The Gram-negative organism Campylobacter jejuni is the major cause of food poisoning. Unlike Escherichia coli, which has two major porins, OmpC and OmpF, C. jejuni has one, termed major outer membrane protein (MOMP) through which nutrients and antibiotics transit. We report the 2.1-Å crystal structure of C. jejuni MOMP expressed in E. coli and a lower resolution but otherwise identical structure purified directly from C. jejuni. The 2.1-Å resolution structure of recombinant MOMP showed that although the protein has timeric arrangement similar to OmpC, it is an 18-stranded, not 16-stranded, β-barrel. The structure has identified a Ca2 + bound at the constriction zone, which is functionally significant as suggested by molecular dynamics and single-channel experiments. The water-filled channel of MOMP has a narrow constriction zone, and single-molecule studies show a monomeric conductivity of 0.7 ± 0.2 nS and a trimeric conductance of 2.2 ± 0.2 nS. The ion neutralizes negative charges at the constriction zone, reducing the transverse electric field and reversing ion selectivity. Modeling of the transit of ciprofloxacin, an antibiotic of choice for treating Campylobacter infection, through the pore of MOMP reveals a trajectory that is dependent upon the presence metal ion.
The synthesis of gold nanoparticles has gained tremendous attention owing to their immense applications in the field of biomedical sciences. Although several chemical procedures are used for the synthesis of nanoparticles, the release of toxic and hazardous by-products restricts their use in biomedical applications. In the present investigation, gold nanoparticles were synthesized biologically using the culture filtrate of the filamentous fungus Alternaria sp. The culture filtrate of the fungus was exposed to three different concentrations of chloroaurate ions. In all cases, the gold ions were reduced to Au(0), leading to the formation of stable gold nanoparticles of variable sizes and shapes. UV-Vis spectroscopy analysis confirmed the formation of nanoparticles by reduction of Au(3+) to Au(0). TEM analysis revealed the presence of spherical, rod, square, pentagonal, and hexagonal morphologies for 1 mM chloroaurate solution. However, quasi-spherical and spherical nanoparticles/heart-like morphologies with size range of about 7-13 and 15-18 nm were observed for lower molar concentrations of 0.3 and 0.5 mM gold chloride solution, respectively. The XRD spectrum revealed the face-centered cubic crystals of synthesized gold nanoparticles. FT-IR spectroscopy analysis confirmed the presence of aromatic primary amines, and the additional SPR bands at 290 and 230 nm further suggested that the presence of amino acids such as tryptophan/tyrosine or phenylalanine acts as the capping agent on the synthesized mycogenic gold nanoparticles.
The OccK protein subfamily located in the outer membrane of Pseudomonas aeruginosa contains dynamic channels with several conformational states that range from open to closed forms. The molecular determinants of the OccK channels that contribute to the diverse gating have, however, remained elusive so far. Performing molecular dynamics (MD) simulations on OccK5 (OpdH) as an example, local fluctuations of loop L7 mediated by a single residue were identified that effectively gate the channel. The features of this gate residue were studied by single-channel electrophysiology and site-directed mutagenesis demonstrating that this gate residue indeed confers unique gating properties to the OccK channels. In support of these functional measurements, MD simulations highlight the correlations between the size of the side-chain belonging to the gate residue on one side and the pore size as well as the L7 flexibility on the other side.
Biomolecular interactions, such as protein-protein, protein-nucleic acid, and protein/nucleic acid-ligand interactions, play crucial roles in various cellular signaling and biological processes, and offer attractive therapeutic targets in numerous human diseases. Currently, drug discovery is limited by the low efficiency and high cost of conventional ensemble-averaging-based techniques for biomolecular interaction analysis and high-throughput drug screening. Nano-pores are an emerging technology for single-molecule sensing of biomolecules. Owing to the notable merits of single-molecule sensing, nanopore sensors have contributed tremendously to nucleic acid sequencing and disease diagnostics. In this minireview, we summarize the recent developments and outlooks in single-molecule sensing of various biomolecular interactions for drug discovery applications using biological and solid-state nanopore sensors.
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