Precursor Protein (APP). (i) Bacterial two-component system PhoQ and bacterial membranes. Two-component systems (TCSs) are signaling complexes essential for bacterial survival and virulence. PhoQ is the histidine kinase chemoreceptor of the PhoQ-PhoP tandem machine that detects the concentration of cationic species at the inner membrane of Gram-negative bacteria. A full understanding of the PhoQ signal transduction mechanism is currently hindered by the lack of a complete atomistic structure. In this thesis project, the first structural model of the transmembrane (TM) portion of PhoQ from Escherichia coli was assembled, by using molecular simulations integrated with cross-linking disulfide scanning data. Its structural and dynamic features induce a concerted displacement of the TM helices at the periplasmic side, which modulates a rotation at the cytoplasmic end. This supports the idea that signal transduction is promoted through a combination of scissoring and rotational movements of the TM helices. Knowledge of this complex mechanism is essential in order to understand how the chemical stimuli sensed by the periplasmic sensor domain trigger, via the relay of the HAMP domain, the histidine auto-phosphorylation and kinase/phosphatase activity at the cytoplasmic end. The PhoQ sensor domain lies in close proximity to the membrane. Interaction with anionic lipids, such as phosphophatidylglycerols (PG) and cardiolipins (CL), are thought to play a key role in PhoQ activity. Present in bacterial and mitochondrial membranes, cardiolipins have a unique dimeric structure, which carries up to two charges, i.e. one per phosphate group, and under physiological conditions, can be unprotonated or singly protonated. Exhaustive models and characterization of cardiolipins are to-date scarce; therefore an ab initio parameterization of cardiolipin species for molecular simulation consistent with commonly used force fields is proposed here. Molecular dynamics (MD) simulations based on these models indicate a protonation-dependent lipid packing. A noteworthy interaction with solvating mono- and divalent cations is also observed. The proposed models will contribute to the biophysical and biochemical characterizations of bacterial and mitochondrial membranes and membrane-embedded proteins. (ii) Structural and dynamic properties of the Amyloid Precursor Protein. The Amyloid Precursor Protein (APP) is a type I membrane glycoprotein present at the neuronal synapsis. The proteolytic cleavage of its C-terminal segment produces amyloid-β (Aβ) peptides of different lengths, the deposition of which is an early indicator of Alzheimer"s disease (AD). Recently, the backbone structure of the APP transmembrane (TM) domain in detergent micelles was determined by nuclear magnetic resonance (NMR, independently by two different experimental groups). The TM conformations of these two structures are however markedly different. One is characterized by a highly kinked α-helix, whereas the other is mainly straight. Molecular dynamics simulations showed that the APP TM region is highly flexible and its secondary structure is influenced by the surrounding lipid environment. The size of the embedding detergent micelles strongly affects the conformation of the APP α-helix, with solvation being the main driving force for the development of a helical curvature. Once embedded in a membrane bilayer, APP systematically prefers a straight helical conformation. This is also confirmed when analyzing in silico the atomistic APP population observed in double electron-electron resonance (DEER) spectroscopy. In summary, the APP transmembrane domain is highly flexible due to the presence of glycine residues and can readily respond to the lipid environment, a property that might be critical for proteolytic processing by γ-secretase enzymes. The presented thesis work clearly shows how molecular simulations and their interplay with available experimental input can help advance the understanding of the mechanism of complex biological systems and processes on a molecular scale. These results, in particular, go well beyond the current understanding of the functioning of two transmembrane proteins relevant for human health. Furthermore, the computational approaches and procedures developed in these projects will hopefully promote novel integrated strategies for investigating biological systems

Diffusion of tin from TEC-8 conductive glass into mesoporous titanium dioxide in dye sensitized solar cells

The photoanode of a dye sensitized solar cell is typically a mesoporous titanium dioxide thin film adhered to a conductive glass plate. In the case of TEC-8 glass, an approximately 500 nm film of tin oxide provides the conductivity of this substrate. During the calcining step of photoanode fabrication, tin diffuses into the titanium dioxide layer. Scanning Electron Microscopy and Electron Dispersion Microscopy are used to analyze quantitatively the diffusion of tin through the photoanode. At temperatures (400 to 600 °C) and times (30 to 90 min) typically employed in the calcinations of titanium dioxide layers for dye sensitized solar cells, tin is observed to diffuse through several micrometers of the photoanode. The transport of tin is reasonably described using Fick\u27s Law of Diffusion through a semi-infinite medium with a fixed tin concentration at the interface. Numerical modeling allows for extraction of mass transport parameters that will be important in assessing the degree to which tin diffusion influences the performance of dye sensitized solar cells

Application of Computational Molecular Biophysics to Problems in Bacterial Chemotaxis

The combination of physics, biology, chemistry, and computer science constitutes the promising field of computational molecular biophysics. This field studies the molecular properties of DNA, protein lipids and biomolecules using computational methods. For this dissertation, I approached four problems involving the chemotaxis pathway, the set of proteins that function as the navigation system of bacteria and lower eukaryotes. In the first chapter, I used a special-purpose machine for molecular dynamics simulations, Anton, to simulate the signaling domain of the chemoreceptor in different signaling states for a total of 6 microseconds. Among other findings, this study provides enough evidence to propose a novel molecular mechanism for the kinase activation by the chemoreceptor and reconcile previously conflicting experimental data. In the second chapter, my molecular dynamics studies of the scaffold protein cheW reveals the existence and role of a conserved salt-bridge that stabilizes the relative position of the two binding sites in the chew surface: the chemoreceptor and the kinase. The results were further confirmed with NMR experiments performed with collaborators at the University of California in Santa Barbara, CA. In the third chapter, my colleagues and I investigate the quality of homology modeled structures with cheW protein as a benchmark. By subjecting the models to molecular dynamics and Monte Carlo simulations, we show that the homology models are snapshots of a larger ensemble of conformations very similar to the one generated by the experimental structures. In the fourth chapter, I use bioinformatics and basic mathematical modeling to predict the specific chemoreceptor(s) expressed in vivo and imaged with electron cryo tomography (ECT) by our collaborators at the California Institute of Technology. The study was essential to validate the argument that the hexagonal arrangement of transmembrane chemoreceptors is universal among bacteria, a major breakthrough in the field of chemotaxis. In summary, this thesis presents a collection of four works in the field of bacterial chemotaxis where either methods of physics or the quantitative approach of physicists were of fundamental importance for the success of the project