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Single-stranded DNA-binding protein (SSB) is a bacterial interaction hub and an appealing target for antimicrobial therapy. Understanding the structural adaptation of the disordered SSB C-terminus (SSB-Ct) to DNA metabolizing enzymes (e.g., ExoI and RecO) is essential for designing high-affinity SSB mimetic inhibitors. Molecular dynamics simulations revealed the transient interactions of SSB-Ct with two hot spots on ExoI and RecO. The residual flexibility of the peptide–protein complexes allows adaptive molecular recognition. Scanning with non-canonical amino acids revealed that modifications at both termini of SSB-Ct could increase the affinity, supporting the two-hot-spot binding model. Combining unnatural amino acid substitutions on both segments of the peptide resulted in enthalpy-enhanced affinity, accompanied by enthalpy–entropy compensation, as determined by isothermal calorimetry. NMR data and molecular modeling confirmed the reduced flexibility of the improved affinity complexes. Our results highlight that the SSB-Ct mimetics bind to the DNA metabolizing targets through the hot spots, interacting with both of segments of the ligands. [ABSTRACT FROM AUTHOR]

With the increasing development of antibiotic resistance among key bacterial pathogens, there is an urgent need to discover novel classes of antibiotics. Although antimicrobial peptides (AMP) with their specific mode of action are considered major candidates for next-generation antibiotics, several challenges limit the use of these peptides for therapeutic applications. In a large body of research, the focus is given to different approaches to the chemical modification of AMPs and how these modifications may improve the stability, antibiotic activity, proteolytic activity and prevent the cytotoxicity and side effects of AMPs. On the other hand, another group of research investigates the delivery of AMPs via nanocarrier systems as strategies used to enhance stability, control the release of peptides and reduce adverse peptide-related side effects, as well as improve their anti-microbial activities. In the present article, we surveyed most recently published researches that provide us with good knowledge on structural features, mechanism of action, therapeutic aim, advantages and limitations, chemical modification approaches and carrying strategies of AMPs. Finally, according to Quality by Design, the most important potential effective factor and potential risk were mentioned in the development of AMP delivery systems. [ABSTRACT FROM AUTHOR]

Additives selectively inhibit or enhance the growth of crystal faces and effectively change the crystal morphology. For the first time, potassium chloride was used as additive during impinging jet crystallization of glycine. The structure of the powder particles was evaluated by X-ray powder diffraction, differential scanning calorimetry, scanning electron microscopy with energy dispersive X-ray spectroscopy, headspace gas chromatography, and flame atomic absorption spectrometry. Even a minor amount of KCl had a significant effect on the crystal roundness and reduced the particle size significantly, despite of the extremely rapid nature of the crystallization process. This method resulted in the α-polymorph of glycine with an appropriate low residual additive content and a minimal residual organic solvent content. The arrangement and the optimal concentration of the additive were determined. [ABSTRACT FROM AUTHOR]

Protein-protein interactions stabilized by multiple separate hot spots are highly challenging targets for synthetic scaffolds. Surface-mimetic foldamers bearing multiple recognition segments are promising candidate inhibitors. In this work, a modular bottom-up approach is implemented by identifying short foldameric recognition segments that interact with the independent hot spots, and connecting them through dynamic covalent library (DCL) optimization. The independent hot spots of a model target (calmodulin) are mapped with hexameric β-peptide helices using a pull-down assay. Recognition segment hits are subjected to a target-templated DCL ligation through thiol-disulfide exchange. The most potent derivative displays low nanomolar affinity towards calmodulin and effectively inhibits the calmodulin-TRPV1 interaction. The DCL assembly of the folded segments offers an efficient approach towards the de novo development of a high-affinity inhibitor of protein-protein interactions. [ABSTRACT FROM AUTHOR]

There is enormous interest toward vanilloid agonists of the pain receptor TRPV1 in analgesic therapy, but the mechanisms of their sensory neuron-blocking effects at high or repeated doses are still a matter of debate. Our results have demonstrated that capsaicin and resiniferatoxin form nanomolar complexes with calmodulin, and competitively inhibit TRPV1-calmodulin interaction. These interactions involve the protein recognition interface of calmodulin, which is responsible for all of the cell-regulatory calmodulin-protein interactions. These results draw attention to a previously unknown vanilloid target, which may contribute to the explanation of the paradoxical pain-modulating behavior of these important pharmacons. [ABSTRACT FROM AUTHOR]

Combined drug products have the advantages of better patient compliance and possible synergic effects. The simultaneous application of several active ingredients at a time is therefore frequently chosen. However, the quantitative analysis of such medicines can be challenging. The aim of this study is to provide a validated method for the investigation of a multidose packed oral powder that contained acetylsalicylic acid, paracetamol and papaverine-HCl. Reversed-phase high-pressure liquid chromatography was used. The Agilent Zorbax SB-C18 column was found to be the most suitable of the three different stationary phases tested for the separation of the components of this sample. The key parameters in the method development (apart from the nature of the column) were the pH of the aqueous phase (set to 3.4) and the ratio of the organic (acetonitrile) and the aqueous (25 mM phosphate buffer) phases, which was varied from 7:93 (v/v) to 25:75 (v/v) in a linear gradient, preceded by an initial hold. The method was validated: linearity, precision (repeatability and intermediate precision), accuracy, specificity and robustness were all tested, and the results met the ICH guidelines. [ABSTRACT FROM PUBLISHER]

