Les quadruplexes de guanines (G4) sont des structures d’ADN non-canoniques qui résultent de l’empilement hydrophobe de tétrades de guanines, stabilisé par des cations métalliques (tels que Na+ et K+). Il existe aujourd’hui un nombre croissant de preuves expérimentales qui attestent de l’implication des G4 dans d’importantes fonctions cellulaires corrélées à leur mécanisme de repliement/dépliement. Toutefois, très peu d’études ont abordé les aspects dynamiques de leur formation. Aussi, nous avons entrepris l’étude de plusieurs G4 mono-moléculaires à l’aide d’une nouvelle extension d’expériences de saut de température, capable de mesurer la dynamique de dénaturation thermique et de renaturation consécutive de l’ADN, sur une fenêtre temporelle allant de quelques millisecondes aux secondes. Les changements conformationnels ont été sondés par dichroïsme circulaire (CD) résolu en temps, connu pour être très sensible à l’arrangement des guanines dans les G4. Au préalable des études résolues en temps, en collaboration avec DISCO/SOLEIL, nous avons mesuré les spectres CD statiques de différentes séquences G4 présentant des topologies distinctes, comme celles des télomères humains, de l’aptamère de la thrombine ou des promoteurs de c-MYC. Nous avons observé des cinétiques de dénaturation et renaturation biphasiques avec des constantes de temps de quelques centaines de millisecondes et quelques secondes. Ces cinétiques dépendent fortement de l’amplitude du saut de température et de la concentration de cations métalliques. L’ensemble de ces observations suggère l’existence de plusieurs voies de repliement/dépliement des G4 sur des surfaces de potentiel très rugueuses. / Guanine-quadruplexes (G4) are non-canonical DNA structures that result from the hydrophobic stacking of guanine quartets stabilized by metal cations (typically Na+ and K+). There is now an increasing body of experimental evidence of their occurrence in important cell functions correlated to their folding/unfolding mechanisms. However, only few studies have addressed the dynamical aspect of their formation. In this context, we have undertaken the study of several intramolecular G4 with a novel extension of temperature-jump experiments capable to measure the thermal denaturation and the consecutive renaturation of DNA over a time window spanning a few ten milliseconds to seconds. Conformational changes have been monitored by time-resolved circular dichroism (CD), which is known to be sensitive to the chiral arrangement of guanines in the G4 scaffolds.Prior to time-resolved measurements, within the frame of a collaboration with DISCO/SOLEIL, we have performed static synchrotron radiation CD measurements on several short G4-forming sequences, such as human telomere, thrombin-binding aptamer and c-MYC promoter sequences, displaying distinct topologies. Denaturation and renaturation kinetics are found to exhibit biphasic decays with time constants of a few hundred milliseconds and a few seconds, respectively. Those kinetics depend strongly on the amplitude of the temperature jump and the concentration of cations. Taken together these observations suggest the existence of multiple folding pathways on extremely rugged landscapes.

