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Designing nanostructured stimuli-responsive amphiphilic polypeptides based on intrinsically disordered elastin-like polypeptides and self-assembled motifs
Date
2025
Authors
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Journal ISSN
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Publisher
University of Delaware
Abstract
Natural proteins employ versatile spatial conformations, including folded domains and intrinsically disordered regions, for their hierarchical organization and biological functions. The strategic combination of ordered and disordered domains in polypeptides has facilitated the design and construction of functional biomaterials and offered unique opportunities for orthogonal control of stimuli-responsiveness and self-assembly properties by fine-tuning different domains independently. This dissertation utilized intrinsically disordered elastin-like polypeptides (ELPs) and multiple self-assembled peptides (bundle-forming peptides, BFPs and collagen-like peptides, CLPs) and designed amphiphilic polypeptides with versatile stimuli-responsiveness and self-assembled structures. Further modulation of inter- and intramolecular interactions of different domains regulated their physicochemical properties and self-assembly process. ☐ The architectural control of the self-assembly of a series of block polypeptides comprising a concatenation of an elastin-like peptide and a coiled-coil, bundle-forming peptide (ELP-BFPs) was first demonstrated. Assembly of the polypeptides was controlled by coacervation of the hydrophobic ELP domain, while the type of coiled-coil assembly of the BFP and the specific placement of short histidine tags significantly tuned assembly behavior. Spectrophotometric analysis of self-assembly demonstrated that the transition temperature of assembly can be controlled by the design of the BFP domain and positioning of the His-tags in the constructs. Cryogenic transmission electron microscopy of assembled polypeptides confirmed distinct morphologies, including core-shell particles and multilayer vesicles, depending on the parallel or antiparallel bundle architecture of the block polypeptide. The designed block polypeptides have applications in materials design and highlight the potential for controlling multi-stimuli responsiveness and morphologies through fine control of the architectural features of the component polypeptide domains. ☐ Modulation of intramolecular interactions successfully altered the self-assembly of ELP-BFP by tuning interactions within the disordered ELP domain. We hypothesized that reinforcing intermolecular ELP interactions via metal-histidine coordination could switch the assembly pathway from disordered phase separation to ordered structures. Nuclear magnetic resonance spectrometry (1H-NMR) confirmed the successful coordination of multivalent metal ions (Cu²⁺, Ni²⁺, Zn²⁺) with histidines, and spectrophotometric analysis of the ELP-BFP phase transition behavior in the presence of different metal ions confirmed the alteration of ELP interactions. This interaction induces a dramatic, pH-switchable, morphological transition confirmed by cryogenic transmission electron microscopy. ELP-antiparallel BFP constructs, which form disordered nanodroplets, are transformed into well-defined nanofibers. Similarly, amphiphilic ELP-parallel BFP constructs transition from bilayers to cylindrical micelles. These results demonstrate that modulation of interactions within the disordered ELP domain is a powerful, non-genetic strategy to program hierarchical self-assembly and switch between distinct nanostructures. ☐ The site-specific modification and its potential application with established poly-lysine/poly-aspartic acid (PLK/PLD) coacervate systems were investigated with block polypeptides comprising elastin-like peptides and collagen-like peptides (ELP-CLPs). The surface charge of ELP-CLP nanovesicles was successfully altered by replacing amide ends with carboxyl groups. Confocal fluorescence microscopy was used to visualize the coating of ELP-CLP vesicles on the surface of coacervates. The coating formation was driven by electrostatic attraction between coacervates and ELP-CLP vesicles, which was confirmed by manipulating the surface charge of different components. The formulation factors, such as mixing protocols and mixing orders, showed influence on the coating process. By testing the diffusibility of model molecules, the resulting ELP-CLP-coated coacervate presented selective permeability of molecules and showed potential to be applied as functional coacervates to mimic membrane organelles and carry out bioreactions in vitro. ☐ In conclusion, these investigations highlight the establishment of a new series of amphiphilic polymers with versatile stimuli-responsiveness and self-assembly behavior. The orthogonal control and fine-tuning of inter- and intramolecular interactions of different domains were successfully accomplished based on designed polypeptide amphiphiles.
Description
"At the request of the author or degree granting institution, this graduate work is not available to view or purchase until January 05 2027."-- ProQuest abstract/details page.
Keywords
Amphiphiles, Coacervate, Elastin-like polypeptides, Metal coordination, Polypeptides, Self-assembly