Mapping the Collagenome with Stapled Collagen-Mimetic Peptides

Tufts University, Medford MA

Investigators

Abstract

With the support of the Chemistry of Life Processes Program in the Chemistry Division, PI Dr. Joshua Kritzer and co-investigators Dr. James Baleja and Dr. Yu-Shan Lin from Tufts University will use a stapling strategy to produce stable collagen-like assemblies that incorporate known interaction sites for human collagen-binding proteins. Collagen is the most abundant protein in mammals, making up 25-35% of all protein in the body. Collagen plays important structural and signaling roles in health, growth, and development, but it is challenging to study because its unique assembled structure is very difficult to produce using defined systems. What is currently known about collagen is largely derived from experiments with processed natural collagens or synthetic peptides, but these systems are a tangle of interconverting structures. This project describes a new and elegant method for locking synthetic peptides into collagen-like structures using chemical bonds or “staples”. The team will use these hyperstable collagen mimics to map protein binding sites and understand cellular signaling of collagens at a much broader scale than previously attainable. The new methods are simple and require only commercially available building blocks, allowing rapid adoption by the larger chemical biology community. The project also supports the development of professional skills workshops for graduate students to prepare them for academic jobs, industry research positions, and other science careers. Intramolecular cross-links (“staples”) have been pursued for decades as a means of restricting peptide conformation. In preliminary work, the Kritzer Lab demonstrated that stapling using the artificial amino acid 4-mercaptoproline (4-MP) provides a solution to the longstanding problem of how to stabilize collagen triple-helical structure. 4-MP staples are powerfully stabilizing for collagen triple helices, producing melting temperatures as high as 85°C. This approach is much more compact, synthetically efficient, high-yielding, and modular than all prior approaches to stabilizing collagen-mimetic peptides. These hyperstable collagen-like assemblies provide new opportunities to address critical questions in collagen biophysics and biology, including the straightforward production of heterotrimeric collagens in a programmable manner, understanding the functional effects of sequence variation among native collagen sequences, and effects of sequence and structure on the binding of collagen receptors including integrins, platelet glycoproteins, and DDR tyrosine kinases. The unprecedented small size and high yields of 4-MP-stapled collagen-mimetic peptides will also allow this project to produce the first-ever collagen arrays. Such arrays could comprehensively expand the field's understanding of the “human collagenome” including features important for protein binding, cell signaling, cell adherence, and cell migration. Overall, this project from PI Dr. Joshua Kritzer and co-PIs Dr. James Baleja and Dr. Yu-Shan Lin seeks to uncover molecular mechanisms of collagen-receptor interactions that will greatly accelerate chemistry and biology research on collagen and the extracellular matrix. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.