Spiders are capable of producing a variety of silk types, each with their own unique protein composition and function. Dragline silk in particular, has been of great interest due to its high tensile strength and extensibility. In the past, synthetically produced dragline fibers have not been able to match the superior properties of natural silk. A recent discovery in the western black widow spider, Latrodectus hesperus, might be the missing link between the current state of synthetic silks and naturally produced fibers. Our research is centered around the discovery a new family of five low-molecular-weight cysteine-rich proteins (CRPs) and their potential function within dragline silk. This study focuses on the characterization of recombinantly expressed CRP1, CRP2, and CRP4. Through structural analysis using circular dichroism, it has been determined that the CRP family members have mostly alpha-helical secondary structure and exhibit small differences in their ability to maintain their structure in the presence of changing environmental conditions. The study also covers the effects of temperature and pH on the folding and unfolding of the CRPs. It appears that pH is the dominant influence on protein unfolding within the major ampullate gland.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第18095号 / 理博第3973号 / 新制||理||1573(附属図書館) / 30953 / 京都大学大学院理学研究科化学専攻 / (主査)教授 三木 邦夫, 教授 杉山 弘, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第20714号 / 工博第4411号 / 新制||工||1685(附属図書館) / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 杉野目 道紀, 教授 村上 正浩, 教授 松田 建児 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

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Molecular recognition, particularly as it applies to strong binding interactions between complementary ligand/receptor molecules in solution, is important in such varied areas as molecular biology, pharmacology, synthetic chemistry, and chemical detection. Strong binding is the additive result of a number of specific, weak, non-covalent interactions occurring between complementary molecules. This dissertation reports on the energetics of forming complexes between small molecules and model DNA constructs. Ligands included cationic and metallated cationic porphyrins and polyheterocyclic ruthenium compounds. DNA receptors included double stranded B-DNAs (hairpin and short linear sequences) as well G-quadruplex DNAs. Thermodynamic data were collected using isothermal titration calorimetry, circular dichroism spectropolarimetry, ultraviolet-visible spectroscopy, and mass spectrometry. The measured thermodynamic parameters included the changes in free energy, enthalpy and entropy for ligand/receptor complex formation as well as the stoichiometry of the stable complexes. The first section of this dissertation reports that the binding of cationic porphyrins to model G-quadruplex DNA may proceed through two pathways, end stacking and intercalation. Modulating the number of pyridinium groups on a pyridinium substituted porphyrin yielded differing binding thermodynamics leading to the understanding that a balance of surface area, charge, and geometry affect the ability of a porphyrin to bind to G-quadruplex DNA. Further investigations into the binding of metallated porphyrins developed the understanding that the geometry of the central metal ion affected not only the thermodynamics but could also inhibit the intercalative mode. It was previously shown that the high affinity binding for binuclear polyheterocyclic ruthenium compounds proceeds through an intercalative mode. To further understand the binding process and the structureunction relationship of the ligand components, the binding of smaller mononuclear complexes that were representative of portions of the binuclear complex was examined in this dissertation. While limiting the intercalative ability lowered the binding affinity, the mononuclear complex with the full intercalating bridge was able bind to DNA with a higher affinity than the binuclear complex. These studies have been successful in part in determining the contributions of numerous weak interactions including: charge (Coulombic interactions), H-bonding, hydrophobic interactions, and solvent structure (solvation changes), to the overall energetics of this molecular recognition process. The first section of this dissertation reports that the binding of cationic porphyrins to model G-quadruplex DNA may proceed through two pathways, end stacking and intercalation. Modulating the number of pyridinium groups on a pyridinium substituted porphyrin yielded differing binding thermodynamics leading to the understanding that a balance of surface area, charge, and geometry affect the ability of a porphyrin to bind to G-quadruplex DNA. Further investigations into the binding of metallated porphyrins developed the understanding that the geometry of the central metal ion affected not only the thermodynamics but could also inhibit the intercalative mode. It was previously shown that the high affinity binding for binuclear polyheterocyclic ruthenium compounds proceeds through intercalation. To further understand the binding process and the structureunction relationship of the ligand components, the binding of smaller mononuclear complexes that were representative of portions of the binuclear complex was examined in this dissertation. While limiting the intercalative ability lowered the binding affinity, the mononuclear complex with the full intercalating bridge was able bind to DNA with a higher affinity than the binuclear complex. These studies have been successful in part in determining the contributions of numerous weak interactions including: charge (Coulombic interactions), H-bonding, hydrophobic interactions, and solvent structure (solvation changes), to the overall energetics of this molecular recognition process.

The covalent attachment of poly(ethylene glycol) (PEG) to a protein surface (known as PEGylation), has been demonstrated to increase the serum half-life of therapeutic proteins by reducing kidney clearance and immunogenicity and by protecting against proteolysis. Theses beneficial effects could be further enhanced if PEGylation consistently increased protein conformational stability (i.e. the difference in free energy between the folded and unfolded states). However, the effects of PEGylation on protein conformational stability are unpredictable; PEGylation has been reported to increase, decrease, or have no effect on the conformational stability of medicinal proteins.This thesis details the results of two studies aimed at discovering the structural determinants which influence the thermodynamic impact of PEGylation on the WW domain, a small model protein. Chapter 1 is a brief introduction to protein therapeutics and protein PEGylation. Chapter 2 describes a study which demonstrates that the thermodynamic impact of PEGylation is strongly dependent on the site to which PEG is conjugated. The studies described in Chapter 3 elaborate on this site dependence, and demonstrate that PEG stabilizes the WW domain through interactions with the surface of the folded peptide, and that two factors – the orientation of the PEG chain (relative to the protein surface) and the identity of nearby side chains – play a critical role in determining the thermodynamic impact of PEGylation.

