Entropic Control of an Excited Folded-Like Conformation in a Disordered Protein Ensemble

Many intrinsically disordered proteins switch between unfolded and folded-like forms in the presence of their binding partner. The possibility of a pre-equilibrium between the two macrostates is challenging to discern given the complex conformational landscape. Here, we show that CytR, a disordered DNA-binding domain, samples a folded-like excited state in its native ensemble through equilibrium multi-probe spectroscopy, kinetics and an Ising-like statistical mechanical model. The population of the excited state increases upon stabilization of the native ensemble with an osmolyte, while decreasing with increasing temperatures. A conserved proline residue, the mutation of which weakens the binding affinity to the target promoter, is found to uniquely control the population of the minor excited state. Semi-quantitative statistical mechanical modeling reveals that the conformational diffusion coefficient of disordered CytR is an order of magnitude slower than the estimates from folded domains. The osmolyte and proline mutation smoothen and roughen up the landscape, respectively, apart from modulation of populations. Our work uncovers general strategies to probe for excited structured states in disordered ensembles, and to measure and modulate the roughness of the disordered landscapes, inter-conversion rates of species and their populations.     

Recent perspectives on the molecular basis of biofilm formation by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> and approaches for treatment and biofilm dispersal

Pseudomonas aeruginosa, a Gram-negative, rod-shaped bacterium causes widespread diseases in humans. This bacterium is frequently related to nosocomial infections such as pneumonia, urinary tract infections (UTIs) and bacteriaemia especially in immunocompromised patients. The current review focuses on the recent perspectives on biofilms formation by these bacteria. Biofilms are communities of microorganisms in which cells stick to each other and often adhere to a surface. These adherent cells are usually embedded within a self-produced matrix of extracellular polymeric substance (EPS). Pel, psl and alg operons present in P. aeruginosa are responsible for the biosynthesis of extracellular polysaccharide which plays an important role in cell surface interactions during biofilm formation. Recent studies suggested that cAMP signalling pathway, quorum-sensing pathway, Gac/Rsm pathway and c-di-GMP signalling pathway are the main mechanism that leads to the biofilm formation. Understanding the bacterial virulence depends on a number of cell-associated and extracellular factors and is very essential for the development of potential drug targets. Thus, the review focuses on the major genes involved in the biofilm formation, the state of art update on the biofilm treatment and the dispersal approaches such as targeting adhesion and maturation, targeting virulence factors and other strategies such as small molecule-based inhibitors, phytochemicals, bacteriophage therapy, photodynamic therapy, antimicrobial peptides and natural therapies and vaccines to curtail the biofilm formation by P. aeruginosa.     

Structural analysis of missense mutations in galactokinase 1 (GALK1) leading to galactosemia type‐2

Galactosemia type 2 is an autosomal recessive disorder characterized by the deficiency of galactokinase (GALK) enzyme due to missense mutations in GALK1 gene, which is associated with various manifestations such as hyper galactosemia and formation of cataracts. GALK enzyme catalyzes the adenosine triphosphate (ATP)–dependent phosphorylation of α‐d ‐galactose to galactose‐1‐phosphate. We searched 4 different literature databases (Google Scholar, PubMed, PubMed Central, and Science Direct) and 3 gene‐variant databases (Online Mendelian Inheritance in Man, Human Gene Mutation Database, and UniProt) to collect all the reported missense mutations associated with GALK deficiency. Our search strategy yielded 32 missense mutations. We used several computational tools (pathogenicity and stability, biophysical characterization, and physiochemical analyses) to prioritize the most significant mutations for further analyses. On the basis of the pathogenicity and stability predictions, 3 mutations (P28T, A198V, and L139P) were chosen to be tested further for physicochemical characterization, molecular docking, and simulation analyses. Molecular docking analysis revealed a decrease in interaction between the protein and ATP in all the 3 mutations, and molecular dynamic simulations of 50 ns showed a loss of stability and compactness in the mutant proteins. As the next step, comparative physicochemical changes of the native and the mutant proteins were carried out using essential dynamics. Overall, P28T and A198V were predicted to alter the structure and function of GALK protein when compared to the mutant L139P. This study demonstrates the power of computational analysis in variant classification and interpretation and provides a platform for developing targeted therapeutics.     

Conformationally constrained peptides target the allosteric kinase dimer interface and inhibit EGFR activation

Although EGFR is a highly sought-after drug target, inhibitor resistance remains a challenge. As an alternative strategy for kinase inhibition, we sought to explore whether allosteric activation mechanisms could effectively be disrupted. The kinase domain of EGFR forms an atypical asymmetric dimer via head-to-tail interactions and serves as a requisite for kinase activation. The kinase dimer interface is primarily formed by the H-helix derived from one kinase monomer and the small lobe of the second monomer. We hypothesized that a peptide designed to resemble the binding surface of the H-helix may serve as an effective disruptor of EGFR dimerization and activation. A library of constrained peptides was designed to mimic the H-helix of the kinase domain and interface side chains were optimized using molecular modeling. Peptides were constrained using peptide “stapling” to structurally reinforce an alpha-helical conformation. Peptide stapling was demonstrated to notably enhance cell permeation of an H-helix derived peptide termed EHBI2. Using cell-based assays, EHBI2 was further shown to significantly reduce EGFR activity as measured by EGFR phosphorylation and phosphorylation of the downstream signaling substrate Akt. To our knowledge, this is the first H-helix-based compound targeting the asymmetric interface of the kinase domain that can successfully inhibit EGFR activation and signaling. This study presents a novel, alternative targeting site for allosteric inhibition of EGFR.     

