INTELLECTUAL MERIT: Fibrous collagen is the most abundant mammalian protein. Fibers of collagen are processed in situ as they form in vivo, and this processing introduces irreversible changes that cannot be undone by any physical or chemical extraction process. Consequently, a synthetic analog is extremely desirable as a route to obtaining useful quantities of this versatile protein for materials or therapeutic applications. This proposal presents a biologically inspired approach whereby collagen triple helices self-assemble and are then guided to fibrillar oligomerization. The key process is an energetically coupled stabilization of the triple helix with concomitant setting of the staggered polypeptide register. The approach uses metal-directed assembly to form two fundamental structures: capping trimers and propagating trimers. The capping trimers are aligned by metal complex formation at either the N-terminal or the C-terminal ends (N-cap and C-cap, respectively). They present "sticky" connections at the other end, in which the terminal residues are offset from each other. The propagating trimers are assembled by metal complexation in the middle of the sequence, and present staggered sequences at either end that are complementary to the N/C caps. Binding of the N-cap and C-cap trimers to the appropriate ends of the propagating species, rather than forming an N-Cap/C-Cap dimer, is driven by electrostatic complementarity. It is possible consequently to access much larger procollagen subunits than has hitherto been possible, and the assembly process itself is subject to a much higher degree of control.
BROADER IMPACTS: There is currently no practical route to a synthetic collagen, and success in the proposed research could have far reaching benefits for many aspects of regenerative medicine. The PI has collaborations with the College of Medicine at the University of Vermont (UVM) with groups working on spinal reconstruction and blood clotting, both of which activities would benefit from the availability of synthetic collagen. The project will support the work of one graduate student in a highly interdisciplinary environment as well as 4 - 5 undergraduates per year. Funds are budgeted for support of the undergraduate research students. As a participant in Project SEED of the American Chemical Society, the PI will provide opportunities for economically disadvantaged high school students to participate in a meaningful research project in his laboratory during the summer months.
Collagen is one of the most abundant proteins in the human body. Collagen serves a range of functions as the key protein anchoring connective tissue. Collagen is a constituent of bone, anchors muscles to bone, and associates with tissues to provide shape and form,including skin. Collagen synthesis is very complex organizing individual proteins into bundles of three, then organizing these molecules into larger structures that make up collagen fibrils. Collagen synthesis has been very difficult to study either in vivo or in vitro. The first step begins with understanding how collagen proteins self-associate into trimers. The PI has developed biochemical/biophysical techniques to study protein self-assembly using smaller peptides that replicate portions of a protein. This approach uses a metal ion center to anchor the peptides that will self-assemble. Using peptide synthesis and combinatorial chemistry we can synthesize a vast number of peptides that contain common collagen features, but have points in the peptides that can be varied with a number of amino acids. Using the combinatorial peptide synthesis technique to generate peptides and our method of assembling these peptides, we have the ability to study in detail nuances of collagen-like molecule self-assembly. The results of this grant have produced four refereed scientific publications that describe a theory for metal ion assembled microcollagen heterotrimers and describe the physical chemistry electrostatic properties governing the self-assembly process. These results will hopefully guide others studying collagen synthesis and the biophysics of collagen. In the process of this work three graduate students in chemistry participated in research on the project and are coauthors on the four publications. All three have completed their theses (on M.S. and two Ph.D. degrees) and have taken full time employment. All three students come from diverse backgrounds including an African-American woman. In addition, a variety of undergraduate biochemistry and chemistry students have worked during the academic year and summer months receiving training and doing research on this project.
