Calculation Of Dissociation Constants Research Articles

Dissociation role on the catalytic activity of organic halides in CO2 conversion to cyclic carbonates: Experimental and computational study

There is a limited number of systematic CO2 conversion studies that provide a clear understanding of the effect of the active sites of catalysts. Hence, this work examines the catalytic activity of 24 organic salts consisting of chloride, bromide or iodide anions and imidazolium, ammonium, or phosphonium-based cations, in the synthesis of hexylene and styrene carbonates from CO2, resulting in a diverse range of yields. The findings revealed that high yields depend heavily on catalyst solubility in the reaction medium, but solubility alone does not guarantee reaction success. This finding supports the new hypothesis that catalyst dissociation, reliant on solubility, is a critical factor in defining the catalytic activity. A strong correlation was observed between carbonate yields and the dissociation constants of catalysts, calculated using the COSMO-RS method. This suggests that greater dissociation, reflecting weaker cation-anion interactions, facilitates the anion nucleophilic attack on the epoxide. Also, the relationship between calculated dissociation constant and experimental ionic conductivity was successfully validated. This highlights the significance of organic salt dissociation on catalytic performance and validates the use of computational tools to predict key operational parameters, enhancing the understanding and optimization of CO2 conversion into cyclic carbonates.

Valacyclovir and Acyclovir Are Substrates of the Guanine Deaminase Cytosolic PSD-95 Interactor (Cypin).

Valacyclovir, enzymatically hydrolyzed in the body to acyclovir, is a guanine-based nucleoside analog commonly prescribed as an antiviral therapy. Previous reports suggest that guanosine analogs bind to guanine deaminase; however, it is unclear whether they act as inhibitors or substrates. Data from our laboratory suggest that inhibition of guanine deaminase by small molecules attenuates spinal cord injury-induced neuropathic pain. Here, we examine whether the guanosine analogs valacyclovir and acyclovir are deaminated by cypin (cytosolic PSD-95 interactor), the major guanine deaminase in the body, or if they act as cypin inhibitors. Using purified Rattus norvegicus cypin, we use NADH-coupled assay to confirm deamination of valacyclovir and determined Michaelis-Menten constants. Subsequently, we use tryptophan fluorescence quenching assay to calculate dissociation constants for valacyclovir and acyclovir and find that inclusion of the valine motif in valacyclovir increases affinity for cypin compared to acyclovir. To our knowledge, neither Km nor KD values for cypin has been previously reported for either compound. We use Amplex Red assay and demonstrate that both valacyclovir and acyclovir are cypin substrates and that their metabolites are further processed by xanthine oxidase and uricase. Using molecular dynamics simulations, we demonstrate that an alpha helix near the active site is displaced when valacyclovir binds to cypin. Furthermore, we used LC-MS-based assay to directly confirm deamination of valacyclovir by cypin. Taken together, our results demonstrate a novel role for cypin in deamination of valacyclovir and acyclovir and suggest that therapeutics based on purine structures may be inactivated by cypin, decreasing inhibitory efficacy.

Хімічні властивості та характеристики поверхневої активності гумінової кислоти з бурого вугілля

The article is devoted to the study of some specific physicochemical properties of humic acids, which are attracting increasing attention due to their potential applications. The unique structural composition, which includes both hydrophilic and hydrophobic sites, makes these materials a versatile agent in various technological processes. It has been shown that humic acid solutions have the properties of surfactants and can serve as a natural alternative to surfactants. The paper presents the results of studies of a sample of humic acid obtained from lignite. Infrared spectroscopy has revealed signs of the aromatic structure of humic acid, the presence of phenolic and other functional groups, such as hydroxyl and nitrile. The presence of carboxylic acids, esters, and complex esters was established. The complex and heterogeneous nature of humic acids has been confirmed, which makes them promising for use as surfactants. No clear evidence of the presence of -NH₂ groups was found in the IR spectrum. This suggests that lignite humic acids generally correspond to the structural formula reported in the literature. The potentiometric titration of the lignite humic acid sample made it possible to determine the dissociation constants of functional groups. The obtained values are close to the calculated dissociation constants of carboxyl groups for the model molecule of lignite humic acid. The analysis of the calculated properties of humic acid based on various structural models showed that lignite is a promising raw material for the production of humic acids with certain surface-active properties. Compared to other structures, humic acid from lignite is more hydrophobic and less soluble in water, which indicates the prospects of its use as a surfactant with better interaction with the oil phase. Keywords: humic acids, lignite, surfactant, infrared spectroscopy, composition, functional groupsб structural model. Corresponding author: Bannikov L.P., e-mail: ukhinbannikov@gmail.com