The B6.Cg-Tg(Thy1-YFP)16Jrs/J transgenic mouse strain, widely used to study neuronal development and regeneration, expresses the yellow fluorescent protein (YFP) in the peripheral nerves and the central nervous system under the control of regulatory sequences of the Thy1 gene. The Thy1 (CD90) cell surface glycoprotein is present on many cell types besides neurons, and is known to be involved in cell adhesion, migration and signal transduction. We hypothesized that Thy1-activating conditions could probably activate the truncated Thy1 regulatory sequences used in the Thy1-YFP construct, resulting in YFP transgene expression outside the nervous system. We demonstrated that the stroma of subcutaneous tumours induced by the injection of 4T1 or MC26 carcinoma cells into BALB/c(Thy1-YFP) mice, carrying the same construct, indeed expressed the YFP transgene. In the tumour mass, the yellow-green fluorescent stromal cells were clearly distinguishable from 4T1 carcinoma cells stably transfected with red fluorescent protein. Local inflammation induced by subcutaneous injection of complete Freund's adjuvant, as well as the experimental wound-healing milieu, also triggered YFP fluorescence in both the BALB/c(Thy1-YFP) and B6.Cg-Tg(Thy1-YFP)16Jrs/J mice, pointing to eventual overlapping pathways of wound-healing, inflammation and tumour growth. [ABSTRACT FROM AUTHOR]

This review aims to create an overview of the currently available results of site-directed mutagenesis studies on transient receptor potential vanilloid type 1 (TRPV1) receptor. Systematization of the vast number of data on the functionally important amino acid mutations of TRPV1 may provide a clearer picture of this field, and may promote a better understanding of the relationship between the structure and function of TRPV1. The review summarizes information on 112 unique mutated sites along the TRPV1, exchanged to multiple different residues in many cases. These mutations influence the effect or binding of different agonists, antagonists, and channel blockers, alter the responsiveness to heat, acid, and voltage dependence, affect the channel pore characteristics, and influence the regulation of the receptor function by phosphorylation, glycosylation, calmodulin, PIP2, ATP, and lipid binding. The main goal of this paper is to publish the above mentioned data in a form that facilitates in silico molecular modelling of the receptor by promoting easier establishment of boundary conditions. The better understanding of the structure-function relationship of TRPV1 may promote discovery of new, promising, more effective and safe drugs for treatment of neurogenic inflammation and pain-related diseases and may offer new opportunities for therapeutic interventions. [ABSTRACT FROM AUTHOR]

YjdL from E. coli is an unusual proton-coupled oligopeptide transporter (POT). Unlike prototypical POTs, dipeptides are preferred over tripeptides, in particular dipeptides with a positively charged C-terminal residue. To further understand this difference in peptide specificity, the sequences of YjdL and YdgR, a prototypical E. coli POT, were compared in light of the crystal structure of a POT from Shewanella oneidensis. Several residues found in the putative active site were mutated and the activities of the mutated variants were assessed in terms of substrate uptake assays, and changes in specificity in terms of uptake inhibition. Most strikingly, changing the YjdL specific Asp392 to the conserved Ser in YjdL obliterated the preference for a positively charged C-terminal residue. Based on this unique finding and previously published results indicating that the dipeptide N-terminus may interact with Glu388, a preliminary orientation model of a dipeptide in the YjdL cavity is presented. Single site mutations of particularly Ala281 and Trp278 support the presented orientation. A dipeptide bound in the cavity of YjdL appears to be oriented such that the N-terminal side chain protrudes into a sub pocket that opens towards the extracellular space. The C-terminal side chain faces in the opposite direction into a sub pocket that faces the cytoplasm. These data indicated a stabilizing effect on a bulky N-terminal residue by an Ala281Phe variant and on the dipeptide backbone by Trp278. In the presented orientation model, Tyr25 and Tyr58 both appear to be in proximity of the dipeptide backbone while Lys117 appears to be in proximity of the peptide C-terminus. Mutational studies of these conserved residues highlight their functional importance. [ABSTRACT FROM AUTHOR]

Abstract: Tripartite efflux systems are responsible for the export of toxins across both the inner and outer membranes of Gram negative bacteria. Previous work has indicated that EmrAB–TolC from Escherichia coli is such a tripartite system, comprised of EmrB an MFS transporter, EmrA, a membrane fusion protein and TolC, an outer membrane channel. The whole complex is predicted to form a continuous channel allowing direct export from the cytoplasm to the exterior of the cell. Little is known, however, about the interactions between the individual components of this system. Reconstitution of EmrA+EmrB resulted in co-elution of the two proteins from a gel filtration column indicating formation of the EmrAB complex. Electron microscopic single particle analysis of the reconstituted EmrAB complex revealed the presence of particles approximately 240×140Å, likely to correspond to two EmrAB dimers in a back-to-back arrangement, suggesting the dimeric EmrAB form is the physiological state contrasting with the trimeric arrangement of the AcrAB–TolC system. [Copyright &y& Elsevier]

Epstein-Barr virus (EBV) infection of B cells is associated with lymphoma and other human cancers. EBV infection is initiated by the binding of the viral envelope glycoprotein (gp350) to the cell surface receptor CR2. We determined the X-ray structure of the highly glycosylated gp350 and defined the CR2 binding site on gp350. Polyglycans shield all but one surface of the gp350 polypeptide, and we demonstrate that this glycan-free surface is the receptor-binding site. Deglycosylated gp350 bound CR2 similarly to the glycosylated form, suggesting that glycosylation is not important for receptor binding. Structure-guided mutagenesis of the glycan-free surface disrupted receptor binding as well as binding by a gp350 monoclonal antibody, a known inhibitor of virus-receptor interactions. These results provide structural information for developing drugs and vaccines to prevent infection by EBV and related viruses. [ABSTRACT FROM AUTHOR]