The BCL-2 family of proteins tightly regulates the delicate balance between life and death. The pore forming Bax is a pro-apoptotic member belonging to this protein family. At the onset of apoptosis, monomeric cytoplasmic Bax translocates to the outer mitochondrial membrane, forms oligomeric pores thereby letting mitochondrial cytochrome c enter the cytosol and initiate the apoptotic cascade. The C-terminal "helix 9" is thought to mediate the membrane binding of BAX. A 20-amino acid peptide corresponding to Bax C-terminus (VTIFVAGVLTASLTIWKKMG) and two mutants where the two lysines are replaced with Glu (charge reversal mutant, EE) or Leu (charge neutralization mutant, LL) have been studied to elucidate the pore formation capabilities of Bax C-terminus and the underlying molecular mechanism. Interactions of the wild-type and the two mutant peptides with zwitterionic and anionic phospholipid membranes caused efficient membrane permeabilization, as documented by release of vesicle-entrapped fluorescent indicator calcein. Light scattering experiments showed that vesicles maintained their integrity upon peptide binding, indicating that the content leakage was due to pore formation and not vesicle degradation. Kinetics of calcein release at various peptide concentrations were used to determine the peptide-peptide association constants and the oligomeric state of the pore. The structure of membrane-bound peptides was analyzed by circular dichroism (CD) and attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. CD data indicated all three peptides reconstituted in lipid vesicles contained [alpha]-helical and [beta]-strand structures. ATR-FTIR experiments indicated that the minimally hydrated samples of peptides in stacked lipid bilayers (absence of bulk water) were mostly [alpha]-helical but adopted mostly [beta]-sheet conformation in the presence of excess water. Finally, the depth of membrane insertion of the peptides was analyzed using tryptophan fluorescence quenching by dibromo-phosphatidylcholines brominated at various positions of their acyl chains. In case of zwitterionc phospholipid membranes, the single Trp¹⁶ was located at ~9 Å from membrane center. In case of membranes containing 30% of an anionic phospholipid, the depth of membrane insertion of the EE mutant was not affected but the wild-type and the LL mutant peptides were embedded much deeper into the membrane, with Trp¹⁶ located at 3-4 Å from membrane center. These results will help achieve a better understanding of the molecular mechanism of membrane pore formation of Bax protein. In addition, they provide insight into the molecular details of membrane pore formation by peptides and could facilitate the design and production of cytotoxic peptides with improved capabilities to lyse cells such as bacteria or cancer cells.

A-B family of toxins consists of plant toxins such as ricin and bacterial toxins such as cholera. The A subunit is the enzymatic domain and the B subunit is the receptor binding domain. Commonly, these toxins bind to the target cell plasma membrane receptors through their B subunit followed by endocytosis and a transport to the endoplasmic reticulum (ER). Inside the ER, the A subunit dissociates from the rest of the toxin, unfolds and triggers the ER quality control mechanism of ER-associated degradation (ERAD). Most ERAD substrates are purged out of the ER into the cytosol for proteasomal degradation. However, the low content of lysine amino acid residues allows the toxin to evade polyubiquitination and subsequent proteasomal degradation. The toxin A subunit refolds into an active conformation in the cytosol, setting off downstream toxic events. In the first part of my thesis, the hypothesis was tested that inhibiting the unfolding of the toxin A subunit inside the ER will prevent ERAD activation, toxin export to the cytosol and intoxication. The chemical chaperones glycerol and sodium 4-phenyl butyrate (PBA) were used to inhibit the toxin A chain unfolding. In vitro biophysical experiments indicated that both chemical chaperones indeed stabilize the cholera toxin A subunit and prevent cytotoxicity. In case of ricin, both chaperones stabilized the toxin A chain but only glycerol prevented cytotoxicity. Additional experiments showed that PBA-treated ricin A chain is destabilized when exposed to anionic lipid membranes mimicking the properties of the ER membrane. In contrast, anionic lipid did not prevent ricin A chain stabilization by glycerol. This explains why glycerol but not PBA blocked ricin intoxication, as only glycerol stabilizes ricin A chain in the presence of ER membranes. Cholera toxin in contrast, remained