A novel inhibitor study on peroxidase activity in the presence of caffeic acid: An in vitro and in silico mechanism research

The present study aims to investigate the substrate specificity of peroxidase (POD), an enzyme held responsible for enzymatic browning, in the presence of caffeic acid, guaiacol, chlorogenic acid and catechol. The use of in silico studies provided additional support for the identification of optimal substrate candidates. Considering the Vmax/Km (EU/mL.min.mM) values obtained, caffeic acid was found to have the highest value (11.85) and to be the best substrate for POD. The inhibitory mechanism of ascorbic acid, citric acid and salicylic acid on the activity of POD was studied in the presence of caffeic acid. Competitive inhibition was observed in the kinetic studies involving ascorbic acid and salicylic acid, whereas non-competitive inhibition was observed in the case of citric acid. The lowest calculated dissociation constant (Ki) of the enzyme-inhibitor complex was that of salicylic acid (0.044 mM). Furthermore, the in vitro Ki results for the inhibitors used were consistent with the in silico studies. The mechanism of POD inhibition by salicylic acid in the presence of a caffeic acid substrate was explained by molecular docking studies. The POD inhibition has been attributed to a decrease in substrate oxidation as a result of ferric (H2O2) interactions with salicylic acid, in addition to a decrease in enzyme stabilization as a result of Pi-lone pair interactions.

Bandaids on broken hearts: Preventing protease-mediated degradation of desmoplakin with small molecules.

The desmosome is an intercellular protein complex crucial for cell-cell adhesion. Found in muscular and cutaneous tissue, the desmosome maintains tissue integrity under significant mechanical stress through the linking of cellular intermediate filament networks. Desmoplakin (DSP) is integral to the desmosome, and functions by connecting desmosomal cadherins to intermediate filaments. Multiple missense mutations in a DSP ‘hotspot region’ (residues 299-515) are causally linked to arrhythmogenic cardiomyopathy. Mutated DSP maintains structural integrity. However, mutations expose a normally-occluded calpain cleavage site (residues 447-451), resulting in decreased protein levels in affected tissues. Calpain is crucial for normal muscle development and maintenance. Therefore, instead of inhibiting calpain function, here we aim to find a “molecular band-aid” for DSP; a molecule that blocks this cleavage site without influencing calpain function. We have partially screened a library of FDA-approved drugs by monitoring fluorescence polarization of FITC-labeled DSP in the presence of protease. Molecules that prevent degradation are subjected to a secondary assay against FITC-BSA to screen for protease-specific inhibition. Molecules that specifically inhibit DSP degradation but not BSA degradation are then subjected to MD-based drug docking studies. Preliminary data reveal that drug ‘hits’ tend to be larger organic molecules. Most, but not all, of these molecules stay bound to DSP over >50 ns of simulation, and in addition have smaller calculated dissociation constants. Together, these preliminary data validate the feasibility of this workflow for identifying promising small molecules that prevent DSP degradation.

Synthesis and in Silico Docking Studies of Ethyl 2-(2-Arylidene-1-alkylhydrazinyl)thiazole-4-carboxylates as Antiglycating Agents.

