Browsing by Author "Pochan, Darrin J."
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Item Computational Design of Single-Peptide Nanocages with Nanoparticle Templating(Molecules, 2022-02-12) Villegas, José A.; Sinha, Nairiti J.; Teramoto, Naozumi; Von Bargen, Christopher D.; Pochan, Darrin J.; Saven, Jeffery G.Protein complexes perform a diversity of functions in natural biological systems. While computational protein design has enabled the development of symmetric protein complexes with spherical shapes and hollow interiors, the individual subunits often comprise large proteins. Peptides have also been applied to self-assembly, and it is of interest to explore such short sequences as building blocks of large, designed complexes. Coiled-coil peptides are promising subunits as they have a symmetric structure that can undergo further assembly. Here, an α-helical 29-residue peptide that forms a tetrameric coiled coil was computationally designed to assemble into a spherical cage that is approximately 9 nm in diameter and presents an interior cavity. The assembly comprises 48 copies of the designed peptide sequence. The design strategy allowed breaking the side chain conformational symmetry within the peptide dimer that formed the building block (asymmetric unit) of the cage. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) techniques showed that one of the seven designed peptide candidates assembled into individual nanocages of the size and shape. The stability of assembled nanocages was found to be sensitive to the assembly pathway and final solution conditions (pH and ionic strength). The nanocages templated the growth of size-specific Au nanoparticles. The computational design serves to illustrate the possibility of designing target assemblies with pre-determined specific dimensions using short, modular coiled-coil forming peptide sequences.Item Genetic Fusion of Thermoresponsive Polypeptides with UCST-type Behavior Mediates 1D Assembly of Coiled-Coil Bundlemers(Angewandte Chemie International Edition, 2023-05-09) Patkar, Sai S.; Tang, Yao; Bisram, Arriana M.; Zhang, Tianren; Saven, Jeffery G.; Pochan, Darrin J.; Kiick, Kristi L.Graphical Abstract: Available at: https://doi.org/10.1002/anie.202301331 Computationally designed coiled coil-forming peptides were functionalized with thermoresponsive resilin-like polypeptides (RLPs) of various lengths and produced via biosynthetic methods in bacterial expression hosts. Interactions between RLPs upon cooling below their upper critical solution temperature (UCST) resulted in nanofibrillar assembly. Abstract Thermoresponsive resilin-like polypeptides (RLPs) of various lengths were genetically fused to two different computationally designed coiled coil-forming peptides with distinct thermal stability, to develop new strategies to assemble coiled coil peptides via temperature-triggered phase separation of the RLP units. Their successful production in bacterial expression hosts was verified via gel electrophoresis, mass spectrometry, and amino acid analysis. Circular dichroism (CD) spectroscopy, ultraviolet-visible (UV/Vis) turbidimetry, and dynamic light scattering (DLS) measurements confirmed the stability of the coiled coils and showed that the thermosensitive phase behavior of the RLPs was preserved in the genetically fused hybrid polypeptides. Cryogenic-transmission electron microscopy and coarse-grained modeling revealed that functionalizing the coiled coils with thermoresponsive RLPs leads to their thermally triggered noncovalent assembly into nanofibrillar assemblies.Item Peptide hydrogels – versatile matrices for 3D cell culture in cancer medicine(Frontiers Media S.A., 2015-04-20) Worthington, Peter; Pochan, Darrin J.; Langhans, Sigrid A.; PeterWorthington, Darrin J. Pochan and Sigrid A. Langhans; Worthington, Peter; Pochan, Darrin J.Traditional two-dimensional (2D) cell culture systems have contributed tremendously to our understanding of cancer biology but have significant limitations in mimicking in vivo conditions such as the tumor microenvironment. In vitro, three-dimensional (3D) cell culture models represent a more accurate, intermediate platform between simplified 2D culture models and complex and expensive in vivo models. 3D in vitro models can overcome 2D in vitro limitations caused by the oversupply of nutrients, and unphysiological cell–cell and cell–material interactions, and allow for dynamic interactions between cells, stroma, and extracellular matrix. In addition, 3D cultures allowfor the development of concentration gradients, including oxygen, metabolites, and growth factors, with chemical gradients playing an integral role in many cellular functions ranging from development to signaling in normal epithelia and cancer environments in vivo. Currently, the most common matrices used for 3D culture are biologically derived materials such as matrigel and collagen. However, in recent years, more defined, synthetic materials have become available as scaffolds for 3D culture with the advantage of forming well-defined, designed, tunable materials to control matrix charge, stiffness, porosity, nanostructure, degradability, and adhesion properties, in addition to other material and biological properties. One important area of synthetic materials currently available for 3D cell culture is short sequence, self-assembling peptide hydrogels. In addition to the review of recent work toward the control of material, structure, and mechanical properties, we will also discuss the biochemical functionalization of peptide hydrogels and how this functionalization, coupled with desired hydrogel material characteristics, affects tumor cell behavior in 3D culture.