M. tuberculosis copper-sensitive operon repressor (Mtb CsoR) is the founding member of a new metalloregulatory protein family in prokaryotes that regulates the transcription of the cso operon in response to copper toxicity. Mtb CsoR tetramer binds 1 monomer mol equiv of Cu(I) with very high affinity (log KCu=18.0) via three conserved residues, Cys36, His61' and Cys65'. Binding of Cu(I) allosterically inhibits the CsoR binding to the DNA operator (CsoO) overlapping the cso promoter (DeltaGc=+3.6 kcal/mol, pH 7.0, 25 oC). These findings are consistent with a role of CsoR as a transcriptional repressor with Cu(I) binding inducing transcriptional derepression. To explore the mechanism of this regulation, His61 was substituted with 1-methylhistidine (MeH) or Beta- (2-thiazolyl)-alanine (Thz) using a native chemical ligation strategy. The CsoO binding affinities of the resultant H61MeH and H61Thz CsoRs are both refractory to inhibition by Cu(I) binding despite the fact that each forms a high affinity 3-coordinate complex with Cu(I). This suggests that while Cu(I) is coordinated by the N?11 atom of His61, the N?22 atom plays an critical role in driving this allosteric switch. Evidence in support of a formation of a hydrogen bonding network involving the N?1 face of His61 and two conserved "second coordination shell" residues, Glu81' and Tyr35, is presented. Remarkably, this mechanism is analogous to that proposed for the Zn(II) sensor CzrA from S. aureus. To test this, we employed the same native chemical ligation approach to substitute the key Zn(II) ligand His97 with 1-methylhistidine; with the preliminary findings fully consistent with an intersubunit allosteric switch involving the N?2 face of this key His97 residue in CzrA. Two predicted homologs of Mtb CsoR were also biochemically characterized to obtain additional support for the hypothesis that CsoR is a key Cu(I) regulatory protein in many bacterial species. B. subtilis CsoR, known to regulate the transcription of the copZA operon, was found to have biochemical properties similar to those of Mtb CsoR as to Cu(I) binding, DNA binding and Cu(I)-dependent allosteric regulation. Interestingly, Bsu CsoR also binds other divalent metal ions (Zn, Ni) with high affinity but with metal coordination geometries distinct from that of Cu(I). Binding of these divalent metal ions only weakly regulates copZA operator binding in vitro, suggesting that coordination number and geometry are most closely related to the allosteric regulation. Finally, a putative CsoR from the pathogenic S. aureus Newman strain was identified and characterized, and was found to exhibit biochemical properties similar to those of Mtb and Bsu CsoRs. Parallels between Cu(I)-sensing CsoRs and functional orthologs in the CsoR/RcnR family are further discussed in the context of the mechanism and evolutionary divergence of this new family of regulatory proteins.

The potential of magnetic nanoparticle-vesicle assemblies (MNP-V) as remote controlled drug delivery platforms capable of inducing cellular responses under magnetic stimuli has been previously demonstrated in the Webb group at the University of Manchester. To create these magnetoresponsive nanomaterials biotin-avidin and Cu-histidinyl multivalent recognition were employed. This thesis describes an exploration of the potential of thiol-thioester exchange reactions (leading to native chemical ligation, NCL) to create magnetoresponsive materials, which potentially have applications in biomedicine. Firstly, iron oxide magnetic nanoparticles have been synthesised using a thermal co-precipitation method followed by chemical modification with sulfhydryl motifs for use as smart biomaterials. Knowing that the behaviour and reactivity of nanoparticles is highly influenced by their physicochemical properties, a thourough characterisation of these particles has been obtained. Secondly, during this project, several thioester derivatives have been synthesised that can be incorporated into the membranes of 800 nm liposomes. Among these, the spectrophotometric properties of synthetic lipid 38 allowed the investigation of trans-thioesterification rates with cysteinyl functionalities, both in solution and at the phospholipid membrane interface of liposomes. Product identification has been achieved using mass spectrometry and 1H-NMR spectroscopy. Finally, the conditions required to induce the release of a dye (e.g. 5(6)-CF) from MNP-V upon exposure to an AMF pulse have been established. Aurintricarboxylic acid (ATA), a general inhibitor of nucleases has been investigated as interesting payload due to its fluorescent and anti-viral properties.

