The broad long term objectives of the grant are to use NMR to provide insight into how variations in amino acid sequence affect conformation, dynamics, and folding of the collagen (Gly-X-Y)n triple helix motif. These studies will be applied to understand biologically significant sites in collagen, in particular those related to recognition and disease.
Specific Aim #1 : Two well-defined interactions sites in the triple helix of fibrillar collagens, the matrix metalloproteinase (MMP) cleavage site, critical to normal degradation of connective tissue, and the a2b1 integrin binding site, critical to cell adhesion processes, will be examined by NMR to determine local conformation and backbone dynamics to elucidate the different modes of recognition and to understand the role of neighboring residues in modulating recognition sites.
Specific Aim #2 : Breaks in the Gly-X-Y repeating pattern are found normally in the triple helix domain of non fibrillar collagens in basement membrane. NMR investigations will elucidate the nature of the interruptions and test the hypothesis that these breaks alter structure and serve as recognition sites.
Specific Aim #3 : The substitution of a single Gly by another amino acid breaks the characteristic repeating (Gly-X-Y)n sequence pattern and results in connective tissue disease that can have lethal to non-lethal phenotypes. Alterations arising from Gly to X mutations may occur at different levels ranging from defective folding of the triple-helix to loss of higher order function such as ligand binding or self recognition. NMR conformation, dynamics and folding studies on model peptides will test the hypothesis that Gly mutations reduce the binding affinities in critical recognition sites by altering the backbone fluctuations.
Specific Aim #4 : Modern NMR techniques including the measurement of dihedral angles and distance restraints will be adapted to the triple helix by use of doubly labeled peptides that will be obtained either by peptide synthesis or through a recombinant bacterial expression system. Understanding structure/function correlation and the mechanism by which enzymes recognize the triple helix may provide information that will aid in in the development of targets for the treatment of several diseases including osteoarthritis and cancer, and will aid in the discovery of new therapies for collagen diseases.
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