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Objective. To study the antibacterial activity of lysostaphin against staphylococci various species, as well as its effect on the viability of Vero cells. Materials and Methods. Lysostaphin was obtained by genetic engineering. Purification of the protein was carried out on SP-sepharose, the purity was determined by electrophoresis in PAGE by Lamley. The susceptibility to lysostaphin of 9 species 175 strains of staphylococci was studied. Identification was performed by MALDI-TOF MS, antibiotic susceptibility by EUCAST (v. 11.0). The MIC of lysostaphin was studied by the method of serial dilutions with concentrations between 0.015 and 512 mg/l. For 72 hours, the viability of Vero cells with lysostaphin at concentrations of 0.5-32.0 mg/l was determined by the MTT method with counting of living cells according to their growth curve. The results were analyzed by ANOVA followed by Dunnett's test. Results. A kinetic study of S. aureus growth in the presence of revealed an inhibitory effect of endopeptidase (MIC 0.06 mg/l). Lysostaphin was characterized by pronounced activity against clinical methicillinsensitive S. aureus. The MIC ranged between 0.03 and 0.5 mg/l and the MIC50/90 was 0.125/0.5 mg/l. For methicillin-resistant S. aureus MIC50/90 0.25/0.5 mg/l. The MIC50 for MRSE was 2 times higher than for MSSE – 1 mg/l. The maximum MIC value was determined against isolates of S. warneri and S. hominis – 64 mg/l, the lowest for S. saprophyticus – 0.5 mg/l. MIC50 of lysostaphin against MRSA was 4 times lower than that of vancomycin, MIC90 was 3 times lower. Differences in viable cells depending on the concentration of lysostaphin were not found. Conclusions. Significant activity of lysostaphin against staphylococci was revealed, which is several times higher than vancomycin against MRSA. Lysostaphin was also effective against methicillin-resistant S. aureus. The high anti-staphylococcal activity and cytocompatibility of lysostaphin are promising for its further study in the prevention and treatment of staphylococcal orthopedic infections.

An H-bond involves the sharing of a hydrogen atom between an electronegative atom to which it is covalently bound (the donor) and another electronegative atom serving as an acceptor. Such bonds represent a critically important geometrical force in biological macromolecules and, as such, have been characterized extensively. H-bond formation invariably leads to a weakening within the acceptor moiety due to the pulling exerted by the donor hydrogen. This phenomenon can be compared to a spring connecting two masses; pulling one mass stretches the spring, similarly affecting the bond between the two masses. Herein, we describe the opposite phenomenon when investigating the energetics of the C–H···O=C bond. This bond underpins the most prevalent protein transmembrane dimerization motif (GxxxG) in which a glycine Cα-H on one helix forms a hydrogen bond with a carbonyl in a nearby helix. We use isotope-edited FT-IR spectroscopy and corroborating computational approaches to demonstrate a surprising strengthening of the acceptor C=O bond upon binding with the glycine Cα-H. We show that electronic factors associated with the Cα-H bond strengthen the C=O oscillator by increasing the s-character of the σ-bond, lowering the hyperconjugative disruption of the π-bond. In addition, a reduction of the acceptor C=O bond's polarity is observed upon the formation of the C–H···O=C bond. Our findings challenge the conventional understanding of H-bond dynamics and provide new insights into the structural stability of inter-helical protein interactions. [ABSTRACT FROM AUTHOR]

Protein methyl groups can participate in multiple motional modes on different time scales. Sub-nanosecond to nano-second time scale motions of methyl axes are particularly challenging to detect for small proteins in solutions. In this work we employ NMR relaxation interference between the methyl H-H/H-C dipole-dipole interactions to characterize methyl axes motions as a function of temperature in a small model protein villin headpiece subdomain (HP36), in which all non-exchangeable protons are deuterated with the exception of methyl groups of leucine and valine residues. The data points to the existence of slow motional modes of methyl axes on sub-nanosecond to nanosecond time scales. Further, at high temperatures for which the overall tumbling of the protein is on the order of 2 ns, we observe a coupling between the slow internal motion and the overall molecular tumbling, based on the anomalous order parameters and their temperature-dependent trends. The addition of 28% (w/w) glycerol-d8 increases the viscosity of the solvent and separates the timescales of internal and overall tumbling, thus permitting for another view of the necessity of the coupling assumption for these sites at high temperatures. [ABSTRACT FROM AUTHOR]

