From peptides to transmembrane proteins : helix versus kink formations in highly dynamical systems

This thesis describes investigations of the relationships between the sequence of small peptides and their folding propensities and the conformational changes of membrane lipids upon interactions with proteins, within the context of varying membrane potentials. In addition, a novel conformational change of a membrane protein will be presented. The determination of structures of folded proteins has progressed remarkably, notably due to outstanding techniques like crystallography, nuclear magnetic resonance or cryo-electron microscopy. However, proteins are highly dynamic and, under physiological conditions, their behavior depends on the chemical and physical environment. On the other hand, a better understanding of the intrinsically disordered proteins requires approaches, which consider their dynamical nature. All-atom molecular dynamics simulations constitute a tool of choice to capture the conformational changes of peptides as well as larger systems involving bilayers and membrane proteins. The first part of this thesis is dedicated to the structural propensities of peptides explored at the amino acid level. The investigations have shown how subtle interactions with the solvent affect their fate towards helical conformations. These findings are further validated through a procedure aimed at reducing the differences between predicted and experimental values while maximizing the entropy of the ensemble. The short-lived conformations found along transition paths are difficult to observe experimentally. Consequently, a statistical approach to investigate at the picosecond timescale the dynamics of the folding events in relation to the surrounding molecules is introduced and successfully tested on a β–hairpin of known structure. These successful results lead to a proposal of a systematic study to elucidate the sequence-conformation(s) relationships at a larger scale. The second project describes the interactions between spider toxins, the cell membrane and a voltage sensor domain in the context of ion channel gating modification. Spider toxins have contributed substantially to the understanding of ion channels. Most of them are gating modifiers, thus affecting the energy level required by ion channels to open or close. Because these molecules are capable of fine-tuning the function of ion channels, they represent very attractive candidates in the field of drug discovery, and some successes have been achieved in this regard. The initial objective of the study was to explore whether the toxin-induced perturbation of the membrane affect consequently the voltage-gated ion channels without any direct binding to the target. A demanding statistical approach was chosen, which takes the high specificity of spider toxins observed in vivo into account. Although the inserted toxins altered noticeably several membrane features, the results support the idea that an indirect, lipid-mediated mode of action of spider toxins on the voltage-sensor domain is not the main driver of the voltage-gated modifier mechanism. However, the investigations led to unexpected discoveries. The strategy employed to investigate an indirect mechanism of spider toxin involved more than 100 replicated simulations of independent bilayers and voltage-sensor domains exposed to a wide range of membrane potentials. The analyses showed surprisingly that the membrane perturbation, induced by the voltage sensor domain, is voltage-dependent. In addition, a novel conformational change of the voltage sensor upon polarization was observed, namely a kink in the S4 helix. The results discussed here aim to contribute to a better understanding in three domains: 1) The interplay between water and the amino acid side chains during conformational changes, precisely the hydration fluctuations of just a few amide or carbonyl functional groups are shown to affect the helix formation propensities of a small peptide. 2) The lipid-mediated gating modifier mechanism is not supported by the simulations. 3) A novel conformational change of the voltage-sensor domain is described as a response to variation of the membrane potential. Precisely, a kink in the middle of the S4 helix occurs only upon polarization. This kink formation allows gating charges to move across the membrane without exposing any hydrophobic residues to the cytoplasm

Deep underground neutrino experiment (DUNE) near detector conceptual design report

The Deep Underground Neutrino Experiment (DUNE) is an international, world-class experiment aimed at exploring fundamental questions about the universe that are at the forefront of astrophysics and particle physics research. DUNE will study questions pertaining to the preponderance of matter over antimatter in the early universe, the dynamics of supernovae, the subtleties of neutrino interaction physics, and a number of beyond the Standard Model topics accessible in a powerful neutrino beam. A critical component of the DUNE physics program involves the study of changes in a powerful beam of neutrinos, i.e., neutrino oscillations, as the neutrinos propagate a long distance. The experiment consists of a near detector, sited close to the source of the beam, and a far detector, sited along the beam at a large distance. This document, the DUNE Near Detector Conceptual Design Report (CDR), describes the design of the DUNE near detector and the science program that drives the design and technology choices. The goals and requirements underlying the design, along with projected performance are given. It serves as a starting point for a more detailed design that will be described in future documents

Deep underground neutrino experiment (DUNE) near detector conceptual design report

