Mineralised collagen displays an improved hydrothermal stability compared to collagen that is unmineralised. The possibility of using in-vitro partial mineralisation of collagen as a method of increasing the hydrothermal stability was investigated. Remineralisation experiments using demineralised turkey leg tendon and chemically modified bovine hide collagen showed that although it was possible to grow hydroxyapatite mineral crystallites on the collagen substrate they were only present at the substrate-solution interface and as such did not give rise to an increase in hydrothermal stability. The morphology of the mineral crystallites produced in-vitro were compared with those in the naturally mineralised tendon using Scanning Electron Microscopy (SEM), Small Angle X-ray scattering (SAXS), X-ray Diffraction (XRD) and Fourier-Transform Infrared Spectroscopy (FT-IR). Differential scanning calorimetry (DSC) studies on demineralised tendon identified a previously unknown high temperature endothermic transition to be present in the thermal scan of both mineralised and unmineralised collagen during denaturation. The position of this transition was found to be affected by hydration, presence of mineral, pH, and crosslinking similar to that of the first transition. Experiments using reagents known to selectively break various non-covalent interactions within collagen indicated that the transition was due to the breaking of covalent bonds via an endothermic chemical reaction, with the most likely candidate being the hydrolysis of peptide bonds within the polypeptide backbone. Optical microscopy of collagen after heating indicated that the fibrillar structure of the collagen was destroyed during the second transition, forming an amorphous gel. Finally, the effect of the mineral phase on the hydrothermal stability of naturally mineralised collagen was discussed in context to its location within the collagen structure. It was postulated that the presence of mineral dehydrates the collagen structure, as well as decreasing the available space within the hole region.

The work described in this thesis is concerned with the development and application of real-time photo-CIDNP (Chemically Induced Dynamic Nuclear Polarisation) to the study of protein structure and folding. Chapters 1 and 2 introduce the protein folding problem, and its study by NMR, then go on to elucidate the mechanisms behind the photo-CIDNP phenomenon. Chapter 3 applies photo-CIDNP spectroscopy to the study of a small cytochrome protein. The difficulties of performing these experiments on chromophore-containing proteins are discussed. Chapter 4 begins with the development of a rapid mixing device for use in real-time NMR and CIDNP studies. Experiments used to characterise the device are presented. This chapter then goes on to describe CIDNP pulse labelling experiments, used to investigate the surface structure of some molten globule states of two a-lactalbumins. This chapter concludes with an application of the rapid mixing device to the real-time refolding of hen egg white lysozyme. Chapter 5 extends the work of the previous chapter, studying the real-time refolding of bovine pancreatic ribonuclease A. Refolding studies are performed from different denaturing conditions, and the effects of sample heating during the real-time CIDNP experiment are discussed. Chapter 6 describes the use of illumination during an NMR experiment to study the conformational changes in a plant blue light receptor protein, phototropin. The structural changes are characterised with 2-dimensional NMR spectroscopy and photo-CIDNP. The kinetics of the ground state recovery are also investigated by real-time NMR spectroscopy. Chapter 7 uses calculated hyperfine coupling constants and a radical pair diffusion model from the literature to simulate the nuclear polarisation obtained for the amino acid tryptophan. Comparisons are made between theory and experiment. Chapter 8 describes the structural characterisation of a homologous series of de novo peptides, designed for subsequent use in EPR experiments when derivatised with a suitable spin label.

Ce travail a été entrepris dans le but de caractériser biochimiquement et biologiquement les collagènes de méduse. l’objectif de notre travail a été d’évaluer ce matériel en vue de sa valorisation pour l’élaboration de biomatériaux à usage médical. dans un premier temps, nous avons optimisé les techniques d’extractions des collagènes de quatre espèces de méduses : rhizostoma pulmo, cotylorhiza tuberculata, pelagia noctiluca et aurelia aurita. l’étude de la stabilité thermique par dichroïsme circulaire des collagènes nous a montré que la température de dénaturation des collagènes de rhizostoma pulmo était de 28,9°c. nous avons choisi de réaliser la réticulation des collagènes de méduse par la méthode des carbodiimides (edc/nhs). cette méthode nous a permis d’augmenter la température de dénaturation jusqu’à 33°c (versus 28,9°c). l’étude de l’interaction entre les cellules d’origine humaine (mg63 et fibroblastes) et les collagènes de méduses, nous a permis de démontrer qu’elles étaient capables d’adhérer aux collagènes de méduses natifs ou dénaturés. des immunomarquages de la vinculine des cellules mg63 et des fibroblastes sur collagènes de méduse natifs ou dénaturés nous ont permis de caractériser les adhésions mises en places par ces deux types cellulaires. l’analyse des milieux de culture des cellules mg63 et des fibroblastes par zymographie sur collagènes de méduse natifs ou dénaturés nous a permis de mettre en évidence que des mmps d’origine humaine, seraient capables de dégrader les collagènes de méduses. nous pouvons dans ce cas estimer que le collagène de méduse serait un bon substrat pour l’élaboration de biomatériaux résorbables / This work was undertaken to characterize biochemically and biologically jellyfish collagen. the aim of our study was to evaluate the material for the development of biomaterials for medical use. initially, we optimized the technical extraction of collagen from four species of jellyfishes: rhizostoma pulmo, cotylorhiza tuberculata, pelagia noctiluca and aurelia aurita. the study of thermal stability by circular dichroism of collagen has shown that the denaturation temperature of rhizostoma pulmo collagens was 28.9 ° c. we chose to achieve crosslinking of collagen of jellyfish by the method of carbodiimides (edc/nhs). this method allowed us to increase the denaturation temperature to 33 ° c (vs. 28.9 ° c). the study of the interaction between human cells (mg63 and fibroblasts) and jellyfish collagen, demonstrated that they were able to adhere native or denatured jellyfish collagen. the immunostains of vinculin of mg63 cells and fibroblasts seeded on native or denatured jellyfish collagen, allowed us to characterize the focal adhesions of these two cell types. analysis of culture media of mg63 cells and fibroblasts by collagen zymography on native or denatured jellyfish collagen gels, allowed us to demonstrate that mmps of human origin, are able to degrade jellyfish collagen. we estimate in this case that the jellyfish collagen is a good substrate for the development of resorbable biomaterials