Antimicrobial activity of <Emphasis Type="Italic">Alcaligenes sp</Emphasis>. HPC 1271 against multidrug resistant bacteria

Alcaligenes sp. HPC 1271 demonstrated antibacterial activity against multidrug resistant bacteria, Enterobacter sp., resistant to sulfamethoxazole, ampicillin, azithromycin, and tetracycline, as well as against Serratia sp. GMX1, resistant to the same antibiotics with the addition of netilmicin. The cell-free culture supernatant was analyzed for possible antibacterials by HPLC, and the active fraction was further identified by LC-MS. Results suggest the production of tunicamycin, a nucleoside antibiotic. The draft genome of this bacterial isolate was analyzed, and the 4.2 Mb sequence data revealed six secondary metabolite-producing clusters, identified using antiSMASH platform as ectoine, butyrolactone, phosphonate, terpene, polyketides, and nonribosomal peptide synthase (NRPS). Additionally, the draft genome demonstrated homology to the tunicamycin-producing gene cluster and also defined 30 ORFs linked to protein secretion that could also play a role in the antibacterial activity observed. Gene expression analysis demonstrated that both NRPS and dTDP-glucose 4,6-dehydratase gene clusters are functional and could be involved in antibacterial biosynthesis.     

Green synthesis of enantiopure quinoxaline alcohols using Daucus carota

Green chemistry comprises a new approach in the synthesis of biologically active compounds using biocatalysts, thus diminishing the hazards for human health and environmental pollution. Asymmetric bioreduction is one of the most widely employed strategies in chemoenzymatic synthesis to produce enantiomerically pure chiral alcohols. The present study highlights the use biocatalyst Daucus carota for selective bioreduction of quinoxaline ketones 1a‐6a to their corresponding optically pure alcohols 1b‐6b in high yields (up to 84%) and good enantioselectivity (up to 98%). The absolute configuration of the chiral product (R)‐1‐(3‐methyl 7‐nitroquinoxalin‐2‐yl) ethan‐1‐ol 2b was confirmed by X‐ray crystallography studies. The chiral R‐configuration of the products obtained was confirmed by absolute configuration studies and was assigned following anti‐Prelogs rule. Quinoxaline pharmacophores form a part of well‐known potent drug molecules; hence, the chiral products were studied for determination of their molecular properties using SwissADME property analyser. All the chiral products show no Lipinski rule violations and are expected to have good oral bioavailability. As per the molecular properties prediction studies, the compound 6b (R)‐1‐(6,7‐dichloro‐3‐ methylquinoxalin‐2‐yl) ethanol is observed to show the best physicochemical properties to be a good lead molecule. Thus, the sustainable methodology was developed, and it confirms the synthesis of novel quinoxaline chiral alcohols in a simple, inexpensive, and eco‐friendly condition using D carota.     

Role of redox imbalance and cytokines in mediating oxidative damage and disease progression of patients with rheumatoid arthritis

Rheumatoid arthritis (RA) is a systemic inflammatory autoimmune disorder wherein the contributory role of oxidative stress has been established in the synovial fluid. As availability of synovial fluid is limited, this study aimed to evaluate in the peripheral blood of patients with RA, the relationship if any, between the extent of oxidative stress in terms of generation of reactive oxygen species (ROS) in neutrophils, plasma NADPH oxidase and myeloperoxidase activity with markers of oxidative damage, circulating cytokines and disease activity score (DAS28). In patients with RA, neutrophils in peripheral blood demonstrated an enhanced generation of ROS, coupled with depletion of free radical scavenging activity. Furthermore, the NADPH oxidase and myeloperoxidase activity was enhanced as were markers of damage. There was a positive correlation between the DAS 28 and generation of ROS, NADPH oxidase and myeloperoxidase activity as also with oxidative stress mediated protein carbonylation. Patients with RA demonstrated an increase in proinflammatory (IL-17, IL-23, and IFN-γ) and some anti-inflammatory (IL-4, IL-5, and TGF-β) cytokines. Although the levels of IL-17 correlated positively with generation of ROS, myeloperoxidase, markers of protein damage and DAS28, IL-23 correlated positively only with protein damage, and negatively with free radical scavenging activity. Importantly, incubation of neutrophils from healthy donors with plasma or SF from patients with RA translated into an enhanced generation of ROS, along with an elevation of intracellular proinflammatory cytokines. Taken together, in patients with RA, circulating neutrophils mediated a shift in the oxidant/antioxidant balance favouring the former, which translated into protein damage and contributed towards disease progression.