Anti-angiogenic therapy is currently a commonly accepted and rapidly developing approach in oncology and other pathologies linked to aberrant neovascularization. Discovery and validation of additional molecular targets in angiogenesis is needed due to the limitations of the existing clinical therapeutics inhibiting activity of vascular endothelial growth factor (VEGF) and its receptors. A brief review of normal and pathological biological functions of the Eph family of receptor tyrosine kinases and their ephrin ligands is presented, and the approaches to developing therapeutics with anti- and pro-angiogenic and anti-tumor activity based on selective molecular modulation of Eph-ephrin signaling pairs are discussed. Functional roles of Eph-kinases and ephrins in such mechanisms of carcinogenesis as cell proliferation and invasion are also addressed. [ABSTRACT FROM AUTHOR]

Genome of the hepatitis C virus (HCV) contains a long open reading frame encoding a polyprotein that is cleaved into 10 proteins. Recently, a novel, so called 'ARFP/F', or 'core+1,' protein, which is expressed through a ribosomal frame shift within the capsid-coding sequence, has been described. Herein, to produce and characterize a recombinant form of this protein, the DNA sequence corresponding to the ARFP/F protein (amino acid 11-161) was amplified using a frame-shifted forward primer exploiting the capsid sequence of the lb-subtype as a template. The amplicon was cloned into the pET-24a vector and expressed in different Escherichia coli strains. The expressed protein (mostly as insoluble inclusion bodies) was purified under denaturing conditions on a nickel-nitrilotriacetic acid (Ni-NTA) affinity column in a single step with a yield of 5 mg/L of culture media. After refolding steps, characterization of expressed ARFP/F was performed by SDS-PAGE and Western blot assay using specific antibodies. Antigenic properties of the protein were verified by ELISA using HCV-infected human sera and by its ability for a strong and specific interaction with sera of mice immunized with the peptide encoding a dominant ARFP/F B-cell epitope. The antigenicity plot revealed 3 major antigenic domains in the first half of the ARFP/F sequence. Immunization of BALB/c mice with the ARFP/F protein elicited high titers of IgG indicating the relevance of produced protein for induction of a humoral response. In conclusion, possibility of ARFP/F expression with a high yield and immunogenic potency of this protein in a mouse model have been demonstrated. [ABSTRACT FROM AUTHOR]

ErbB is a family of epidermal growth factor receptors representing an important class of receptor tyrosine kinases that play a leading role in cellular growth, development, and differentiation. Transmembrane domains of these receptors transduce biochemical signals across the plasma membrane via lateral homo- and heterodimerization. The relatively small size of ErbB transmembrane domain complexes with detergents or lipids makes it possible to study their detailed spatial structure using three-dimensional heteronuclear high-resolution NMR spectroscopy. Here, we describe an efficient expression system and a purification procedure for preparative-scale production of transmembrane peptides from all four ErbB proteins-ErbB1, ErbB2, ErbB3, and ErbB4-for the purpose of structural studies. The recombinant peptides were produced in Escherichia coli BL21(DE3)pLysS cells as N-terminal extensions of thioredoxin A. The fusion proteins were cleaved with the light chain of human enterokinase. Several (10-30) milligrams of purified isotope-labeled transmembrane peptides were isolated using a simple and convenient procedure, which consists of consecutive steps of immobilized metal affinity chromatography and cation-exchange chromatography. The purified peptides were reconstituted in a lipid/detergent environment (micelles or bicelles) and characterized using dynamic light scattering and CD and NMR spectroscopy. The data obtained indicate that purified ErbB transmembrane peptides are suitable for structural and dynamic studies of their homo- and heterodimer complexes using high resolution NMR spectroscopy. [ABSTRACT FROM AUTHOR]

