Peptidic "Molecular Beacon" for Collagen

Abstract

Collagen-mimetic peptides (CMP) have been invaluable tools for understanding the structure and function of collagen, which is the most abundant protein in animals. CMPs have also been developed as probes that detect damaged collagen because of the specificity required to form a collagen triple helix. These probes are not, however, ratiometric. Here, we used EPR spectroscopy to determine the end-to-end distances of CMPs that do not form stable homotrimeric helices. We found that those distances are shorter than the distances in the context of a collagen triple helix, suggesting their potential utility as a "molecular beacon" and guiding the choice and location of a pendant fluorophore-quencher pair. We then showed that a molecular beacon based on a glycine-(2S,4S)-4-fluoroproline-(2S,4R)-4-hydroxyproline tripeptide repeat and EDANS-DABCYL pair enabled the ratiometric detection of its binding to both other CMPs and natural mammalian collagen. These results provide guidance for the development of a new modality for detecting damaged collagen in physiological settings.

Figure 1.

Structures of short (n = 3 or 5), monomeric CMPs used in this work.

Figure 2.

End-to-end distance probability distributions of short, monomeric CMPs determined with EPR spectroscopy. All peptides were prepared at 300 μM in 60:40 glycerol-d₈/D₂O containing K₃[Fe(CN)₆] (0.2 equiv). Data were obtained at 20 K and analyzed with DeerAnalysis 2019 software. The distance probability distributions were obtained by Tikhonov regularization in the distance domain. The dotted green vertical lines denote the expected C^α–C^α distance of TOAC residues in the context of a collagen triple helix.

Figure 3.

Structures of long, monomeric CMPs used in this work.

Figure 4.

Analyses of CMP-based molecular beacons (0.8 mM) in 0.1% v/v acetic acid with circular dichroism spectroscopy. (A) Graph showing the effect of temperature on the molar ellipticity at 228 nm of 1:1 mixtures of ^E(PPG)₇^D or ^E(PPG)₇ with (fOG)₇, and of ^E(PPG)₇^D alone. (B) Graph showing the effect of temperature on the molar ellipticity at 228 nm of 1:1 mixtures of ^E(fOG)₇^D or ^E(fOG)₇ with (PPG)₇, and of ^E(fOG)₇^D alone.

Figure 5.

Fluorescence CMP-based molecular beacons (2 μM) were used with natural type I collagen (RatCol) at increasing concentrations (0–2.0 mg/mL). Mixtures were prepared in 0.1% v/v acetic acid, incubated at 55 °C for 10 min, and then equilibrated at 4 °C overnight. The change in relative fluorescence (F/F₀) was calculated with respect to the fluorescence (λ_ex = 336 nm, λ_em = 490 nm) of molecular beacons in the absence of type I collagen.