Microarrays enable high throughput analysis with minute amounts of analyte. They are widely used in the ’omics’ fields both as diagnostic and analytical tools. Their ability to dramatically impact an entire field of research has focused our attention on the development of novel methods for the formation, analysis and applications of microarrays to study carbohydrate-protein interactions and the analysis of glycosylation patterns of biomolecules. Availability of appropriately modified ligands is often a limiting factor in the preparation of microarrays. To address this issue robust routes for the synthesis of nine amino ethylglycosides were developed that can be employed for microarray formation. The syntheses of more complex ligands typically deliver small quantities of material despite the requirements for special skills, equipment and long preparation times. Considering the number of complex oligosaccharides that are necessary for systematic microarray studies, the problem of availability of these complex structures is difficult to address solely with synthetic ligands. A modified native chemical ligation (NCL) strategy, in which a surface bound oxo-ester is used instead of a thioester, was optimised and used for efficient chemoselective immobilisation of sugars and peptides carrying N-terminal cysteines. The reaction proceeds under physiological conditions and has the potential to become a valuable tool for immobilisation of N-terminal cysteine-containing molecules from biological samples. The new NCL coupling methodology was developed on gold surfaces and analysed by MALDI-ToF MS. The majority of array systems, however, rely on secondary protein interactions on glass or polystyrene surfaces. A direct, more accurate analytical tool could ease the analysis and significantly improve the quality of data read-out from glass microarrays. MALDI-ToF MS that is applicable to gold microarrays cannot be used on surfaces that do not provide the necessary electrical conductivity. The undertaken experiments indicated that application of conductive tape to the back of glass or polystyrene slides made MALDI-ToF analysis on poorly conducting surfaces possible. Furthermore, the triphenylmethyl (trityl) groups attached to the surface-molecules were shown to act as ’internal-matrix’ and enable the direct MALDI analysis. Once the new array formation and analysis techniques were developed, we turned our attention towards the application of microarrays to analyse carbohydrate-protein interactions. The tools for analysis of glycosylation of biomolecules are laborious and can only be used in specialised labs. As glycosylated biomolecules gain prominence in research, clinical and industrial settings, high throughput analysis of glycosylation patterns is becoming a requirement for quality control. A technique for screening of glycosylation patterns in glycopeptides on microarrays was developed based on biophotonic scattering. This technique enables the detection of glycosylation patterns by screening immobilised glycoproteins with a range of lectins. To study the interactions between enzymes and carbohydrates, a chemoenzymatic synthesis of a mannopeptide, which consisted of four carbohydrate units, was shown in solution and on chip. Three different glycosyl transferases were successfully employed. New methods for microarray formation and analysis were developed and applied to carbohydrate-protein interaction studies. This yielded a new technique to determine protein glycosylation patterns and to produce complex glycans by enzymatic synthesis.

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Current single molecule localisation microscopy methods allow for multicolour imaging of macromolecules in cells, and for a degree quantification on molecule numbers in one colour. However, that has not yet been an attempt to develop tools capable of quantitative imaging with multiple colours in cells. This work addressed this challenge by designing linker peptides with chemospecific groups to allow attachment of activator and emitter dyes for STORM imaging, and a targeting module. The design ensured a stoichiometric ratio of targeting module to activator and emitter dyes. Peptides with HaloTag ligands attached were labelled with various activator and emitter pairs and used to label HaloTag fusions of S. pombe and mouse embryonic stem cells. These peptides were found to bind non-specifically to various areas of both cell types, and did not localise to HaloTag protein, whereas controls did. Another peptide was also labelled with activator-emitter pairs and attached to expressed anti-GFP and ant-mCherry nanobodies via native chemical ligation. The labelled anti-GFP nanobody was to demonstrate ensemble and single molecule imaging in S. pombe, as well as characterisation on single molecule surfaces in comparison to a conventional randomly labelled antibody. The stoichiometrically labelled nanobody had a more consistent number of photons detected per localisation, number of localisation per molecule and number of blinks per molecule, which implied that it could be more useful than randomly labelled nanobodies for counting experiments. It was also shown to be capable of specific laser activation for STORM imaging with both an Alexa405Cy5 and Cy3Cy5 pairs. These anti-GFP and anti-mCherry nanobodies and peptide linker are new tools for both counting and multicolour imaging in super-resolution, which could be widely applied to constructs that are already tagged with GFP or mCherry.