Synthesis of amine libraries from aldehydes via the liquid-phase parallel reductive amination with a simple scavenger (a Dowex resin) is described. Three parallel protocols are compared: reactions in well plates, SynCore apparatus, glass vials (without specific equipment). The presented detailed protocols can be useful for practical training of students. [ABSTRACT FROM AUTHOR]

coupled cell-free expression system (CECF) for the production of the transmembrane domain of the human receptor tyrosine kinase ErbB3 (residues from 632 to 675) has been developed based on the Escherichia coli S30 extract. The synthesis of the domain in the soluble form in the presence of various detergents and in the form of an insoluble precipitate of the reaction mixture has been examined. The conditions for the purification of the recombinant domain obtained using the two approaches have been determined. The final yield of the target protein under optimal conditions was 1.8-2.0 mg per 1 ml of the reaction mixture. [ABSTRACT FROM AUTHOR]

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Abstract  More than half of the mutations of the amyloid precursor protein (APP) discovered in familiar forms of Alzheimer’s disease are located in the transmembrane domain. The pathogenic mutations presumably affect the lateral dimerization of the APP transmembrane domain in the membrane and change the dimer conformation and/or stability. Thus, the mutations cause an alternative APP digestion pattern in the membrane and neurotoxic amyloid β-peptide generation. For the detailed study of the specific protein-protein and protein-lipid interactions of the APP transmembrane domain, an E. coli recombinant expression construct was made. The recombinant protein contains an APP transmembrane domain (APPtm(686–726)) with adjacent extramembrane N and C ends. Here, we report the method of isotope-labeled APPtm expression and purification in quantities necessary for a heteronuclear NMR spectroscopy structure and dynamics study. On the basis of the 1H-15N-HSQC spectra, we developed APPtm(686–726) solubilization conditions in the membrane-emulated milieu detergent micelles and lipid bicelles. [ABSTRACT FROM AUTHOR]

Abstract  Knowledge of the spatial structure of proteins is a prerequisite for both awareness of their functional mechanisms and the framework for rational drug discovery and design. Meanwhile, direct structural determination is often hampered or impractical due to the complexity, expensiveness, and limited capabilities of experimental techniques. These issues are especially pronounced for integral membrane proteins. On numerous occasions, the theoretical prediction of protein structures may facilitate the process by exploiting physical or empirical principles. This paper surveys modern techniques for the prediction of the spatial structure of proteins using computer algorithms, and the main emphasis is placed on the most “complex” targets—membrane proteins (MPs). The first part of the review describes de novo methods based on empirical physical principles; in the second part, a comparative modeling philosophy, which accounts for the structure of related proteins, is described. Special focus is made regarding pharmacologically relevant classes of G-coupled receptors, receptor tyrosine kinases, and other MPs. Algorithms for the assessment of the models’ quality and potential fields of application of computer models are discussed. [ABSTRACT FROM AUTHOR]

Simple Summary: Receptor tyrosine kinases probably constitute the most important subfamily of transmembrane receptors with respect to their role in regulating the balance between cell proliferation and cell death. Their activation involves ligand-induced conformational changes followed by their dimerization. Although this simple mechanism is still known to lie at the root of the process, the picture is complicated by the involvement of several receptor domains in the dimerization and the formation of larger receptor aggregates. Both clustering and activation are influenced by lipid-mediated interactions of the plasma membrane with the receptors. The intricate regulation of receptor activation is subverted in cancer that involves not only alterations in receptor structure and expression but also changes in lipid composition of the cell membrane. This paper provides a concise overview of how these biophysical aspects of transmembrane signaling regulate this important process in health and disease. The search for an understanding of how cell fate and motility are regulated is not a purely scientific undertaking, but it can also lead to rationally designed therapies against cancer. The discovery of tyrosine kinases about half a century ago, the subsequent characterization of certain transmembrane receptors harboring tyrosine kinase activity, and their connection to the development of human cancer ushered in a new age with the hope of finding a treatment for malignant diseases in the foreseeable future. However, painstaking efforts were required to uncover the principles of how these receptors with intrinsic tyrosine kinase activity are regulated. Developments in molecular and structural biology and biophysical approaches paved the way towards better understanding of these pathways. Discoveries in the past twenty years first resulted in the formulation of textbook dogmas, such as dimerization-driven receptor association, which were followed by fine-tuning the model. In this review, the role of molecular interactions taking place during the activation of receptor tyrosine kinases, with special attention to the epidermal growth factor receptor family, will be discussed. The fact that these receptors are anchored in the membrane provides ample opportunities for modulatory lipid–protein interactions that will be considered in detail in the second part of the manuscript. Although qualitative and quantitative alterations in lipids in cancer are not sufficient in their own right to drive the malignant transformation, they both contribute to tumor formation and also provide ways to treat cancer. The review will be concluded with a summary of these medical aspects of lipid–protein interactions. [ABSTRACT FROM AUTHOR]

