Biotinylation

of peptides was found to be effectively 100%. Peptides, listed in Table 1, were checked for helicity [23]. Samples of Toolkit peptide III-24 and CRPcys were dissolved at 2.5 mg mL−1 in cold 10 mM acetic acid. Peptides were held at 4 °C for 2 h, 1 d, 3 d, 14 d, or frozen (−20 °C) for 2 h, 1 d, 3 d, 14 d, or 80 d before immediate gel filtration analysis; a final sample was frozen for 14 d but thawed ten times during that period, mimicking sequential sampling. The experiment was repeated, except that nitrogen was bubbled through the acetic acid for 2 min before using it to dissolve peptide. As peptides are normally stored at 4 °C, we also retrieved 9–48-month-old stock samples of CRPcys, GPPcys, Baf-A1 GFOGERcys, II-56, III-04 and III-24, all kept at 1 mg mL−1 in 10 mM acetic acid. Samples were analyzed using mass-spectrometry, gel filtration, and

reduced cysteine quantified using Ellman’s reagent, 5,5′-dithio-bis(2-nitrobenzoic acid) (Sigma D8130) [2]. The heterobifunctional reagent SPDP (Sigma P3415) was dissolved learn more in dry ethanol (50 mM), added to 3 mM peptide pre-dissolved in 0.1 M NaHCO3 (1.5 equiv.), and the mixture flushed with N2 gas. After 1 h, peptide was dialyzed overnight at 4 °C in 0.01 M acetic acid (one change), stored at 4 °C or freeze-dried and stored at −80 °C. Peptide III-24 (2.5 mg mL−1) was dissolved in 10 mM phosphate-buffered saline pH 7.4 containing 2 mM TCEP, heated briefly to 70 °C and allowed to fold for 18 h overnight at 4 °C. It was filtered and loaded into a DynaPro Titan DLS instrument pre-equilibrated at 4 °C. The sample was probed at 4–50 °C, being equilibrated at each temperature for 5 min. Data was handled as previously described [17] and the hydrodynamic radius in nm used to calculate a predicted molecular

weight as appropriate for different polymers: a rod-like triple helix, an aggregate of triple helices, or a denatured single chain. We did not observe any collagenous gel formation. Peptide cross-linking and helicity was measured by preparing 800 μL samples at 0.25 mg mL−1 in 10 mM phosphate buffered saline (pH 7.4) and loading onto a Bio-sep Sec-S2000 Gel filtration column (300 mm × 21.2 mm, 5 μM bead size, 14.5 nm average pore size) at 10 °C, equilibrated in the same buffer. Running isocratically, the eluant was monitored at 214 nm. PDK4 For peptide III-24, the column was additionally run at 40 °C to investigate the increased stability conferred by cross-linking, and peptide III-24, III-04, GPPcys, and GFOGERcys, were additionally sampled at 60 °C to obtain a peptide polymer profile (Suppl. Table S1b). Overlapping gel filtration sample peaks derived from different peptide polymers require mathematical deconvolution into components. Three major effects describe a gel filtration peak: first, bead pore size and homogeneity (r ± σ, Fig. 2a, Suppl. Section 2.10); second, diffusion and inhomogeneity of flow, using the axial dispersion coefficient, L ( Fig. 2b and Suppl.