NMR And Fluorescence Characterization Of Collagen Mimic Peptides

As the most abundant protein in mammals, collagen is the main component of skin, bone, tendon,cornea, cartilage and ligament. Collagen provides a molecular scaffold for connective tissues, and is highly bioactive, mediating a variety of biological functions including cell adhesion, migration and division. The mutation or degradation of collagen is involved in various diseases such as Osteogenesis Imperfecta, arthritis, thrombosis and tumor. Therefore, investigation of collagen structure and novel detection methodologies will help us develop improved diagnosis and treatment for collagen-related diseases. In this thesis, we have utilized NMR techniques to investigate the structure of peptides mimicking the biologically important sites in collagen, and have developed novel fluorescence approaches to detect the characteristic triple helix structure of collagen. These studies have greatly advanced our understanding of collagen structure, function and pathology.First, we have characterized the structure and dynamics of the biologically important sites in collagen using NMR techniques. Fibrillar collagens perfectly maintain the repetitive(Gly-X-Y)n sequence pattern, and even a single Gly substitution leads to pathological conditions. However,nonfibrillar collagens contain plenty natural interruption sites, which are considered to play important biological functions. It is still not clear why natural interruptions and Gly mutations result in totally contradictory consequences, though both of them have disrupted the the repetitive(Gly-X-Y)n sequence pattern. We have designed two groups of peptides to mimic typical Gly-Ala and Gly-Ser mutations and similar natural interruption sites in collagen, respectively. We have integrated NMR,CD and computational simulation techniques to compare the the differences of stability, conformation,dynamics and folding between the two peptides mimicking Gly mutation and interruption sites,contributing to our understanding of the molecular mechanism underlying their different biological roles.In addition, we have established novel fluorescence assays to detect collagen triple helix structure.The characterization of collagen triple helix structure traditionally relies on NMR and CD techniques,which require a large amount of samples as well as expensive isotopic labeling or distinct thermalstabilities. The fluorescence assay only require a tiny amount of unlabeled peptides, and it is highly sensitive to detect collagen triple helix. We have designed a dye-labeled collagen-like peptide, which can form both homotrimer and heterotrimer structures with totally different fluorescence properties.We can detect the formation of heterotrimers at nM level and determine their exact helical composition using this fluorescence self-quenching assay. Meanwhile, we have created a graphene oxide-peptide platform, which can specifically identify unfolded collagen fragments. This conformation-sensitive, sequence-specific platform provides a novel effective approach to sense collagen.