Ethyl 2-(2-arylidene-1-alkylhydrazinyl)thiazole-4-carboxylates (1a-k) were synthesized by alkylation on HN- of ethyl 2-(2-arylidenehydrazinyl)thiazole-4-carboxylates. The proposed structures (1a-k) are corroborated by spectro-analytical techniques like UV, FT-IR, 1 H-, 13 C-NMR and HR-MS. All synthesized compounds were screened for their antiglycation and antioxidant assays. The in vitro antiglycation results revealed promising activity of compounds 1a, 1b, 1d, 1e, 1f, 1g, 1j and 1k with IC50 values 0.0004±1.097-17.22±0.538 μM when compared to standard, aminoguanidine (IC50 =25.50±0.337 μM). Among all tested compounds 1j and 1k are the best antiglycating agents with IC50 values 1.848±0.646 and 0.0004±1.097 μM, respectively. The in-silico studies also agree with these results where binding energy for 1j and 1k was found to be -9.25 and -8.42 kcal/mol with calculated dissociation constants of 0.16 and 0.67 μM, respectively. The antiglycation results demonstrate the application of these compounds in reducing diabetic complications.

Selection and Characterization of ssDNA Aptamers Targeting Largemouth Bass Virus Infected Cells With Antiviral Activities.

Largemouth bass virus (LMBV) is one of the most devastating viral pathogens in farmed Largemouth bass. Aptamers are novel molecule probes and have been widely applied in the field of efficient therapeutic and diagnostic agents development. LMBV-infected fathead minnow cells (LMBV-FHM) served as target cells in this study, and three DNA aptamers (LBVA1, LBVA2, and LBVA3) were generated against target cells by SELEX technology. The selected aptamers could specifically bind to LMBV-FHM cells, with rather high calculated dissociation constants (Kd) of 890.09, 517.22, and 249.31 nM for aptamers LBVA1, LBVA2, and LBVA3, respectively. Three aptamers displayed efficient antiviral activities in vitro. It indicates that the selected aptamers have great potentials in developing efficient anti-viruses treatments. The targets of aptamers LBVA1, LBVA2, and LBVA3 could be membrane proteins on host cells. The targets of aptamers (LBVA1, LBVA2, and LBVA3) come out on the cells surface at 8, 10, 8 h post-infection. As novel molecular probes for accurate recognition, aptamer LBVA3 could detect LMBV infection in vitro and in vivo, it indicates that the selected aptamers could be applied in the development of rapid detective technologies, which are characterized by high sensitivity, accuracy, and easy operation.

Fluorescence Spectroscopic Analysis of ppGpp Binding to cAMP Receptor Protein and Histone-Like Nucleoid Structuring Protein

The cyclic AMP receptor protein (CRP) is one of the best-known transcription factors, regulating about 400 genes. The histone-like nucleoid structuring protein (H-NS) is one of the nucleoid-forming proteins and is responsible for DNA packaging and gene repression in prokaryotes. In this study, the binding of ppGpp to CRP and H-NS was determined by fluorescence spectroscopy. CRP from Escherichia coli exhibited intrinsic fluorescence at 341 nm when excited at 280 nm. The fluorescence intensity decreased in the presence of ppGpp. The dissociation constant of 35 ± 3 µM suggests that ppGpp binds to CRP with a similar affinity to cAMP. H-NS also shows intrinsic fluorescence at 329 nm. The fluorescence intensity was decreased by various ligands and the calculated dissociation constant for ppGpp was 80 ± 11 µM, which suggests that the binding site was occupied fully by ppGpp under starvation conditions. This study suggests the modulatory effects of ppGpp in gene expression regulated by CRP and H-NS. The method described here may be applicable to many other proteins.

