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Linker histones (H1) are the basic proteins in higher eukaryotes that are responsible for the final condensation of chromatin. H1 also plays an important role in regulating gene expression. H1 has been described as a transcriptional repressor as it limits the access of transcriptional factors to DNA. Linker histone binds to DNA that enters or exits the nucleosome. Several crystal structures have been published for the nucleosome (histone core/DNA complex), and the interactions of the core histone proteins with DNA are well understood. In contrast the location of the linker histone and its interactions with ds-DNA are poorly understood. In this study we have used isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and CD spectropolarimetry to determine the thermodynamic signatures and structural changes that accompany H1 binding to ds-DNA. The thermodynamic parameters for the binding of intact linker histones (H1.1, H1.4, and H10) to highly polymerized calf-thymus DNA and to short double stranded DNA oligomers have been determined. We have also determined the thermodynamics for binding of H10 C-terminal tail (H10-C) and globular domain (H10-G) to calf-thymus DNA. The real surprise in the energetics is that the enthalpy change for formation of the H1/DNA complex is very unfavorable and that H1/DNA complex formation is driven by very large positive changes in entropy. The binding site sizes for H1.1, H1.4, and H10 were determined to be 36bp, 32bp, and 36bp respectively. CD results indicate that CT-DNA is restructured upon complexation with either the full length H1 protein (H10) or its C-terminal domain (H10-C). In contrast, the structure of H10 is largely unchanged in the DNA complex. Temperature dependence of enthalpy change, osmotic stress and ionic strength dependence of Ka were tested using ITC. These results indicate that the entropy driven H1/DNA complexes are a result primarily from the expulsion of bound water molecules from the binding interface. This study provides new insights into the binding of linker Histone H1 to DNA. A better understanding of the functional properties of H1 and its interactions with DNA could provide new insights in understanding the role H1 in DNA condensation and transcriptional regulation.

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.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

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Les homéoprotéines sont des facteurs de transcription importants au cours du développement des organismes vivants, capables notamment de voyager de cellule en cellule. Ces protéines comportent une longue extrémité N-terminale désordonnée, suivie de trois hélices α séparées par une boucle et un tour. Des études de relations structure-activité ont montré que des domaines cationiques (riches en K et R) particuliers dans ces protéines sont responsables de ces propriétés de transfert cellulaire leur permettant d’être secrétées et internalisées dans les cellules. Ces processus impliquent que ces protéines hydrophiles soient capables de franchir la membrane plasmique composée d'un coeur hydrophobe. La membrane plasmique est en effet composée d’une bicouche lipidique, dans laquelle sont insérées de nombreuses protéines, telles que les protéoglycanes portant des ramifications de glycosaminoglycanes (GAG), polysaccharides anioniques. Dans le but de comprendre au niveau moléculaire le processus d'entrée des homéoprotéines dans des cellules eucaryotes, différentes constructions protéiques ont été produites et étudiées : le peptide pénétrant les cellules correspondant à l'hélice 3 (H3), la séquence correspondant à l'homéodomaine (HD), l'homéodomaine étendu d'une séquence putative de liaison aux GAG (NLS-HD) et la protéine entière (En2). La quantification absolue de l’entrée de ces constructions dans des cellules CHO-K1 par spectrométrie de masse a mis en évidence une efficacité d'entrée meilleure pour H3 > NLS-HD > HD, ainsi que l’importance des GAG de surface dans le processus et plus particulièrement celui des héparanes sulfates (HS). Des expériences complémentaires d’ITC, de dichroïsme circulaire et de RMN ont permis d'identifier deux sites d’interaction avec l’héparine (un site principal de haute affinité et un site secondaire de plus basse affinité), interagissant principalement avec le polysaccharide par interactions électrostatiques. In fine, ces études conduisent à une meilleure compréhension moléculaire du processus d'internalisation des homéoprotéines dans des cellules eucaryotes. / Homeoproteins are important transcription factors during the development of living organisms, and are able to travel from cell to cell. These proteins contain a long N-terminal extremity structurally disordered, followed by three α helices separated by a U-turn. Structure-activity relation studies have shown that in these proteins, some cationic domains (rich in K and R) confer them the cellular transfer properties, allowing them to be secreted by and internalized into cells. These processes imply that the hydrophilic proteins are able to cross plasma membrane. Indeed, the plasma membrane possess a hydrophobic heart and is composed by a lipidic bilayer, in which numerous proteins are inserted, such as proteoglycans carrying glycosaminoglycan (GAG) ramifications, that belong to anionic polysaccharids. In order to understand the entry process of homeoproteins into eukaryotic cells at a molecular level, different proteic constructions have been produced and studied: the cell penetrating peptide corresponding to the third α helix (H3), the sequence corresponding to its homeodomain (HD), the homeodomain with an added putative GAG-binding domain (NLS-HD), and the wild-type protein Engrailed 2 (En2). The absolute mass spectrometry quantification of the peptide and proteins in cells shows a range of internalization efficiency as follows: H3 > NLS - HD > HD. It also highlights the importance of cell-surface GAGs in the internalization and more particularly that of heparan sulfates (HS). Complementary experiments of ITC, circular dichroism and NMR have shown two interaction sites for the heparin (one principal site of high affinity and a secondary site showing a lower affinity) both interacting mainly with polysaccharidic residues using electrostatic interactions. In fine, these studies lead to a better molecular understanding of homeoproteins internalization process in eukaryotic cells.