Background: Functional collagen fibrils are formed through the self-assembly of collagen triple helices. Results: A designed triple helical peptide self-assembles into collagen-like mini-fibrils. Conclusion:The sequence of the triple helix alone is sufficient to "code" for the axial, periodic structure of the mini-fibrils. Significance: The work demonstrates an approach to achieve collagen-like fibrils through the self-assembly of designed triple helices.

It has proven challenging to obtain collagen-mimetic fibrils by protein design. We recently reported the self-assembly of a mini-fibril showing a 35 nm, D-period like, axially repeating structure using the designed triple helix Col108. Peptide Col108 was made by bacterial expression using a synthetic gene; its triple helix domain consists of three pseudo-identical units of amino acid sequence arranged in tandem. It was postulated that the 35 nm d-period of Col108 mini-fibrils originates from the periodicity of the Col108 primary structure. A mutual staggering of one sequence unit of the associating Col108 triple helices can maximize the inter-helical interactions and produce the observed 35 nm d-period. Based on this unit-staggered model, a triple helix consisting of only two sequence units is expected to have the potential to form the same d-periodic mini-fibrils. Indeed, when such a peptide, peptide 2U108, was made it was found to self-assemble into mini-fibrils having the same d-period of 35 nm. In contrast, no d-periodic mini-fibrils were observed for peptide 1U108, which does not have long-range repeating sequences in its primary structure. The findings of the periodic mini-fibrils of Col108 and 2U108 suggest a way forward to create collagen-mimetic fibrils for biomedical and industrial applications.

Collagen fibrils represent a unique case of protein folding and self‐association. We have recently successfully developed triple‐helical peptides that can further self‐assemble into collagen‐mimetic mini‐fibrils. The 35 nm axially repeating structure of the mini‐fibrils, which is designated the d ‐period, is highly reminiscent of the well‐known 67 nm D ‐period of native collagens when examined using TEM and atomic force spectroscopy. We postulate that it is the pseudo‐identical repeating sequence units in the primary structure of the designed peptides that give rise to the d ‐period of the quaternary structure of the mini‐fibrils. In this work, we characterize the self‐assembly of two additional designed peptides: peptide Col877 and peptide Col108rr. The triple‐helix domain of Col877 consists of three pseudo‐identical amino acid sequence units arranged in tandem, whereas that of Col108rr consists of three sequence units identical in amino acid composition but different in sequence. Both peptides form stable collagen triple helices, but only triple helices Col877 self‐associate laterally under fibril forming conditions to form mini‐fibrils having the predicted d ‐period. The Co108rr triple helices, however, only form nonspecific aggregates having no identifiable structural features. These results further accentuate the critical involvement of the repeating sequence units in the self‐assembly of collagen mini‐fibrils; the actual amino acid sequence of each unit has only secondary effects. Collagen is essential for tissue development and function. This novel approach to creating collagen‐mimetic fibrils can potentially impact fundamental research and have a wide range of biomedical and industrial applications.

An elegantly simple and probably ancient molecular mechanism of allostery is described for the Escherichia coli arginine repressor ArgR, the master feedback regulator of transcription in L-arginine metabolism. Molecular dynamics simulations with ArgRC, the hexameric domain that binds L-arginine with negative cooperativity, reveal that conserved arginine and aspartate residues in each ligand-binding pocket promote rotational oscillation of apoArgRC trimers by engagement and release of hydrogen-bonded salt bridges. Binding of exogenous L-arginine displaces resident arginine residues and arrests oscillation, shifting the equilibrium quaternary ensemble and promoting motions that maintain the configurational entropy of the system. A single L-arg ligand is necessary and sufficient to arrest oscillation, and enables formation of a cooperative hydrogen-bond network at the subunit interface. The results are used to construct a free-energy reaction coordinate that accounts for the negative cooperativity and distinctive thermodynamic signature of L-arginine binding detected by calorimetry. The symmetry of the hexamer is maintained as each ligand binds, despite the conceptual asymmetry of partially-liganded states. The results thus offer the first opportunity to describe in structural and thermodynamic terms the symmetric relaxed state predicted by the concerted allostery model of Monod, Wyman, and Changeux, revealing that this state is achieved by exploiting the dynamics of the assembly and the distributed nature of its cohesive free energy. The ArgR example reveals that symmetry can be maintained even when binding sites fill sequentially due to negative cooperativity, which was not anticipated by the Monod, Wyman, and Changeux model. The molecular mechanism identified here neither specifies nor requires a pathway for transmission of the allosteric signal through the protein, and it suggests the possibility that binding of free amino acids was an early innovation in the evolution of allostery.