Design and Analysis of De Novo Collagen-Like Heterotrimers using Charge Repulsion-Based Negative Design

Abstract

Collagen, the most ubiquitous protein in the body, is the primary component of the extracellular matrix (ECM). The collagen molecule's folding mechanics have not yet been fully elucidated, which has hindered the progress of de novo synthetic collagen matrices and biomaterials. Our lab seeks to construct rationally-designed collagen-related peptides (CRPs) for structural mimetic studies, allowing for further analysis of the interactions guiding the self-assembly of collagen's signature triple helix. We have designed a set of three polypeptides A, B, and C, with positively charged arginines placed at the N-terminus, center, and C-terminus respectively. The rest of the polypeptide is made of highly stable Pro-Hyp-Gly repeats. The design was conceived such that an A-B-C heterotrimer will be favored due to the presence of repulsive charges on adjacent positions in all competing products. These peptides, after being combined in solution, were tested for the presence and stability of formed triple helices. Consistent with previous work, CRP folding may be non-cooperative and nucleated, processively leading from the C-terminus to the N-terminus. Interestingly, repulsive interactions do not effectively determine specificity of assembly. To further understand the role of unfavorable interactions in specificity of assembly, we are constructing negatively-charged aspartate analogs of the current system. Structural modeling suggests aspartates should have a larger impact on stability and specificity of assembly.