posted on 2020-08-21, 13:06authored bySarah
A. H. Hulgan, Abhishek A. Jalan, I-Che Li, Douglas R. Walker, Mitchell D. Miller, Abigael J. Kosgei, Weijun Xu, George N. Phillips, Jeffrey D. Hartgerink
Collagen
mimetic peptides (CMPs) self-assemble into a triple helix
reproducing the most fundamental aspect of the collagen structural
hierarchy. They are therefore important for both further understanding
this complex family of proteins and use in a wide range of biomaterials
and biomedical applications. CMP self-assembly is complicated by a
number of factors which limit the use of CMPs including their slow
rate of folding, relatively poor monomer–trimer equilibrium,
and the large number of competing species possible in heterotrimeric
helices. All of these problems can be solved through the formation
of isopeptide bonds between lysine and either aspartate or glutamate.
These amino acids serve two purposes: they first direct self-assemble,
allowing for composition and register control within the triple helix,
and subsequently can be covalently linked, fixing the composition
and register of the assembled structure without perturbing the triple
helical conformation. This self-assembly and covalent capture are
demonstrated here with four different triple helices. The formation
of an isopeptide bond between lysine and glutamate (K–E) is
shown to be a faster and higher yielding reaction than lysine with
aspartate (K–D). Additionally, K–E amide bonds increase
the thermal stability, improve the refolding capabilities, and enhance
the triple helical structure as compared to K–E supramolecular
interactions, observed by circular dichroism. In contrast, covalent
capture of triple helices with K–D amide bonds occurs slower,
and the captured triple helices do not have enhanced helical structure.
The crystal structure of a triple helix captured through the formation
of three K–E isopeptide bonds unequivocally demonstrates the
connectivity of the amide bonds formed while also confirming the preservation
of the canonical triple helix. The rate of reaction and yield for
covalently captured K–E triple helices along with the excellent
preservation of triple helical structure demonstrate that this approach
can be used to effectively capture and stabilize this important biological
motif for biological and biomedical applications.