Fluorophores Nucleate and Stabilize Collagen Triple Helices – <i>En Route</i> to Better Diagnostic Tools for Fibrosis and Tissue Repair

Abstract Fluorescently labeled collagen model peptides (CMPs) are useful tools for monitoring fibrotic processes and tissue repair. Fine‐tuning the propensity of the CMP to form a collagen triple helix is crucial since only single‐stranded but not triple‐helical CMPs integrate efficiently into damaged or remodeling tissue. Yet the impact of fluorophores on the folding behavior of CMPs and triple helix stability has so far been underappreciated. Here, we show that N‐terminal fluorophores enhance triple helix stability and nucleation. Thermal denaturation and temperature jump experiments showed that triple helix stability and nucleation are proportionally affected by the accessible surface area of the fluorophore. For biological applications, fluorophores with a small surface area are therefore beneficial. Furthermore, we show that fluorophores bearing ionizable groups with an appropriate p K a value, such as commonly used fluorescein, can be used to create pH‐responsive assemblies. Our findings highlight the importance of carefully selecting appropriate fluorophores and spacers when developing fluorescent probes for biological applications.