either unaffected or slightly stabilized in presence of anionic lipids both in presence and absence of PBA. This shows that destabilization by anionic lipids is a toxin-specific rather than a general effect. In the second part of my thesis, the effect of inner leaflet of plasma membrane on the structure of cholera toxin A chain (CTA1) was studied. Since CTA1 refolds into an active conformation in the cytosol in association with unidentified host factors, I hypothesized that inner leaflet of the plasma membrane might play a role to stabilization and/or refolding of CTA1. CTA1 was shown to be a membrane interacting protein, and membranes mimicking lipid rafts had a significant stabilizing effect on its structure. Lipid rafts helped in the regaining of the tertiary and secondary structure of CTA1, while non-raft lipids had a smaller stabilizing effect on CTA1 structure. In the next part of my thesis, I studied the effect of membrane binding on the structure and function of human pancreatic phospholipase A₂ (PLA₂). Lipid thermal phase transition was found to have a dramatic effect on PLA₂ activity. It was also established that although membrane binding and insertion was essential for of PLA₂ activity, lipid structural heterogeneity was more important than the depth of membrane insertion for enzyme activation. Most importantly, significant changes in PLA₂ secondary and tertiary structures were identified that evidently contribute to the interfacial activation of PLA₂. Overall, we conclude that the function of membrane binding enzymes can be significantly modulated via conformational changes induced by interactions with membranes. Thus, we have elucidated various roles of membrane lipids from unfolding and refolding to activation and modulation of membrane binding enzymes. Physical properties of lipids help in regulating various aspects of protein structure and function and their analysis helped us in appreciating the influence wielded by the membrane lipids in the enzyme's surrounding environment.

Currently the treatment of alcohol use disorder is very difficult and often requires the combination of therapy and medications, with many who undertake treatment experiencing relapse over time. There is also no treatment in use to prevent the development of alcohol use disorder. It is the aim of this work to provide information that may be useful for the development of a preventative treatment for developing alcohol use disorder by elucidating more of the acute effects of alcohol use. It is known that these effects originate in the brain. Within the brain are circuits made up of neurons that communicate with each other through chemical synapses. These chemical synapses involve the release of neurotransmitters from one neuron that are detected by another neuron, which initiates its own response. It is known that ethanol can change how much neurotransmitter is released from a neuron, depending on the specific neuron tested, and many researchers have implicated the "release machinery" as a target. It is also known that alcohol can affect lipid membrane properties that are important for the fusion of the vesicle membrane, encapsulating the neurotransmitter, with the cell membrane for release of the neurotransmitter outside of the neuron. It is not known if alcohol directly affects the SNARE proteins ("release machinery") or the lipid membranes to initiate the change in neurotransmitter release previously observed. Within this work you will find a discussion of the steps of neurotransmitter release and the known effects of anesthetics on components of this process, as an introduction to the topic (Chapters 1 and 2). In Chapters 3-5 you will find studies that successively dive deeper and deeper into the effects of alcohol on the SNARE proteins and lipid membranes. We show that ethanol is effective at a dose of 0.4% v/v or 64 mM at increasing fusion probability in a model of neurotransmitter release that uses the 3 SNARE proteins to drive fusion of a vesicle with a supported membrane. We also show that alcohol has little direct effect on the SNARE proteins themselves. In addition, we provide evidence that alcohol alters fusion oppositely, depending on which membrane leaflet it has most direct access to. In Chapter 5 we show that alcohol increases the probability of lipid tail protrusion in silico. Previously it has been shown that protrusion of one fatty acid tail of one lipid can initiate fusion of that membrane with an apposing membrane. These data provide further insight into the effects of alcohol on a neuron and we would argue are valuable to research pursuing treatment and prevention of alcohol use disorder.