La maturation des collagènes fibrillaires est régulée par la libération protéolytique des propeptides qui conduit à la formation spontanée de fibres, à partir des molécules de collagène mature. Les fibres de collagène ainsi formées confèrent résistance et solidité aux tissus. Les métalloprotéases Tolloïdes sont les principales enzymes responsables de la maturation C-terminale des procollagènes. Ces protéases possèdent de nombreux autres substrats et se distinguent par un mode de régulation original, l’utilisation de régulateurs « substrats-spécifiques », qui leur permet de moduler leur activité vis-à-vis de ces différents substrats. Parmi ces régulateurs, les Procollagen C-Proteinase Enhancers (PCPE-1 et 2) semblent jouer un rôle important car ils augmentent l’activité de clivage du C-propeptide des procollagènes jusqu’à 20 fois. Même si PCPE-1 a été découvert en 1985, son mode d’action n’est toujours pas compris. Le principal objectif de cette thèse était donc de mieux comprendre le mécanisme de l’activation. Nous avons commencé par identifier la région minimale de PCPE-1 capable d’activer les Tolloïdes et démontré une très forte coopérativité entre les domaines de cette région. Par ailleurs, nous avons mis en évidence, pour la première fois, un nouveau rôle physiologique pour le domaine C-terminal de PCPE-1 (NTR). Concernant les autres partenaires du complexe de maturation, nous avons observé une interaction d’affinité modérée entre PCPE-1 et la protéase Tolloïde appelée BMP-1 et montré que PCPE-1 se fixe uniquement sur la partie C-terminale du procollagène. Enfin, nous avons mis en évidence que d’autres métalloprotéases, les Méprines, pourraient également jouer un rôle dans la maturation de collagènes fibrillaires en étant régulées négativement par les PCPEs. L’ensemble de ces résultats nous a permis de proposer un nouveau schéma d’interaction pour les PCPEs et de faire de nouvelles hypothèses concernant leur mécanisme d’action / The maturation of fibrillar collagens is a tightly regulated process controlled by two proteolytic cleavages that remove the propeptide regions from procollagen precursors leading to spontaneous assembly of mature collagen molecules into fibrils. These fibrils provide tensile strength and toughness to connective tissues and consequently to the organism itself. The group of extracellular metalloproteinases mostly responsible for procollagen processing are the tolloids. As these enzymes have several functions other than procollagen processing, their activities on different substrates are controlled by a growing number of substrate-specific regulators. The most prominent of these regulators are the procollagen C-proteinase enhancers (PCPEs), of which PCPE-1 is a 55 kDa glycoprotein composed of two CUB and a C-terminal NTR domain, which is capable of enhancing the proteolytic activity of tolloids up to 20-fold. Even though PCPE-1 has been known since 1985 the molecular mechanism of enhancement is still unclear. The aim of this thesis was to understand and characterize this mechanism with the aid of biochemical and biophysical methods. We have identified the minimal unit responsible for enhancing activity. In addition, we propose a mechanism for how the subdomains responsible for enhancement cooperatively bind to procollagen substrates. Furthermore, we have for the first time been able to identify a possible physiological function of the NTR domain. Also, we have identified meprins as new players involved in procollagen processing and this has given valuable insights in the mechanism of action of PCPEs. Finally, we have been able to demonstrate that the interaction of PCPE-1 with procollagen is mostly limited to the C-propeptide region. Based on these findings we propose a new hypothetical interaction mechanism for PCPE-1