Concentration- and pH-Dependent Oligomerization of the Thrombin-Derived C-Terminal Peptide TCP-25

Abstract

Peptide oligomerization dynamics affects peptide structure, activity, and pharmacodynamic properties. The thrombin C-terminal peptide, TCP-25 (GKYGFYTHVFRLKKWIQKVIDQFGE), is currently in preclinical development for improved wound healing and infection prevention. It exhibits turbidity when formulated at pH 7.4, particularly at concentrations of 0.3 mM or more. We used biochemical and biophysical approaches to explore whether the peptide self-associates and forms oligomers. The peptide showed a dose-dependent increase in turbidity as well as α-helical structure at pH 7.4, a phenomenon not observed at pH 5.0. By analyzing the intrinsic tryptophan fluorescence, we demonstrate that TCP-25 is more stable at high concentrations (0.3 mM) when exposed to high temperatures or a high concentration of denaturant agents, which is compatible with oligomer formation. The denaturation process was reversible above 100 µM of peptide. Dynamic light scattering demonstrated that TCP-25 oligomerization is sensitive to changes in pH, time, and temperature. Computational modeling with an active 18-mer region of TCP-25 showed that the peptide can form pH-dependent higher-order end-to-end oligomers and micelle-like structures, which is in agreement with the experimental data. Thus, TCP-25 exhibits pH- and temperature-dependent dynamic changes involving helical induction and reversible oligomerization, which explains the observed turbidity of the pharmacologically developed formulation.

The role of full-length apoE in clearance of Gram-negative bacteria and their endotoxins

“…The signal obtained for the protein alone was subtracted from all spectra. The results are expressed as an average of three independent experiments ± standard error of the mean (SEM) (30).…”

High-throughput prediction and characterization of antimicrobial peptides from multi-omics datasets of Chinese tubular cone snail (Conus betulinus)

“…We manually collected 11 putative AMPs from the multi-omics datasets (Supplementary Table 5). Four candidate peptides (TCP-1, UBI_31-38, cbTbeta and Histone_H4) were truncated according to those verified active AMP truncations that were reported in previous studies, such as 31-38 residues of UBI (Brouwer et al, 2006), and 25amino-acid thrombin-derived C-terminal peptide (TCP-25) (Petruk et al, 2020;Dahlman et al, 2021).…”

Differential Internalization of Thrombin-Derived Host Defense Peptides into Monocytes and Macrophages

“…As we show that TCPs are detectable in macrophages for at least 10 h, internalized TCPs may enhance bacterial killing in macrophages as well. Indeed, TCPs may switch their mode of action from mainly anti-inflammatory at neutral pH to antibacterial at acidic pH [ 40 ] due to differences in peptide oligomerization [ 41 ]. However, as pre-incubation of E. coli BioParticles with TCPs does not alter phagocytosis by human and murine macrophages, and neither does pre-incubation of macrophages with the peptides prior to adding bacteria [ 12 ], potential changes in intracellular killing due to the presence of peptides may not reflect the major physiological role of TCPs.…”