The long term objectives of this proposal are to develop NMR techniques to probe the folding of collagen-like triple helical peptides, and to use the resulting structural and dynamical information to understand the basis for collagen folding diseases that arise from mutations in the triple helix motif. The substitution of a single Gly by another amino acid breaks the characteristic (Gly-X-Y) repeat in the sequence of the collagen triple helix and results in connective tissue disease. Defective folding has been implicated in the etiology of the disease and the nature of the folding defect remains to be defined. A detailed structural and kinetic picture of the folding pathway of the triple helix will be obtained with NMR structure, dynamics and folding experiments. Equilibrium NMR studies of the native trimer form and the unfolded monomer form and kinetic NMR experiments, including real time folding experiments and characterization of a long lived transient intermediate(s), will be combined to provide a detailed view of the folding mechanism. Examination of the sequence dependence of folding of the native triple helix will serve as a basis for studying how Gly/X mutations result in abnormal folding. It is hypothesized that the local amino acid sequence surrounding the mutation site and the identity of the substituted residue are determining factors in """"""""misfolding"""""""" of the triple helix. NMR experiments will be applied to collagen-like peptides that are designed to model the sequence features found at mutation sites in order to define the nature of the folding defect at the substitution site and at remote positions. These NMR-based studies may pave the way to an understanding of the mechanism by which a Gly substitution can lead to pathological consequences and may shed light on the relationship between folding mechanisms and clinical phenotype.
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