1H and 19F NMR study of the processes of dissociation of complexes Tm3+ and Ho3+ with ciprofloxacin

The thermodynamics and kinetics of the dissociation processes of the Tm3+ and complexes with ciprofloxacin were studied by 1H and 19F NMR (at different temperatures). This is one of the few studies on the dynamics of lanthanide complexes on 19F nuclei.The Gibbs free energy of activation of the dissociation processes (ΔG≠298) was found to be 60±3 and 58±3 kJ/mol for the Tm3+ and Ho3+ complexes, respectively. The calculated dissociation constant Kdis for the Ho3+ complex with ciprofloxacin turned out to be 0.02 mol/L (which is consistent with the corresponding Kdis values ​​for the Al3+ and Fe3+ complexes, which were determined earlier). Complexes of lanthanides with ciprofloxacin are kinetically and thermodynamically unstable, and are in solution in a partially dissociated state {in the "physiological" temperature range (35–42 ° C)}.

Phyto-Compounds from a Rather Poisonous Plant, Strychnos nuxvomica, Show High Potency Against SARS-CoV-2 RNA-Dependent RNA Polymerase.

The establishment of strategy to inhibit the virus replication is an attractive means in combating SARS-CoV-2 infection. We studied phyto-compounds from Strychnos nux-vomica (a poisonous plant) against SARS-CoV-2 RNA-dependent RNA polymerase by computational methods. Molecular docking, molecular dynamics (MD) simulation and energetics calculations were employed to elucidate the role of the phyto-compounds. Ergotamine with a binding free energy of -14.39 kcal/mol showed a promising capability in terms of binding affinity and the interaction to conserved motifs, especially the SDD signature sequence. The calculated dissociation constants for ATP, ergotamine, isosungucine and sungucine were 12 μM, 0.072 nM, 0.011 nM and 0.152 nM, respectively. The exhibited kd by these phyto-compounds reflected tens of thousands fold potency as compared to ATP. The binding free energies of sungucine and isosungucine were much lower (-13.93 and -15.55 kcal/mol, respectively) compared to that of ATP (-6.98 kcal/mol). Sharing the same binding location as that of ATP and having high binding affinities, Ergotamine, Isosungucine, Sungucine and Strychnine N-oxide could be effective in controlling the SARS-CoV-2 virus replication by blocking the ATP and inhibiting the enzyme function.

Generating aptamers for specific recognition against soft-shelled turtle iridovirus infection

Soft-shelled turtle iridovirus (STIV) causes devastating virus disease in farmed soft-shelled turtles (Trionyx sinensis). Aptamers with remarkably high specificity and affinity have been widely applied in diseases diagnosis and therapies. In this study, single-stranded DNA (ssDNA) aptamers were generated against STIV-infected Fathead minnow (FHM) cells via Cell-SELEX (Cell based-Systematic Evolution of Ligands by Exponential enrichment technology). Aptamers could form stable stem-loop structures and specifically recognize STIV-infected FHM cells, with calculated dissociation constants (Kd) of 191 ± 12 and 497 ± 39 nM for aptamers LYST1and LYST2, respectively, which indicated that they could serve as specific delivery system for antiviral therapies. Both aptamers displayed antiviral activities in vitro. The targets of aptamers LYST1and LYST2 in cell membranes could be membrane proteins, which were trypsin-sensitive. FAM-labeled aptamer LYST1 could identify STIV infection in diseased turtle tissues. It is the first time that aptamers against STIV-infected cells have been selected and characterized. The aptamers have great potential in development of rapid diagnostic assays and targeted antiviral drugs against STIV in aquaculture.

NMR spectroscopy uncovers direct interaction between BAF60A and p53

The BRG1-associated factor 60A (BAF60A), an SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily D member 1, has been known to be important for transcriptional activation and inhibition through the alteration of the DNA nucleosome. Although the association between BAF60A and p53 plays a critical role in tumor suppression, the interaction mode is still unclear. Here, we report the detailed interactions between BAF60A and p53 by both NMR spectroscopy and pull-down analysis. Both N-terminal region (BAF60ANR) and the SWIB domain (BAF60ASWIB) of BAF60A directly interact with the tetramerization domain of p53 (p53TET). NMR data show that Ile315, Met366, Ala388, and Tyr390 of BAF60ASWIB are mostly involved in p53TET binding. The calculated dissociation constant (KD) value between BAF60ASWIB and p53TET revealed relatively weak binding affinity, at approximately 0.3 ± 0.065 mM. Our data will enhance detailed interaction mechanism to elucidate the molecular basis of p53-mediated integration via BAF60A interaction.

