Plant virus nucleoproteins, through self-assembly, form monodisperse, nanoscale structures with high symmetry and multiple binding functionalities. The filamentous plant viruses, which generate uniform high aspect ratio nanostructures, are of specific interest, as purely synthetic techniques face significant hurdles. Interest in Potato virus X (PVX), characterized by its filamentous structure of 515 ± 13 nm, has been growing among materials scientists. Both genetic modification and chemical coupling have been described for enhancing PVX's functionalities and for creating PVX-based nanomaterials to serve applications in health and materials science. In the context of environmentally safe materials, particularly those that are not harmful to crops like potatoes, we outlined methods to inactivate PVX. This chapter introduces three means of inactivating PVX, ensuring its non-infectivity to plants, whilst preserving both its structural form and functional properties.

To ascertain the charge transfer (CT) mechanisms in biomolecular tunnel junctions, the establishment of electrical contacts using a non-invasive method that maintains the integrity of the biomolecules is crucial. Despite the presence of multiple techniques for establishing biomolecular junctions, we explain the EGaIn method, which provides the capacity for easy formation of electrical contacts with biomolecule monolayers under typical lab conditions, enabling the exploration of CT as a function of voltage, temperature, or magnetic field. 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.

Protein cages are increasingly being utilized to formulate Pickering emulsions, highlighting their utility in molecular delivery. Despite the growing curiosity, the approaches to examine the liquid-liquid interface are few in number. This chapter's focus is on the standard methods for developing and analyzing protein cage-stabilized emulsions. Small-angle X-ray scattering (SAXS), in conjunction with dynamic light scattering (DLS), intrinsic fluorescence spectroscopy (TF), and circular dichroism (CD), serve as characterization methods. Through the integration of these methods, the precise nanoscale configuration of the protein cage at the oil-water interface is revealed.

Millisecond time-resolved small-angle X-ray scattering (TR-SAXS) is now achievable owing to recent advancements in X-ray detectors and synchrotron light sources. quality use of medicine The beamline setup, experimental strategy, and important observations for stopped-flow TR-SAXS experiments investigating ferritin assembly are outlined in this chapter.

Protein cages, objects of intense scrutiny in cryogenic electron microscopy, include both naturally occurring and synthetic constructs; chaperonins, which aid in protein folding, and virus capsids are prime examples. A wide range of protein morphologies and functions are apparent, with certain proteins being nearly universal, and others restricted to a small number of organisms. To achieve better resolution in cryo-electron microscopy (cryo-EM), protein cages often display high symmetry. To image biological subjects, cryo-electron microscopy employs an electron probe on meticulously vitrified samples. To preserve the sample's native state as closely as possible, a porous grid is employed for rapid freezing in a thin layer. During electron microscope imaging, the grid is perpetually maintained at cryogenic temperatures. After image acquisition is finalized, a selection of software tools can be engaged for the purpose of analyzing and reconstructing three-dimensional structures from the two-dimensional micrograph images. Cryo-EM's adaptability extends to samples of substantial size or complex composition, rendering it a superior technique to NMR or X-ray crystallography for structural biology investigations. Significant enhancements to cryo-EM results in recent years have been driven by concurrent hardware and software advancements, culminating in the attainment of true atomic resolution from vitrified aqueous specimens. Cryo-EM advancements, especially concerning protein cages, are discussed here, accompanied by insights drawn from our work.

E. coli expression systems facilitate the straightforward production and engineering of bacterial encapsulins, protein nanocages. Well-characterized encapsulin, originating from Thermotoga maritima (Tm), boasts a known three-dimensional structure. Unsurprisingly, without modification, cell penetration is negligible, making it an alluring candidate for targeted drug delivery applications. The potential applications of encapsulins as drug delivery vehicles, imaging agents, and nanoreactors have recently prompted their engineering and study. Thus, the significance of the capability to alter the surface of these encapsulins, such as by the addition of a targeting peptide sequence or other functional characteristics, is apparent. High production yields and straightforward purification methods are, ideally, integrated with this. Genetically modifying the surfaces of Tm and Brevibacterium linens (Bl) encapsulins, considered model systems, is described in this chapter as a means to purify and characterize the resultant nanocages.

