Self-assembly of plant virus nucleoproteins results in monodisperse, nanoscale structures with a high degree of symmetry and polyvalency. Uniform high aspect ratio nanostructures, a notable feature of filamentous plant viruses, present a significant hurdle to purely synthetic approaches. The materials science community has shown interest in Potato virus X (PVX) due to its filamentous structure, which measures approximately 515 ± 13 nanometers. Both genetic engineering and chemical conjugation techniques have been documented as ways to enhance the functionalities of PVX and generate PVX-based nanomaterials for use in healthcare and material science applications. To develop environmentally safe materials—meaning materials not harmful to crops like potatoes—we outlined methods for inactivating PVX. This chapter explores three approaches to inactivating PVX, achieving non-infectiousness in plants, all while preserving its structure and function.

In order to study the mechanisms of charge movement (CT) in biomolecular tunnel junctions, it is required to fabricate electrical contacts using a non-invasive technique that leaves the biomolecules unmodified. Diverse approaches to biomolecular junction formation exist; however, this paper focuses on the EGaIn method, which facilitates the straightforward creation of electrical contacts to biomolecule monolayers in typical laboratory setups, allowing for the exploration of CT dependent on voltage, temperature, or magnetic field parameters. This non-Newtonian liquid metal, an alloy of gallium and indium, gains its shapeable properties through a thin surface layer of gallium oxide (GaOx) – allowing for the creation of cone-shaped tips or stabilization within microchannels. EGaIn structures, which make stable contacts with monolayers, offer the opportunity for a highly detailed investigation of CT mechanisms across biomolecules.

Applications in molecular delivery are prompting further investigation into Pickering emulsions stabilized by protein cages. Despite the rising fascination, the procedures to investigate the liquid-liquid interface are limited. This chapter presents the standard practices for crafting and evaluating the properties of protein-cage-stabilized emulsions. Circular dichroism (CD), coupled with dynamic light scattering (DLS), intrinsic fluorescence spectroscopy (TF), and small-angle X-ray scattering (SAXS), constitutes the characterization methodology. Understanding the protein cage's nanostructure at the oil-water boundary is enabled by the application of these combined methods.

Recent advancements in synchrotron light sources and X-ray detectors have unlocked the ability for millisecond-resolution time-resolved small-angle X-ray scattering (TR-SAXS). antitumor immunity This chapter's focus is on stopped-flow TR-SAXS experiments to study ferritin assembly, with specific details on the beamline layout, experimental protocol, and points requiring attention.

Protein cages, a subject of widespread investigation in cryogenic electron microscopy, demonstrate a fascinating array of natural and synthetic variations, from enzymes like chaperonins assisting protein folding to the protective shells of viruses, virus capsids. Protein structures and functionalities demonstrate a vast diversity, with some being nearly universally found, and others restricted to only a few organisms. To achieve better resolution in cryo-electron microscopy (cryo-EM), protein cages often display high symmetry. Employing electron probes on vitrified samples, cryo-electron microscopy (cryo-EM) is the technique for imaging biological subjects. A thin, porous grid rapidly freezes a sample in a layer, aiming to maintain its native state as closely as possible. During electron microscope imaging, the grid is perpetually maintained at cryogenic temperatures. Following the acquisition of images, a range of software programs can be used to analyze and reconstruct three-dimensional structures from the two-dimensional micrograph data. Cryo-EM provides a valuable methodology for structural biology studies by enabling the examination of samples that are either too extensive in size or heterogeneous in composition for techniques like NMR or X-ray crystallography. The past few years have witnessed substantial progress in cryo-EM, spurred by innovations in both hardware and software, culminating in the ability to achieve true atomic resolution using vitrified aqueous samples. Cryo-EM advancements, especially concerning protein cages, are discussed here, accompanied by insights drawn from our work.

Bacterial encapsulins, being a class of protein nanocages, are readily produced and engineered within E. coli expression systems. Encapsulin from Thermotoga maritima (Tm) is well-understood in terms of its structure, and, without any modifications, it is not readily incorporated by cells. This characteristic makes it a prime candidate for targeted pharmaceutical delivery. Recent engineering and study of encapsulins indicate their potential for use as drug delivery carriers, imaging agents, and nanoreactors. Consequently, the potential to alter the exterior of these encapsulins, including the addition of a peptide sequence for targeting or other functions, is critical. Ideally, this should be coupled with high production yields and straightforward purification methods. This chapter details a method for genetically altering the surfaces of Tm and Brevibacterium linens (Bl) encapsulins, using them as models, to achieve purification and subsequently characterize the resulting nanocages.