The Deep Underground Neutrino Experiment (DUNE) is an international, world-class experiment aimed at exploring fundamental questions about the universe that are at the forefront of astrophysics and particle physics research. DUNE will study questions pertaining to the preponderance of matter over antimatter in the early universe, the dynamics of supernovae, the subtleties of neutrino interaction physics, and a number of beyond the Standard Model topics accessible in a powerful neutrino beam. A critical component of the DUNE physics program involves the study of changes in a powerful beam of neutrinos, i.e., neutrino oscillations, as the neutrinos propagate a long distance. The experiment consists of a near detector, sited close to the source of the beam, and a far detector, sited along the beam at a large distance. This document, the DUNE Near Detector Conceptual Design Report (CDR), describes the design of the DUNE near detector and the science program that drives the design and technology choices. The goals and requirements underlying the design, along with projected performance are given. It serves as a starting point for a more detailed design that will be described in future documents

Deep Underground Neutrino Experiment (DUNE) near detector conceptual design report

The Deep Underground Neutrino Experiment (DUNE) is an international, world-class experiment aimed at exploring fundamental questions about the universe that are at the forefront of astrophysics and particle physics research. DUNE will study questions pertaining to the preponderance of matter over antimatter in the early universe, the dynamics of supernovae, the subtleties of neutrino interaction physics, and a number of beyond the Standard Model topics accessible in a powerful neutrino beam. A critical component of the DUNE physics program involves the study of changes in a powerful beam of neutrinos, i.e., neutrino oscillations, as the neutrinos propagate a long distance. The experiment consists of a near detector, sited close to the source of the beam, and a far detector, sited along the beam at a large distance. This document, the DUNE Near Detector Conceptual Design Report (CDR), describes the design of the DUNE near detector and the science program that drives the design and technology choices. The goals and requirements underlying the design, along with projected performance are given. It serves as a starting point for a more detailed design that will be described in future documents

Observing muon neutrino to electron neutrino oscillations in the NOvA Experiment

Neutrino oscillations offers an insight on new physics beyond the Standard Model. The three mixing angles ($\theta_{12}$, $\theta_{13}$ and $\theta_{23}$) and the two mass splittings ($\Delta m^2_{12}$ and $\Delta m^2_{23}$) have been measured by different neutrino oscillation experiments. Some other parameters including the mass ordering of different neutrino mass eigenstates and the CP violation phase are still unknown. \nova~is a long-baseline accelerator neutrino experiment, using neutrinos from the NuMI beam at Fermilab. The experiment is equipped with two functionally identical detectors about 810 kilometers apart and 14 mrad off the beam axis. In this configuration, the muon neutrinos from the NuMI beam reach the disappearance maximum in the far detector and a small fraction of that oscillates into electron neutrinos. The sensitivity to the mass ordering and CP violation phase determination is greately enhanced. This thesis presents the \nue appearance analysis using the neutrino data collected with the \nova~experiment between February 2014 and May 2015, which corresponds to 3.45 $\times 10^{20}$ protons-on-target (POT). The $\nu_e$ appearance analysis is performed by comparing the observed \nuecc-like events to the estimated background at the far detector. The total background is predicted to be 0.95 events with 0.89 originated from beam events and 0.06 from cosmic ray events. The beam background is obtained by extrapolating near detector data through different oscillation channels, while the cosmic ray background is calculated based on out-of-time NuMI trigger data. A total of 6 electron neutrino candidates are observed in the end at the far detector which represents 3.3 $\sigma$ excess over the predicted background. The \nova~ result disfavors inverted mass hierarchy for $\delta_{cp} \in [0,0.6\pi]$ at $90 \%$ C.L

Single-Molecule FRET Guided Modeling of RNA Structure and Dynamics

Dynamics are central to the function of biomolecules. In the field of RNA, riboswitches are a prime example where regulatory function is encoded by structural transitions. In this work, we used single-molecule fluorescence spectroscopy together with molecular simulations to probe such onformational dynamics. The first part of this thesis reviews established and novel labeling approaches to site-specifically tag nucleic acids with fluorescent markers for Förster resonance energy transfer (FRET) applications. We characterize bioconjugated dyes in terms of their photophysics and introduce computational tools that help in selecting informative distance coordinates. Biologically active RNA molecules are composed of recurrent, well conserved modules connecting secondary and tertiary structure. Their systematic annotation over several decades led to the notion that RNA folding can be understood by the thermodynamics and kinetics of the constituting building blocks. Here, we chose a long-range tertiary contact reaching from the core of a group II intron to its anking 50-exon. The structure of the isolated contact was previously solved by NMR and allowed us to link chemical features of the ribose backbone and metal ion coordination with dissociation rates measured by single-molecule FRET. We speculate that kinetic heterogeneity in exon recognition has important implications on ribozyme catalysis. Finally, we turn to a coenzyme B12 riboswitch whose mechanism has remained elusive owing to its structural complexity. Based on the consensus sequence and fragments of other cobalamin riboswitches we built a homology model of the E. coli btuB RNA and probed its dynamics by single-molecule FRET. We found a Mg2+ dependent conformational equilibrium which is thought to coordinate folding of the metabolite binding aptamer with the peripheral expression platform