Positive and Negative Design Leads to Compositional Control in AAB Collagen Heterotrimers

Although collagen is the most abundant protein in the human body and has at least 28 types, research involving collagen mimetic systems only recently began to consider the innate ability of collagen to control helix composition and register. Collagen triple helices can be homotrimeric or heterotrimeric, and while some types of natural collagen form only one specific composition of helix, others can form multiple compositions. It is critical to fully understand and, if possible, reproduce the control that native collagen has on helix composition and register. In this Article, we utilize both positive and negative design for the assembly of specific AAB heterotrimers using charged amino acids to form intrahelix electrostatic interactions, which promote heterotrimer formation and simultaneously discourage homotrimers. Homotrimers are further discouraged by reducing hydroxyproline content, which would otherwise lead to nonspecific promotion of triple helix formation. We combine peptides in a 2:1 ratio in which the more abundant peptide has a charge 1/2 and opposite of the less abundant peptide, which can result in the formation of a zwitterionically neutral AAB heterotrimer. Using this approach, we are able to design collagen mimetic systems with full control over the composition of the resulting triple helix. All previous reports on synthetic collagen heterotrimers have shown mixed populations with respect to composition due to varying amounts of residual homotrimers. Our results yield a greater understanding of the self-assembly of collagenous sequences as well as provide a novel design scheme, both positive and negative, for the synthesis of extracellular matrix mimetics.