Stuctural studies of triple-helical proteins

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DESCRIPTION (provided by applicant): Collagen is the most abundant protein in the body, defined by its unique triple-helical conformation and repeating sequence pattern with glycine as every third residue. Biophysical studies are proposed to relate the (Gly-X-Y)n amino acid sequence and breaks in this pattern with molecular features and higher order structure of collagens, which are directly related to their function and pathology. Our studies on the classic triple helix will be extended to characterization of the consequences of natural breaks in the (Gly-X-Y)n pattern found in non-fibrillar collagens, such as type IV collagen in basement membranes and type VII collagen that mediates dermal-epidermal attachment. The effects of the length and sequence of such breaks on triple-helix stability, folding and conformation will be investigated using model peptides. To complement peptide studies, flexibility, folding and enzyme susceptibility will be examined on an expressed bacterial product where a break is introduced between two tandem triple-helix modules. These studies will provide information about the structural consequences of breaks and their biological role. The association of collagen molecules to higher order structures is essential to their mechanical and biological function. The relation between sequence, the process of triple-helix association to higher order structures, and the morphology of the final product will be defined. Studies will be carried out to further characterize non- specific lateral assembly observed for collagen peptides and to introduce electrostatic and hydrophobic residues in peptide sequences to produce more specific axial interactions and defined higher order structures. Small natural breaks in the (Gly-X-Y)n repeat appear to put flanking triple-helix regions out of register, and their impact on self-association will be investigated. Gly missense mutations in fibrillar and non-fibrillar collagen lead to a variety of hereditary diseases. The folding, stability, hydrodynamic and conformational consequences of Gly missense mutations will be characterized in peptides and in an expressed bacterial construct. In non-fibrillar collagens, it is hypothesized that missense mutations interfere with the renucleation mechanism needed to fold through natural breaks. Definition of the fundamental principles of collagen triple-helix molecular structure and association into higher order structures will further our understanding of normal matrix structure/function relationships and enhance the development of collagen-based biomaterials. In addition, it will provide a basis for defining extracellular matrix alterations in disease and for developing drugs which could inhibit the breakdown of collagens in cancer and osteoarthritis.