Utiliser la liaison peptidique dans des systèmes dynamiques covalents est très difficile en raison de sa stabilité intrinsèque. Dans ce travail, une nouvelle méthodologie pour échanger fragments peptidiques dans des conditions biocompatibles est décrite. Légères modifications du groupe amine d'un résidu de cystéine en peptides modèle permettent l'activation spécifique de cette jonction peptidique pour des réactions d'échange covalent. Grâce à un mécanisme de ligation chimique native réversible, fragments peptidiques sont échangés en solution aqueuse à pH physiologique et en présence de dithiothréitol (DTT), avec des demi-temps d'équilibration de 2 à 10 heures. Différentes possibles applications biologiques de cette nouvelle réaction réversible à peptides et glycopeptides sont aussi proposées. / The possibility to use the peptide bond in dynamic covalent systems is very challenging because of its intrinsic stability. In this work, a novel methodology to exchange peptide fragments in bio-compatible conditions is described. The introduction of small modifications to the N-terminus of a cysteine residue in model peptides allows for the specific activation of that peptide bond for exchange reactions. Through a reverse Native Chemical Ligation (NCL) mechanism, peptide fragments were scrambled in aqueous solution at physiological pH and in the presence of dithiothreitol (DTT), with half-times of equilibration in the 2-10 h range. Additionally, possible biological applications of this new reversible reaction to both peptides and glycopeptides are proposed.

Les protéines jouent un rôle essentiel dans le fonctionnement des organismes vivants et sont au cœur de nombreux mécanismes biologiques. La synthèse totale chimique des protéines permet un contrôle précis de leur composition et constitue donc un outil puissant d’investigation des processus biologiques impliquant ces molécules. La synthèse peptidique en phase solide (SPPS) décrite par B. Merrifield en 1963 a révolutionné le domaine. Quelques années plus tard, les méthodes de ligations chimiques et le développement de stratégies d’assemblage de segments peptidiques ont permis la synthèse de nombreuses protéines par voie chimique. Cependant, la difficulté des chimistes à produire de grandes protéines traduit l’absence de méthodes robustes pour la synthèse en routine de tels macromolécules.De nouvelles méthodologies de synthèse totale basées sur la Native Chemical Ligation et les ligations SEA et SeEA ont été développées dans le cadre de cette thèse. En particulier, l’utilisation de sélénoesters latents ou formés in situ a été étudiée pour faciliter l’accès à des protéines de grande taille.La première partie de cette thèse porte sur le potentiel du groupement bis(2-sélényléthyl)amido SeEA, à savoir l’analogue sélénié du groupement bis(2-sulfanyléthyl)amido SEA, pour accélérer la formation de liaisons peptidiques par formation d’intermédiaires sélénoesters. Cette méthode a permis la synthèse de la protéine NK1 (variant naturel du facteur de croissance des hépatocytes HGF) constituée de 180 acides aminés.La seconde partie décrit la formation in situ de sélénoesters à partir de segments peptidiques SEA grâce à la conception de nouveaux catalyseurs à base de sélénium. Ces catalyseurs ont été utilisés pour accélérer deux réactions essentielles dans les procédés de synthèse totale développés dans cette thèse, à savoir la ligation SEA ainsi que la synthèse de peptides thioesters à partir de peptides SEA. L’efficacité de l’un de ces catalyseurs a été illustrée par la synthèse totale de la granulysine.La dernière partie de cette thèse décrit une stratégie d’assemblage séquentielle de segments peptidiques en phase solide, qui constitue une alternative aux méthodes d’assemblage en phase liquide. Ce procédé a été automatisé et présente un grand potentiel pour la synthèse automatisée de protéines par voie chimique. / Proteins play a crucial role in living organisms and in almost all biological mechanisms. The total chemical synthesis of proteins allows an atom by atom control of their structure and thus constitutes a powerful tool of investigation of biological processes involving these molecules. The solid phase peptide synthesis (SPPS) introduced by B. Merrifield in 1963 has revolutionized the field. Few years later, the discovery of chemical ligation methods and the development of peptide segment assembling strategies has been applied to the synthesis of many proteins. However, the difficulty in accessing large proteins reflects the absence of robust methods for the routine synthesis of such macromolecules.Novel synthetic methods based on Native Chemical Ligation and SEA/SeEA ligations have been developed in the frame of this thesis. In particular, I explored the interest of latent or in situ formed selenesters for facilitating the access to large proteins.The first part of this thesis describes the chemical properties of the bis(2-selenylethyl)amido (SeEA) group, i.e. the selenium analog of the bis(2-sulfanylethyl)amido (SEA) group, and its usefulness for accelerating peptide bond formation. This method was used for the synthesis of the NK1 protein (natural variant of the hepatocyte growth factor, HGF) constituted of 180 amino acids.The second part describes the in situ formation of selenesters through the design of novel selenium catalysts. These catalysts were used to accelerate the SEA ligation as well as the synthesis of thioester peptides from SEA peptides. Both reactions are central in the total synthesis processes developed in this thesis. Their usefulness is illustrated by the total synthesis of granulysin.The last part describes a method for the sequential ligation of peptide segments on a water compatible solid support which is complementary to the solution methods discussed above. This process has been automated and has a great potential for the automated chemical synthesis of proteins.

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