Chemical alterations in protein structure either produce new functions or influence their inherent functions. Various strategies for protein modification have been created, yet selective alteration of two distinct reactive sites with varying chemical agents remains a complex undertaking. This chapter details a straightforward method for selectively modifying the inner and outer surfaces of protein nanocages using two distinct chemicals, leveraging the molecular size-filtering properties of the surface pores.

Using the naturally occurring iron storage protein, ferritin, as a template, the fixation of metal ions and metal complexes within its cage structure has enabled the development of inorganic nanomaterials. In fields such as bioimaging, drug delivery, catalysis, and biotechnology, ferritin-based biomaterials show significant promise. The ferritin cage's structural distinctiveness, allowing exceptional stability at elevated temperatures (approximately up to 100°C) and a vast pH adaptability (2-11), facilitates its use in a multitude of interesting applications. The infiltration of metals within the ferritin structure is a key operation in the production of ferritin-based inorganic bionanomaterials. In applications, metal-immobilized ferritin cages can be employed directly or as precursors to create uniformly sized and water-soluble nanoparticles. Neuroscience Equipment Considering this approach, we provide a detailed protocol for the immobilization of metals within ferritin cages, and the ensuing crystallization procedure for the metal-ferritin composite to facilitate structural determination.

Within the realm of iron biochemistry/biomineralization, deciphering the iron accumulation processes within ferritin protein nanocages has been a key focus, directly relevant to health and disease states. Although the mechanisms of iron acquisition and mineralization vary among ferritin proteins within the superfamily, we present methodologies for exploring iron accumulation in all ferritin proteins via an in vitro iron mineralization process. The in-gel assay, combining non-denaturing polyacrylamide gel electrophoresis with Prussian blue staining, is reported in this chapter as a valuable technique for evaluating the loading efficiency of iron within ferritin protein nanocages by quantifying the relative iron content. Likewise, the electron microscopy technique allows for the determination of the iron mineral core's absolute dimensions, while the spectrophotometric method quantifies the total iron within its nanocystic interior.

Three-dimensional (3D) array materials, built from nanoscale building blocks, have attracted significant interest because of their potential to exhibit collective properties and functionalities stemming from the interactions of their constituent components. Homogeneity of size and the capacity for chemical or genetic engineering of novel functionalities make protein cages, particularly virus-like particles (VLPs), outstanding components for the fabrication of higher-order assemblies. This chapter elucidates a protocol for the creation of a novel class of protein-based superlattices, designated protein macromolecular frameworks (PMFs). Moreover, we present a showcase method for evaluating the catalytic activity of enzyme-enclosed PMFs, whose catalytic efficacy is elevated by the favored localization of charged substrates within the PMF compartment.

The self-assembly of proteins in nature has motivated scientists to develop large-scale supramolecular architectures incorporating a variety of protein modules. Menadione Artificial assemblies of hemoproteins, with heme acting as a cofactor, have been reported using several methods, yielding diverse structures such as fibers, sheets, networks, and cages. In this chapter, the design, preparation, and characterization of cage-like micellar assemblies for chemically modified hemoproteins are presented, demonstrating the attachment of hydrophilic protein units to hydrophobic molecules. The specific systems' construction using cytochrome b562 and hexameric tyrosine-coordinated heme protein as hemoprotein units, incorporating heme-azobenzene conjugate and poly-N-isopropylacrylamide as attached components, are detailed in the procedures.

As promising biocompatible medical materials, protein cages and nanostructures are well-suited for applications like vaccines and drug carriers. Recent innovations in the design and creation of protein nanocages and nanostructures have created groundbreaking opportunities for novel applications in synthetic biology and biopharmaceuticals. To create self-assembling protein nanocages and nanostructures, a simple approach is to design a fusion protein comprised of two diverse proteins which organize into symmetrical oligomeric units.