This application is based on a series of new technologies that now make it possible --- for the first time --- to begin true protein engineering of type I collagen, the most abundant structural protein of higher organisms. Therefore, it is possible to resolve a series of fundamental questions about the role of collagen in normal biology and its role in a series of human diseases caused by mutations in the structure of the protein. The new technologies that now make this proposal feasible are: (a) A highly reproducible system whereby we can examine assembly of type I collagen into fibrils by enzymic cleavage of partially processed procollagen in a physiological buffer and over a physiological range of temperatures. (b) An efficient host-vector system in which milligram quantities of recombinant procollagen can be generated. (c) An experimental design whereby we overcome the technical problems posed by the large and repetitive coding sequences of type I procollagen by preparing gene constructs that consist of exchangeable DNA cassettes coding for the 4 D- periods, the telopeptides, and the propeptides of the protein. (d) Synthesis of most of the DNA cassettes required and successful expression of a parent construct that can readily be modified to obtain expression of a series of highly informative hybrid, chimeric, and mutated recombinant procollagens. We will employ these experimental tools and strategies to pursue the following Specific Aims: A. Define the structural domains and then the amino acid sequences of the type I collagen molecule that are critical for normal assembly of the protein into fibrils. B. Define the high- and low-melting domains in the triple helix of type I collagen and then define the amino acid sequences that characterize the domains. C. Define the structural domains and then the amino acid sequences of type I collagen that are binding sites for cells and cell receptors. D. Define the structural domains and then the amino acid sequences of type I collagen that contain binding sites for fibromodulin and decorin. E. Define how the binding of fibromodulin or decorin alters the assembly of type I collagen into fibrils in terms of the kinetics and thermodynamics of the assembly process and the morphology of the fibrils formed.
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| Adachi, E; Katsumata, O; Yamashina, S et al. (1999) Collagen II containing a Cys substitution for Arg-alpha1-519. Analysis by atomic force microscopy demonstrates that mutated monomers alter the topography of the surface of collagen II fibrils. Matrix Biol 18:189-96 |
| Prockop, D J; Fertala, A (1998) The collagen fibril: the almost crystalline structure. J Struct Biol 122:111-8 |
