Nothing is solid about proteins. Governing rules and established laws are constantly broken. As an example, the last decade and a half have witnessed the fall of one of the major paradigms in structural biology. Contrarily to the more than a century-old belief that the unique function of a protein is determined by its unique structure, which, in its turn, is defined by the unique amino acid sequence, many biologically active proteins lack stable tertiary and/or secondary structure either entirely or at their significant parts. Such intrinsically disordered proteins (IDPs) and hybrid proteins containing ordered domains and functional IDP regions (IDPRs) are highly abundant in nature, and many of them are associated with various human diseases. Such disordered proteins and regions are very different from ordered and well-structured proteins and domains at a variety of levels and possess well-recognizable biases in their amino acid compositions and amino acid sequences. A characteristic feature of these proteins is their exceptional structural heterogeneity, where different parts of a given polypeptide chain can be ordered (or disordered) to different degrees. As a result, a typical IDP/IDPR contains a multitude of potentially foldable, partially foldable, differently foldable or not foldable structural segments. This distribution of conformers is constantly changing in time, where a given segment of a protein molecule has different structures at different time points. The distribution is also constantly changing in response to changes in the environment. This mosaic structural organization is crucial for their functions and many IDPs are engaged in biological functions that rely on high conformational flexibility and that are not accessible to proteins with unique and fixed structures. As a result, the functional repertoire of IDPs complements that of ordered proteins, with IDPs/IDPRs being often involved in regulation, signaling and control. This Sprenger Briefs volume is dedicated to IDPs and IDPRs and an attempt is made to compress a massive amount of knowledge and into a digest that aims to be of use to those wishing a fast entry into this promising field of structural biology.

Intrinsically Disordered Proteins

Characterization and environmental applications of clay–biochar composites

Removal of methylene blue from water with montmorillonite nanosheets/chitosan hydrogels as adsorbent

Clay nanomaterials are an emerging class of 2D biomaterials of interest due to their atomically thin layered structure, charged characteristics, and well-defined composition. Synthetic nanoclays are plate-like polyions composed of simple or complex salts of silicic acids with a heterogeneous charge distribution and patchy interactions. Due to their biocompatible characteristics, unique shape, high surface-to-volume ratio, and charge, nanoclays are investigated for various biomedical applications. Here, a critical overview of the physical, chemical, and physiological interactions of nanoclay with biological moieties, including cells, proteins, and polymers, is provided. The state-of-the-art biomedical applications of 2D nanoclay in regenerative medicine, therapeutic delivery, and additive manufacturing are reviewed. In addition, recent developments that are shaping this emerging field are discussed and promising new research directions for 2D nanoclay-based biomaterials are identified.

2D Nanoclay for Biomedical Applications: Regenerative Medicine, Therapeutic Delivery, and Additive Manufacturing

Montmorillonite nanoclay as a multifaceted drug-delivery carrier: A review

Ca/Na montmorillonite and natural Wyoming bentonite (MX-80) have been studied experimentally and theoretically. For a clay system in equilibrium with pure water, Monte Carlo simulations predict a large swelling when the clay counterions are monovalent, while in presence of divalent counterions a limited swelling is obtained with an aqueous layer between the clay platelets of about 10 A. This latter result is in excellent agreement with X-ray scattering data, while dialysis experiments give a significantly larger swelling for Ca montmorillonite in pure water. Obviously, there is one "intra-lamellar" and a second "extra-lamellar" swelling. Montmorillonite in contact with a salt reservoir containing both Na(+) and Ca(2+) counterions will only show a modest swelling unless the Na(+) concentration in the bulk is several orders of magnitude larger than the Ca(2+) concentration. The limited swelling of clay in presence of divalent counterions is a consequence of ion-ion correlations, which reduce the entropic repulsion as well as give rise to an attractive component in the total osmotic pressure. Ion-ion correlations also favor divalent counterions in a situation with a competition with monovalent ones. A more fundamental result of ion-ion correlations is that the osmotic pressure as a function of clay sheet separation becomes nonmonotonic, which indicates the possibility of a phase separation into a concentrated and a dilute clay phase, which would correspond to the "extra-lamellar" swelling found in dialysis experiments. This idea also finds support in the X-ray scattering spectra, where sometimes two peaks corresponding to different lamellar spacings appear.

Ca/Na Montmorillonite: Structure, Forces and Swelling Properties.