Lung cancer has a high prevalence, with a growing number of new cases and mortality every year. Furthermore, the survival rate of patients with non-small-cell lung carcinoma (NSCLC) is still quite low in the majority of cases. Despite the use of conventional therapy such as tyrosine kinase inhibitor for Epidermal Growth Factor Receptor (EGFR), which is highly expressed in most NSCLC cases, there was still no substantial improvement in patient survival. This is due to the drug's ineffectiveness and high rate of resistance among individuals with mutant EGFR. Therefore, the development of new inhibitors is urgently needed. Understanding the EGFR structure, including its kinase domain and other parts of the protein, and its activation mechanism can accelerate the discovery of novel compounds targeting this protein. This study described the structure of the extracellular, transmembrane, and intracellular domains of EGFR. This was carried out along with identifying the binding pose of commercially available inhibitors in the ATP-binding and allosteric sites, thereby clarifying the research gaps that can be filled. The binding mechanism of inhibitors that have been used clinically was also explained, thereby aiding the structure-based development of new drugs. [ABSTRACT FROM AUTHOR]

Small cationic guanyl-preferring ribonucleases (RNases) produced by the Bacillus species share a similar protein tertiary structure with a high degree of amino acid sequence conservation. However, they form dimers that differ in conformation and stability. Here, we have addressed the issues (1) whether the homologous RNases also have distinctions in catalytic activity towards different RNA substrates and interactions with the inhibitor protein barstar, and (2) whether these differences correlate with structural features of the proteins. Circular dichroism and dynamic light scattering assays revealed distinctions in the structures of homologous RNases. The activity levels of the RNases towards natural RNA substrates, as measured spectrometrically by acid-soluble hydrolysis products, were similar and decreased in the row high-polymeric RNA >>> transport RNA > double-stranded RNA. However, stopped flow kinetic studies on model RNA substrates containing the guanosine residue in a hairpin stem or a loop showed that the cleavage rates of these enzymes were different. Moreover, homologous RNases were inhibited by the barstar with diverse efficiency. Therefore, minor changes in structure elements of homologous proteins have a potential to significantly effect molecule stability and functional activities, such as catalysis or ligand binding. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is classified as an amyloid-related disease. Amyloid beta (Aβ) is a transmembrane protein known to play a major role in the pathogenesis of AD. These Aβ proteins can form ion channels or pores in the cell membrane. Studies have elucidated the structure of the transmembrane domain of Aβ ion channels. In addition, various studies have investigated substances that block or inhibit the formation of Aβ ion channels. Zinc ions are considered as potential inhibitors of AD. In this study, we focused on the transmembrane domain and some external domains of the Aβ protein (hAPP-TM), and solution-state NMR was used to confirm the effect on residues of the protein in the presence of zinc ions. In addition, we sought to confirm the structure and orientation of the protein in the presence of the bicelle using solid-state NMR. [ABSTRACT FROM AUTHOR]

Membrane proteins evolved to reside in the hydrophobic lipid bilayers of cellular membranes. Therefore, membrane proteins bridge the different aqueous compartments separated by the membrane, and furthermore, dynamically interact with their surrounding lipid environment. The latter not only stabilizes membrane proteins, but directly impacts their folding, structure and function. In order to be characterized with biophysical and structural biological methods, membrane proteins are typically extracted and subsequently purified from their native lipid environment. This approach requires that lipid membranes are replaced by suitable surrogates, which ideally closely mimic the native bilayer, in order to maintain the membrane proteins structural and functional integrity. In this review, we survey the currently available membrane mimetic environments ranging from detergent micelles to bicelles, nanodiscs, lipidic-cubic phase (LCP), liposomes, and polymersomes. We discuss their respective advantages and disadvantages as well as their suitability for downstream biophysical and structural characterization. Finally, we take a look at ongoing methodological developments, which aim for direct in-situ characterization of membrane proteins within native membranes instead of relying on membrane mimetics. [ABSTRACT FROM AUTHOR]