Generation and characterization of aptamers against grass carp reovirus infection for the development of rapid detection assay

Grass carp reovirus (GCRV) causes devastating viral haemorrhagic disease in farmed grass carp (Ctenopharyngon idellus). As novel molecular probes, aptamers have been widely applied in rapid diagnosis and efficient therapies against virus or diseases. In this study, three single-stranded DNA (ssDNA) aptamers were selected against GCRV-infected CIK cells via SELEX (systematic evolution of ligands by exponential enrichment technology). Secondary structures predicted by MFOLD indicated that aptamers formed stem-loop structures, and GVI-11 had the lowest ΔG value of -30.84 KJ/mol. Three aptamers could specifically recognize GCRV-infected CIK cells, with calculated dissociation constants (Kd) of 220.86, 176.63 and 278.66nM for aptamers GVI-1, GVI-7 and GVI-11, respectively, which indicated that they could serve as specific delivery system for antiviral therapies. The targets of aptamers GVI-1, GVI-7 and GVI-11 on the surface of GCRV-infected cells could be membrane proteins, which were trypsin-sensitive. Furthermore, FAM-labelled aptamer GVI-7 could be applied to detect GCRV infection in vivo. It is the first time to generate and characterize aptamers against GCRV-infected cells. These aptamers have great potentials in development of rapid diagnosis technology and antiviral agents against GCRV infection in aquaculture.

The leucine-rich repeat domain of human peroxidasin 1 promotes binding to laminin in basement membranes

Human peroxidasin 1 (PXDN) is a homotrimeric multidomain heme peroxidase and essential for tissue development and architecture. It has a biosynthetic function and catalyses the hypobromous acid-mediated formation of specific covalent sulfilimine (SN) bonds, which cross-link type IV collagen chains in basement membranes. Currently, it is unknown whether and which domain(s) [i.e. leucine-rich repeat domain (LRR), immunoglobulin domains, peroxidase domain, von Willebrand factor type C domain] of PXDN interact with the polymeric networks of the extracellular matrix (ECM), and how these interactions integrate and regulate the enzyme's cross-linking activity, without imparting oxidative damage to the ECM. In this study, we probed the interactions of four PXDN constructs with different domain compositions with components of a basement membrane extract by immunoprecipitation. Strong binding of the LRR-containing construct was detected with the major ECM protein laminin. Analysis of these interactions by surface plasmon resonance spectroscopy revealed similar kinetics and affinities of binding of the LRR-containing construct to human and murine laminin-111, with calculated dissociation constants of 1.0 and 1.5 μM, respectively. The findings are discussed with respect to the recently published in-solution structures of the PXDN constructs and the proposed biological role of this peroxidase.

Characterization of Novel Aptamers Specifically Directed to Red-Spotted Grouper Nervous Necrosis Virus (RGNNV)-Infected Cells for Mediating Targeted siRNA Delivery.

Nervous necrosis virus (NNV) causes viral nervous necrosis, the most devastating disease in more than 50 fish species worldwide, with massive mortality rates up to 100%, resulting in great economic losses to mariculture. However, few methods are available for the efficient diagnosis and treatment of viral nervous necrosis. Aptamers are molecular recognition ligands characterized by their remarkably high specificity and affinity, great stability, and ease of synthesis, and have been widely studied in application of disease diagnosis and therapies. In this study, we generated three aptamers against red-spotted grouper nervous necrosis virus (RGNNV)-infected grouper brain (GB) cells using the Cell-SELEX (cell based-systematic evolution of ligands by exponential enrichment) technology. The selected aptamers formed stable stem-loop structures, and could specifically recognize RGNNV-infected GB cells, with calculated dissociation constants (Kd) of 27.96, 29.3, and 59.5 nM for aptamers GBN2, GBN10, and GBN34, respectively. They also recognized RGNNV-infected brain tissues. The three aptamers were non-toxic and showed antiviral activities both in vitro and in vivo. Fluorescence microscopy and flow cytometry also demonstrated that aptamer GBN34 could be efficiently and specifically internalized into RGNNV-infected GB cells. The targeted cellular delivery of aptamer-small interfering RNA (siRNA) conjugates remarkably inhibited RGNNV infection in GB cells. The efficiency of the aptamer-based targeted delivery system was about 75% reduction in infection after 48 h, which was similar to that of transfection. These aptamers have great potential utility in the rapid diagnosis and inhibition of RGNNV infection in mariculture.