Aqueous dispersions of pure sodium and calcium smectite clays with platelet sizes on the order of a few hundred nanometers were characterized using a combination of cryo-transmission electron microscopy (cryo-TEM) and small-angle X-ray scattering (SAXS). With monovalent sodium counterions the clay is dispersed as individual platelets, as seen by cryo-TEM, that order into a nematic phase. From SAXS a one-dimensional swelling of the day in water is observed with the characteristic spacing h(s) = delta/phi(c), where h(s) is the separation between the platelets, delta = 1 nm is the effective platelet thickness, and phi(c) is the clay volume fraction in the sample. In calcium montmorillonite, on the other hand, cryo-TEM images dearly show the presence of tactoids, where the platelets have aggregated into stacks with a periodic spacing of 2 nm. From imaging a large number of tactoids the distribution function f(N) far the number of platelets per tactoid was estimated, and the average number (N) approximate to 10. The characteristic 2 nm spacing as well as the small number of platelets per tactoid was also confirmed by SAXS. The present study demonstrates that cryo-TEM, with carefully prepared specimen, is a very useful technique to characterize clay dispersions, particularly in aggregated systems. (Less)

Microstructural and Swelling Properties of Ca and Na Montmorillonite: (In Situ) Observations with Cryo-TEM and SAXS

Aqueous dispersions of Ca montmorillonite contain small clusters of clay platelets, often named "tactoids". In these tactoids, the platelets are arranged parallel to each other with a constant spacing of 1 nm. We have used small-angle X-ray scattering (SAXS) to determine the average number of platelets per tactoid, . We found that this number depends on the platelet size, with larger platelets yielding larger tactoids. For a dispersion in equilibrium with a mixed electrolyte solution, the tactoid size also depends on the ratio of divalent to monovalent cations in the reservoir. Divalent counterions are strongly favored in this competition and will accumulate in the tactoids. In dispersions of pure sodium montmorillonite, that are equilibrated with a mixture of Na+ and Ca2+ cations, the Na+ cations initially cause a repulsion between the platelets, but the divalent ions rapidly replace the monovalent ones and lead to the formation of tactoids, typically within less than one hour based on the divalent to monovalent ratio. This cation exchange as well as tactoid formation can be semiquantitatively predicted from Monte Carlo simulations. (Less)

Tactoid Formation in Montmorillonite

Studying the freezing of saltwater on a molecular level is of fundamental importance for improving freeze desalination techniques. In this study, we investigate the freezing process of NaCl solutions using a combination of X-ray diffraction and molecular dynamics simulations (MD) for different salt-water concentrations, ranging from seawater conditions to saturation. A linear superposition model reproduces well the brine rejection due to hexagonal ice Ih formation and allows us to quantify the fraction of ice and brine. Furthermore, upon cooling at T = 233 K, we observe the formation of NaCl·2H2O hydrates (hydrohalites), which coexist with ice Ih. MD simulations are utilized to model the formation of NaCl crystal hydrates. From the simulations, we estimate that the salinity of the newly produced ice is 0.5% mass percent (m/m) due to ion inclusions, which is within the salinity limits of fresh water. In addition, we show the effect of ions on the local ice structure using the tetrahedrality parameter and follow the crystallite formation using the ion coordination parameter and cluster analysis.

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Brine rejection and hydrate formation upon freezing of NaCl aqueous solutions.

Understanding and controlling defect formation during the assembly of nanoparticles is crucial for fabrication of self-assembled nanostructured materials with predictable properties. Here, time-resolved small-angle X-ray scattering was used to probe the temporal evolution of strain and lattice contraction during evaporation-induced self-assembly of oleate-capped iron oxide nanocubes in a levitating drop. We show that the evolution of the strain and structure of the growing mesocrystals is related to the formation of defects as the solvent evaporated and the assembly process progressed. Superlattice contraction during the mesocrystal growth stage is responsible for the rapidly increasing isotropic strain and the introduction of point defects. The crystal strain, quantified by the Williamson-Hall analysis, became more anisotropic due to the formation of stress-relieving dislocations as the mesocrystal growth was approaching completion. Understanding the formation of the transformation of defects in mesocrystals and superlattices could assist in the development of optimized assembly processes of nanoparticles with multifunctional properties.

Mo Segad

Papers

Ca/Na Montmorillonite: Structure, Forces and Swelling Properties.

Microstructural and Swelling Properties of Ca and Na Montmorillonite: (In Situ) Observations with Cryo-TEM and SAXS

Tactoid Formation in Montmorillonite

Brine rejection and hydrate formation upon freezing of NaCl aqueous solutions.

Temporal Evolution of Superlattice Contraction and Defect-Induced Strain Anisotropy in Mesocrystals during Nanocube Self-Assembly