Peptoid Residues Make Diverse, Hyperstable Collagen Triple-Helices

Abstract

The triple-helical structure of collagen, responsible for collagen's remarkable biological and mechanical properties, has inspired both basic and applied research in synthetic peptide mimetics for decades. Since non-proline amino acids weaken the triple helix, the cyclic structure of proline has been considered necessary, and functional collagen mimetic peptides (CMPs) with diverse sidechains have been difficult to produce. Here we show that N-substituted glycines (N-glys), also known as peptoid residues, exhibit a general triple-helical propensity similar to or greater than proline, allowing synthesis of thermally stable triple-helical CMPs with unprecedented sidechain diversity. We found that the N-glys stabilize the triple helix by sterically promoting the preorganization of individual CMP chains into the polyproline-II helix conformation. Our findings were supported by the crystal structures of two atomic-resolution N-gly-containing CMPs, as well as experimental and computational studies spanning more than 30 N-gly-containing peptides. We demonstrated that N-gly sidechains with diverse exotic moieties including a 'click'-able alkyne and a photo-sensitive sidechain can be incorporated into stable triple helices, enabling functional applications such spatio-temporal control of cell adhesion and migration on a gelatin matrix. The folding principles discovered in this study open up opportunities for a new generation of collagen mimetic therapeutics and materials with extraordinary properties. design principles uncovered in this study drastically expand the library of residues with high triple-helical propensity, which has immense implications for a new generation of collagenmimetic therapeutics and materials. Results and DiscussionTriple-helical stability of CMPs with N-glys. To investigate the triple-helical folding propensity of peptoids residues, we inserted a series of N-gly guests into the central X position of a conventional CMP host peptide with the sequence: Ac-(GlyProHyp)3-Gly-X-Hyp-(GlyProHyp)3-NH2 (designated as X-CMP, Fig. 1b, and supplementary Materials and Methods) 34 . We measured the X-CMPs' triple-helical stability via thermal unfolding experiments monitored under circular dichroism (CD, Fig. 1c, Supplementary Section 3 and Table S1), first in a group of N-glys with sidechains selected from the canonical amino acids (Fig. 1d). It is known that Pro is the most stabilizing amino acid at position X, and substitution from Pro to another canonical amino acid clearly reduces Tm by 4-17 °C (Fig. 1d, column AA) 14 . Surprisingly, we found that many of the N-glys with canonical sidechains were at least as stable as Pro, and almost all N-gly residues were more stable than their amino acid counterparts (Fig. 1d, exception: Nval), with the biggest Tm difference seen between Nphe-and Phe-CMP (Fig. 1c). These results demonstrate that, in the X position of the CMP, shifting the sidechain from the Cα carbon to the nitrogen (i.e., transforming a canonical amino acid to its peptoid analogue) may improve triple-he...

Terminal Repeats in Collagen Triple-Helices

“…(GPO)7: 55 °C]. 24,27 The N-termini of most existing POG-based crystal structures are disordered (Figure S3), probably because those N-terminal Pro residues are not acetylated (Table S2), resulting in charge repulsion disrupting the H-bonding (Figure 6a). Meanwhile, it was recently reported that the positive charges of ammonium groups destabilize the triple-helix [e.g., H-(POG)7-NH2, pH 7.4 v.s.…”

“…(GPO)8: 44.5 °C ] 25,26 , though sometimes significant [(POG)7: 43 °C v.s. (GPO)7: 55 °C] 24,27 , are often attributed to terminal functional groups and charges, 27 peptide concentrations, as well as methods and errors from different measurements, including heating rates. 18,28 So, are these collagen repeats indeed structurally equivalent, or can they make terminal cappings with different characteristics?…”

“…Next, we surveyed existing crystal structures of CMP triple-helices in the Protein Data Bank (PDB, see Table S3) to search for evidence of structural differences between CMPs with POGand GPO-terminal repeats. 23,24,[32][33][34][35][36][37][38][39][40][41] We analyzed the B-factor of each CMP structure as it often correlates with the flexibility and internal motion in protein crystallography. 42 We plotted normalized B-factors of all non-hydrogen atoms along each CMP triple-helix: while all structures have elevated structural flexibility at the termini, a general trend of higher terminal B-factor was noted for the POG-sequences (Figure 3a and Figure S3,4).…”

“…Of these, only (POG)n and (GPO)n are traditionally used in collagen research. 18,21,23,24 Interestingly, in the UniProt database, the recognized triple-helix regions of most types of human collagen chains are both initiated and terminated as GXY, rather than XYG (Table S1).…”

Application of (4R)-aminoproline in peptide engineering: conformational bias and pH-responsiveness revisited

“…Decoration of the proline residues may endow collagen mimics with additional functions. [38][39][40] The incorporation of Amp, for instance, may create a highly polar collagen triple helix owing to the high polarity of the individual Amp residues (Fig. 3).…”