Design and Optimization of Tropomyosin Fragments for Tropomodulin Interaction Studies

Monomeric actin, or G-actin, polymerizes into filamentous actin, or F-actin which is an important component of cellular structures controlling many processes including vesicle trafficking, cell motility, inter and intracellular transport, and muscle contraction. Actin filaments are composed of a pointed end (slow growing) and a barbed end (fast growing). Interactions at each end enables control of the length of actin filaments. Tropomodulin (Tmod1-4) modulates actin filament length by interacting with tropomyosin (Tpm) and actin at the pointed end of the filament. Tmod binds two Tpm molecules through two distinct Tpm binding sites. Establishing the structure of each Tmod-Tpm binding interface is critical to understand the thin filament length regulation mechanism of Tmod. To facilitate NMR structure determination, we have established the optimal size of Tpm fragment necessary to form respective stable complexes. The four fragments we designed were residues 1-43, 1-33, 1-26 or 1-19 of Tpm1.8 fused with a GCN4 sequence promoting coiled-coil formation. We used NMR to determine and compare spectral effects between the optimized Tpm fragments and Tpm-binding sites of Tmod2. A significant change in spectra occurred when length of Tpm sequence changed from 19 to 26 residues and with almost no change with further increase in Tpm sequence length. Stability of the designed Tpm fragments and their affinity for Tpm-binding sites on Tmod2 were evaluated by CD spectroscopy. Our results revealed that Tpm1.8 fragments containing residues 1-19 and 1-43 were more stable with a melting temperature of 44 and 52oC, respectively, however, the 1-19 fragment was not long enough to represent the whole binding interface. The calculated dissociation constants showed strong affinity of Tmod2 for the Tpm1.8 1-43. Therefore, Tpm1.8-1-43 fragment is the most optimal for use in NMR studies to further study its interaction with Tmod.

Potential novel inhibitors against emerging zoonotic pathogen Nipah virus: a virtual screening and molecular dynamics approach

Nipah virus is a pathogen considered highly infectious, and its lethality can cause between 40% and 70% of deaths in those infected. At present, no effective treatment is available which results in an imperative need to explore new approaches to the search for drugs. Through virtual screening techniques, docking and molecular dynamics, 183 ligands were evaluated against the Nipah virus glycoprotein (NiV-G), involved throughout the process of virus entry to the host cell, resulting in a good target for blocking the infection. Of the 183 drugs computationally screened, three of them (MMV020537, MMV688888 and MMV019838) were found to be potential inhibitors of NiV-G. Their calculated dissociation constants were 0.03 nM, 2.18 nM and 31.61 nM, respectively. Molecular dynamics studies confirm their stability binding modes in the active site of the protein. These potential inhibitors can be used later as leads for the development of new drugs that allow effective treatment of the disease.Communicated by Ramaswamy H. Sarma

Upgrading and Validation of the AMBER Force Field for Histidine and Cysteine Zinc(II)-Binding Residues in Sites with Four Protein Ligands.