Protein chemical modifications bestow novel functionalities or fine-tune pre-existing roles. Although methods for protein modification have proliferated, the selective modification of two different reactive protein sites with unique chemical reagents presents a persistent difficulty. A simple method for selectively modifying the internal and external surfaces of protein nanocages using two distinct chemical agents, leveraging the molecular size filtering of the surface pores, is highlighted in this chapter.

Ferritin, the naturally occurring iron storage protein, is a widely recognized template for the preparation of inorganic nanomaterials, achieved through the sequestration of metal ions and complexes within its cage. Applications for ferritin-based biomaterials span a wide range of fields, including bioimaging, drug delivery, catalysis, and biotechnology. Applications of the ferritin cage are enabled by its unique structural features, which exhibit remarkable stability at elevated temperatures (up to approximately 100°C), and its adaptability across a broad pH range (2-11). The infiltration of metals within the ferritin structure is a key operation in the production of ferritin-based inorganic bionanomaterials. A metal-immobilized ferritin cage is directly applicable in various situations, or it can be used as a starting point for making uniformly sized, water-soluble nanoparticles. bioactive properties This protocol outlines the procedure for trapping metal ions inside ferritin shells and subsequently crystallizing the resulting metal-ferritin complex for structural investigation.

The fundamental understanding of iron incorporation into ferritin protein nanocages is essential in the field of iron biochemistry/biomineralization and its bearing on human health and disease. Although the acquisition and mineralization of iron differ mechanistically within the ferritin superfamily, we describe the techniques suitable for investigating iron accumulation in all ferritin proteins through in vitro iron mineralization. In this chapter, we detail how the non-denaturing polyacrylamide gel electrophoresis, coupled with Prussian blue staining (in-gel assay), proves useful for evaluating the iron-loading efficiency of ferritin protein nanocages, determined by the relative quantity of incorporated iron. Similarly, the absolute size of the iron mineral core and the aggregate iron within its nanoscale cavity are both determinable, the former by transmission electron microscopy, and the latter via spectrophotometry.

The interactions between individual building blocks within three-dimensional (3D) array materials constructed from nanoscale components are a primary focus of significant interest, owing to the potential for emergent collective properties and functions. Virus-like particles (VLPs), protein cages, exhibit a distinctive advantage as building blocks for intricate higher-order assemblies, owing to their exceptional uniformity in size and the capacity for tailoring novel functionalities through chemical and/or genetic modifications. This chapter details a protocol for developing a novel class of protein-based superlattices, termed protein macromolecular frameworks (PMFs). A method for assessing the catalytic activity of enzyme-enclosed PMFs, demonstrating improved catalytic performance due to the preferential partitioning of charged substrates into the PMF, is also outlined in this work.

The self-organization of proteins in nature has been a source of inspiration for researchers to create vast supramolecular systems built from a spectrum of protein motifs. Aminocaproic In the context of hemoproteins utilizing heme as a cofactor, several reported approaches exist for the fabrication of artificial assemblies, taking on forms like fibers, sheets, networks, and cages. This chapter explores the design, preparation, and characterization of cage-like micellar assemblies, where chemically modified hemoproteins possess hydrophilic protein units linked to hydrophobic molecules. Procedures for the construction of specific systems utilizing cytochrome b562 and hexameric tyrosine-coordinated heme protein as hemoprotein units are outlined, including heme-azobenzene conjugate and poly-N-isopropylacrylamide molecules.

Protein cages and nanostructures, emerging as promising biocompatible medical materials, hold great potential as vaccines and drug carriers. The recent emergence of engineered protein nanocages and nanostructures has paved the way for leading-edge applications in the fields of synthetic biology and biopharmaceuticals. A simple method of constructing self-assembling protein nanocages and nanostructures is the creation of a fusion protein. This fusion protein, composed of two distinct proteins, results in the formation of symmetric oligomers.