Cryo-EM Structure of Mechanosensitive Channel YnaI Using SMA2000: Challenges and Opportunities

Mechanosensitive channels respond to mechanical forces exerted on the cell membrane and play vital roles in regulating the chemical equilibrium within cells and their environment. High-resolution structural information is required to understand the gating mechanisms of mechanosensitive channels. Protein-lipid interactions are essential for the structural and functional integrity of mechanosensitive channels, but detergents cannot maintain the crucial native lipid environment for purified mechanosensitive channels. Recently, detergent-free systems have emerged as alternatives for membrane protein structural biology. This report shows that while membrane-active polymer, SMA2000, could retain some native cell membrane lipids on the transmembrane domain of the mechanosensitive-like YnaI channel, the complete structure of the transmembrane domain of YnaI was not resolved. This reveals a significant limitation of SMA2000 or similar membrane-active copolymers. This limitation may come from the heterogeneity of the polymers and nonspecific interactions between the polymers and the relatively large hydrophobic pockets within the transmembrane domain of YnaI. However, this limitation offers development opportunities for detergent-free technology for challenging membrane proteins.

Cloning, expression, and purification of chaperone AcrH from Aeromonas hydrophilausing Escherichia coli host cells

Aeromonas hydrophila (A. hydrophila) is a gram-negativebacterium, using the type III secretion system (T3SS). In the T3SS, a key structure is a translocon that inserts into the target membrane and forms a channel for bacterial toxins into the host cell. A. hydrophila is pathogenic to different organisms, including humans and aquatic animals (especially domestic animals with high economic value in Vietnam and the world, such as fishes, shrimps, amphibians). The pore completes the channel from bacteria to host, is composed of a major translocator (AopB) and minor translocator (AopD). These translocators are bound by a small chaperone (AcrH) in bacterial cytosol. AcrH chaperone plays an important role in keeping the high stability of translocators and prevents nonspecific interactions of hydrophobic domains before the pore formed in the host cell membrane. Previous studies only analysed the structure of the AcrH in combination with the AopB, but in a non-binding form with the AopB has not been elucidated. That limits the understanding of the formation mechanism of T3SS. Therefore, the authors aimed to clone, express, and purify the AcrH recombinant protein which can be used for the structural study and elucidation of T3SS pore formation. In this study, the authors cloned a fragment of the gene encoding for AcrH chaperone from A. hydrophila and inserted the gene into the pET-28a expression vector. AcrH protein from amino acids 21 to 158 was expressed in E. coli BL21 (DE3) and purified using a nickel bead column with high purity (over 99%). As a result, the obtained AcrH protein can be used for studies of structure and function that contribute to perfecting the pathogenesis of gram-negative bacteria and developing research on the treatment mechanism caused by these bacteria.

Weak Intermolecular Interactions in a Series of Bioactive Oxazoles

The intermolecular interactions in a series of nine similar 4,5-phenyl-oxazoles were studied on the basis of crystal structures determined by X-ray diffraction. The crystal architectures were analyzed for the importance and hierarchies of different, weak intermolecular interactions using three approaches: the geometrical characteristics, topological analysis (for the model based on the transfer of multipolar parameters), and energetics of the molecule–molecule interactions. The geometries of the molecules were quite similar and close to the typical values. The results of the analysis of the interactions suggest that the number of nonspecific interactions is more important than the apparent strength of the specific interactions. The interactions involving covalently bound bromine and divalent sulfur atoms were classified as secondary, they certainly did not define the crystal packing, and they played a minor role in the overall crystal cohesion energies. Incidentally, another method for confirming the degree of isostructurality, according to the topologies of the interactions, is described.

A Bit Stickier, a Bit Slower, a Lot Stiffer: Specific vs. Nonspecific Binding of Gal4 to DNA

Transcription factors regulate gene activity by binding specific regions of genomic DNA thanks to a subtle interplay of specific and nonspecific interactions that is challenging to quantify. Here, we exploit Reflective Phantom Interface (RPI), a label-free biosensor based on optical reflectivity, to investigate the binding of the N-terminal domain of Gal4, a well-known gene regulator, to double-stranded DNA fragments containing or not its consensus sequence. The analysis of RPI-binding curves provides interaction strength and kinetics and their dependence on temperature and ionic strength. We found that the binding of Gal4 to its cognate site is stronger, as expected, but also markedly slower. We performed a combined analysis of specific and nonspecific binding—equilibrium and kinetics—by means of a simple model based on nested potential wells and found that the free energy gap between specific and nonspecific binding is of the order of one kcal/mol only. We investigated the origin of such a small value by performing all-atom molecular dynamics simulations of Gal4–DNA interactions. We found a strong enthalpy–entropy compensation, by which the binding of Gal4 to its cognate sequence entails a DNA bending and a striking conformational freezing, which could be instrumental in the biological function of Gal4.

Encapsulation of benzene carboxylic acids using cyclodextrins

The encapsulation of medicinal substances in various polymers is a common way to increase their thermal, hydrolytic, and chemical stability. However, the bioavailability of the encapsulated drugs decreases. The solution to this important problem can be the preparation of nanocapsules of medicinal substances using complexing agents, for example, cyclodextrins. The purpose of this work is to study the possibility of encapsulation of benzoic, salicylic, and β-resorcylic acids using α- and β-cyclodextrins. The interaction of benzene carboxylic acids with cyclodextrins in aqueous solutions at 20–30 оС has been studied by spectrophotometry. The formation of complexes with a composition of 1:1 in the cyclodextrin – benzene carboxylic acid systems has been proven. The stability constants of the complexes and the main thermodynamic parameters of complex formation have been calculated. It has been shown that the nature of changes in the spectral characteristics of benzene carboxylic acids in the presence of cyclodextrins can be used to predict the possibility of aromatic organic compounds encapsulation. The determining role of the complementarity of geometric parameters of cyclodextrins and benzene carboxylic acids in the preparation of inclusion complexes with features of nanocapsules has been found. The prospects of using β-cyclodextrin for encapsulation of benzene carboxylic acids have been demonstrated. The complexes of β-cyclodextrin with benzene carboxylic acids were synthesized and studied by IR spectroscopy, X-ray analysis and derivatography. The formation of two types of complexes in the β-cyclodextrin – benzene carboxylic acid system was established. The first type of complexes is formed due to nonspecific interactions between the hydrophobic cavity of β-cyclodextrin and the benzene carboxylic acid molecule, the second type is due to specific interactions between the functional groups of molecules. Benzoic and salicylic acids form nanocapsules with β-cyclodextrin, and their hydrolytic and thermal stability increases. Complexes of the second type acquire the properties of a new compound: β-resorcylic acid loses its individuality, forming strong supramolecular structures with β-cyclodextrin.

Nonspecific interactions between SpCas9 and dsDNA sites located downstream of the PAM mediate facilitated diffusion to accelerate target search

Nonspecific interactions between DNA ∼8 bp downstream of the PAM and lysines within residues 1151–1156 of Cas9 mediate one-dimensional diffusion and cause asymmetric target search regions flanking the PAM.

Nanoprobes to interrogate nonspecific interactions in lipid bilayers: from defect-mediated adhesion to membrane disruption

When a lipid membrane approaches a material/nanomaterial, nonspecific adhesion may occur. The interactions responsible for nonspecific adhesions can either preserve the membrane integrity or lead to its disruption. Despite the...