Influence of linker design on the stability, folding, and assembly of tethered collagen-mimetic peptides

Covalently tethered collagen-mimetic peptides (CMPs) serve as synthetically programmable molecules for studying the collagen triple helix fold. Tethered CMPs, which overcome limitations that are inherent to their untethered counterparts (e.g., decreased stability, concentration-dependent folding, and slow folding kinetics), are constructed using a variety of templating strategies. Despite the plethora of reports of tethered CMPs in literature, there has been little exploration in determining the effects that the linker region, which connects the CMP sequence to the trivalent scaffold, has on the stability and assembly of tethered CMP triple helices. Here, we systematically study the influence of linker length and composition on the stability, folding, and assembly of covalently tethered CMPs. We synthesized a family of tethered CMPs comprising PEGylated linkers of different lengths (CTH-PEG2, CTH-PEG4, CTH-PEG6) to assess how linker length influences the properties of CMP triple helices. Moreover, we synthesized tethered CMPs comprising hydrophobic (CTH-HEX) and peptide-based linkers (CTH-GSG). All tethered CMPs possess a triblock sequence architecture that directs the assembly of resulting triple helices into nanostructures. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) confirm that tethered CMPs assemble into nanosheets and nanoribbons. Circular dichroism (CD) spectroscopy reveals that increasing the length of the flexible linker systematically decreases the thermal stability of tethered CMP triple helices and alters their folding kinetics. Furthermore, CD data of CTH-HEX and CTH-GSG indicate that linker composition can play a role, though limited, in influencing the stability and folding properties of CMP triple helices. The presented work highlights how tuning the linker design - both length and composition - serves as a facile route towards fine-tuning the properties of CMP triple helices and their assemblies without perturbing the CMP sequence architecture, and will provide guidance to future researchers in choosing appropriate linkers for their own applications.