Programmable nanomaterials via hierarchical assembly of computationally designed parallel folding coiled coil peptide bundlemers

Abstract

Peptide-based biomaterials offer an exceptional opportunity to bridge the precision of molecular design with the emergent order of hierarchical nanostructures. The overarching goal of this thesis is to establish a unified framework for the programmable design of peptide materials through computationally engineered coiled coil bundlemers, which function as supermolecular building blocks for both physical self-assembly and covalent network formation. By integrating molecular modeling, experimental synthesis, and advanced structural characterization, this work explores how to control sequence parameters and crosslinking chemistry to achieve tunable structural, mechanical, and liquid-crystalline properties in peptide systems. ☐ At the foundation of this research is the computational design of parallel coiled-coil bundlemers, tetrameric peptide nanoparticles with C₂ symmetry, that display all N-termini on one end and all C-termini on the opposite end. This unique conformation allows controlled end-to-end assembly and selective chemical modification. The first part of this work demonstrates that these bundlemers can be covalently conjugated through thiol-maleimide click reactions to form discrete, monodisperse “dibundlemer” rods as confirmed by small-angle X-ray scattering (SAXS). Spectroscopic analyses, including circular dichroism (CD) and Förster resonance energy transfer (FRET), validated the parallel configuration of the α helix peptides and confirmed agreement with computational predictions. Concentrated dibundlemer solutions exhibited optical birefringence and nematic liquid crystalline behavior, revealing that even short, rigid peptide rods could form an ordered liquid crystalline phase. This study establishes a bottom-up route for constructing anisotropic peptide nanostructures. ☐ Building upon these findings, the second part investigates how sequence length and net charge states regulate the assembly of parallel coiled coil bundlemers into nanofibrillar and liquid crystalline architectures. By varying the net charge state of the designed peptides, noncovalent interactions, including the dipolar interaction, hydrogen bonding, and electrostatic interactions, collectively guide hierarchical assembly. Transmission electron microscopy (TEM) and SAXS confirmed that bundlemers assemble end-to-end into uniform fibrils, whose morphologies depend strongly on pH conditions. At moderate concentrations, the system exhibits liquid crystalline and hexagonally packed phases, while at higher concentrations, chain entanglement and increased viscosity induce kinetic trapping and disorder. Controlled sonication disrupts this metastable state, reconfiguring the system into a lamellar-hexagonal composite phase consistent with smectic B (Sm-B) liquid crystalline phase. These findings underscore the critical roles of surface charge distribution and sequence design in governing nanofibril assembly, liquid-crystal formation, and crystal-like hierarchical organization in peptide-based biomaterials. ☐ The final part focuses on reinforcing self-assembled peptide architectures through covalent crosslinking. Covalently linked parallel bundlemer systems were developed by incorporating thiol-maleimide and thiol-ene click chemistry pairs into the parallel bundlemer sequences. These modifications enabled the creation of mechanically robust networks while preserving the inherent α-helical structure, as verified by circular dichroism spectroscopy. TEM imaging showed that both designs maintained fibrillar assembly, while SAXS revealed consistent cylindrical geometries corresponding to fibril dimensions. The thiol-maleimide design (Prll_1c_NMCC) exhibited simultaneous conjugation and assembly, forming sheet-like or film-like morphologies, whereas the thiol-ene design (Prll_1c_TE) generated fibrils that transformed into large aggregated clusters upon UV irradiation. MALDI analysis confirmed successful crosslinking reactions. Rheological measurements revealed dramatic improvements in mechanical strength for both systems. The storage modulus exceeded the loss modulus, signifying gel-like behavior. Together, these results demonstrate that introducing covalent conjugation pathways into peptide assemblies significantly enhances structural stability and mechanical tunability without compromising their native folding or self-assembly behavior. ☐ Collectively, this work demonstrates that precise molecular design enables the creation of programmable peptide materials that span the nanoscale to the macroscale by integrating computational modeling, controlled self-assembly, and covalent chemistry.