Coiled Coil Peptide Tiles (CCPTs): Expanding the Peptide Building Block Design with Multivalent Peptide Macrocycles

Abstract

Owing to their synthetic accessibility and protein-mimetic features, peptides represent an attractive biomolecular building block for the fabrication of artificial biomimetic materials with emergent properties and functions. Here, we expand the peptide building block design space through unveiling the design, synthesis, and characterization of novel, multivalent peptide macrocycles (96mers), termed coiled coil peptide tiles (CCPTs). CCPTs comprise multiple orthogonal coiled coil peptide domains that are separated by flexible linkers. The constraints, imposed by cyclization, confer CCPTs with the ability to direct programmable, multidirectional interactions between coiled coil-forming "edge" domains of CCPTs and their free peptide binding partners. These fully synthetic constructs are assembled using a convergent synthetic strategy via a combination of native chemical ligation and Sortase A-mediated cyclization. Circular dichroism (CD) studies reveal the increased helical stability associated with cyclization and subsequent coiled coil formation along the CCPT edges. Size-exclusion chromatography (SEC), analytical high-performance liquid chromatography (HPLC), and fluorescence quenching assays provide a comprehensive biophysical characterization of various assembled CCPT complexes and confirm the orthogonal colocalization between coiled coil domains within CCPTs and their designed on-target free peptide partners. Lastly, we employ molecular dynamics (MD) simulations, which provide molecular-level insights into experimental results, as a supporting method for understanding the structural dynamics of CCPTs and their complexes. MD analysis of the simulated CCPT architectures reveals the rigidification and expansion of CCPTs upon complexation, i.e., coiled coil formation with their designed binding partners, and provides insights for guiding the designs of future generations of CCPTs. The addition of CCPTs into the repertoire of coiled coil-based building blocks has the potential for expanding the coiled coil assembly landscape by unlocking new topologies having designable intermolecular interfaces.

Figure 1

(a) Sequence length of CCPTs lies between the lengths typically associated with traditional, short peptides and recombinantly expressed proteins. (b) CCPTs exhibit multidirectional interactions with the potential for building programmable, predetermined architectures (e.g., discrete 3D polyhedral architectures and extended nanostructures) via controlled associations along CCPT edges.

Figure 2

Peptide sequences. (a) Sequences of T_AAA and T_ABC. (b) Sequences of de novo peptides that form orthogonal parallel coiled coil heterodimers. (c) A, A’, B, B’, C, and C’ form three on-target pairings: AA’, BB’, and CC’.

Figure 3

CCPT synthesis. (a) Convergent synthesis of T_AAA (i: thioester formation, ii: NCL, iii: desulfurization, iv: SrtA-mediated cyclization). (b) Linear precursor, L_AAA, is assembled from the NCL of F1 and F2, which are synthesized using SPPS. The synthesis and purification of T_AAA were confirmed via (c) analytical high-performance liquid chromatography (HPLC) and (d) liquid chromatography mass spectrometry (LCMS).

Figure 4

Biophysical characterization of A', L_AAA, T_AAA, and their complexes. (a) CD spectra of A, L_AAA, and T_AAA. (b) CD thermal denaturation plots of A, L_AAA, T_AAA, AA’, L_3AA’, and T_3AA’. CD spectra of (c) A’, L_AAA, and L_3AA’ and (d) A’, T_AAA, and T_3AA’. (e) SEC traces of A, L_AAA, and T_AAA prior to the addition of A’ (bottom) and after the addition of A’ (top).

Figure 5

Characterization of T_ABC and T_ABC complexes. (a) LCMS spectrum of T_ABC. (b) CD spectra of T_ABC, A’, B’, C’, and T_{AA’BB’CC’}. (c) CD thermal denaturation plots of T_ABC and T_{AA’BB’CC’}. (d) Visual representation of the various dimeric and multimeric T_ABC complexes that were prepared. (e) SEC traces of T_{AA’BB’CC’}, T_ABCC’, T_ABB’C, T_AA’BC, and T_ABC. (f) Analytical HPLC traces of SEC fractions containing the major species in solution. Integration of the peaks confirm the expected stoichiometric ratios of T_ABC and free peptides (see Table S4).

Figure 6

Fluorescence quenching assays. Fluorescence quenching data for complexes comprising (a) T_A*BC, (b) T_AB*C, and (c) T_ABC*. Yellow stars and gray pentagons represent the sites of 4CF and Mse, respectively, within the peptide sequences.

Figure 7

MD simulation analysis of CCPTs and CCPT complexes. (a) Representative final MD simulation trajectories (400th ns) for all seven CCPTs and CCPT complexes. (b) Overlayed MD trajectories of 200–395 ns structures (Δt = 5 ns). (c) Calculated fraction of helical residues (out of the expected number of helical residues) within T_AAA or T_ABC for all CCPTs and CCPT complexes studied. Average SASA for (d) T_AAA (black line) and T_AAA within T_3AA’ (red line) and (e) T_ABC (black line) and T_ABC within T_{AA’BB’CC’} (blue line).