We developed and validated a novel force field in the context of the AMBER parameterization for the simulation of zinc(II)-binding proteins. The proposed force field assumes nonbonded spherical interactions between the central zinc(II) and the coordinating residues. A crucial innovative aspect of our approach is to account for the polarization effects of the cation by redefining the atomic charges of the coordinating residues and an adjustment of Lennard-Jones parameters of Zn-interacting atoms to reproduce mean distance distributions. The optimal transferable parametrization was obtained by performing accurate quantum mechanical calculations on a training set of high-quality protein structures, encompassing the most common folds of zinc(II) sites. The addressed sites contain a zinc(II) ion tetra-coordinated by histidine and cysteine residues and represent about 70% of all physiologically relevant zinc(II) sites in the Protein Data Bank. Molecular dynamics simulations with explicit solvent, carried out on several zinc(II)-binding proteins not included in the training set, show that our model for zinc(II) sites preserves the tetra-coordination of the metal site with remarkable stability, yielding zinc(II)-X mean distances similar to experimental data. Finally, the model was tested by evaluating the zinc(II)-binding affinities, using the alchemical free energy perturbation approach. The calculated dissociation constants correlate satisfactorily with the experimental counterpart demonstrating the validity and transferability of the proposed parameterization for zinc(II)-binding proteins.

Study of G-Protein Coupled Receptor Signaling in Membrane Environment by Plasmon Waveguide Resonance.

Here we describe an experimental technique, termed plasmon waveguide resonance (PWR) spectroscopy that enables the characterization of molecular interactions occurring at the level of anisotropic thin films as lipid membranes and therein inserted or interacting molecules. PWR allows one to characterize such molecular interactions at different levels: (1) acquire binding curves and calculate dissociation constants; (2) obtain kinetic information; (3) obtain information about associated anisotropy changes and changes in membrane thickness; (4) obtain insight about lateral homogeneity (formation of domains). Points 1, 2, and 4 can be directly obtained from the data. Point 3 requires spectral fitting procedures so that the different optical parameters characterizing thin films as proteolipid membranes, namely refractive index and extinction coefficient for both p- (TM component of light that is parallel to the incident light) and s- (TE component of light that is perpendicular to the incident light) polarizations and thickness, can be determined. When applied to membrane proteins as the G-protein coupled receptor (GPCR) family, both ligand-induced conformational changes of the receptor can be followed as well as interactions with effectors (e.g., G-proteins). Additionally, by either altering the lipid composition in cellular membranes or specifically controlling its composition in the case of lipid model membranes with reconstituted proteins, the role of the lipid environment in receptor activation and signaling can be determined. Additionally, the eventual partition of receptors in different lipid microdomains (e.g., lipid rafts) can be followed. Such information can be obtained ex cellulo with mammalian cell membrane fragments expressing the protein of interest and/or in vitro with lipid model systems where the protein under investigation has been reconstituted. Moreover, PWR can also be applied to directly follow the reconstitution of membrane proteins in lipid model membranes. The measurements are performed directly (no labeling of molecular partners), in real time and with very high sensitivity. Here we will discuss different aspects of GPCR activation and signaling where PWR brought important information in parallel with other approaches. The utility of PWR is not limited to GPCRs but can be applied to any membrane protein. PWR is also an excellent tool to characterize the interaction of membrane active molecules (as cell penetrating, antimicrobial, viral and amyloid peptides) with lipids. A brief section is dedicated to such applications, with particular emphasis on amyloid peptides. To finalize, as PWR is a homemade technology, ongoing instrument developments aiming at breaking current experimental limitations are briefly discussed, namely, the coupling of PWR with electrochemical measurements and the expansion of measurements from the visible to the infrared region.

Investigation of the calcium-induced activation of the bacteriophage T5 peptidoglycan hydrolase promoting host cell lysis.

Peptidoglycan hydrolase of bacteriophage T5 (EndoT5) is a Ca2+-dependent l-alanyl-d-glutamate peptidase, although the mode of Ca2+ binding and its physiological significance remain obscure. Site-directed mutagenesis was used to elucidate the role of the polar amino acids of the mobile loop of EndoT5 (111-130) in Ca2+ binding. The mutant proteins were purified to electrophoretic homogeneity, the overall structures were characterized by circular dichroism, and the calcium dissociation constants were determined via NMR spectroscopy. The data suggest that polar amino acids D113, N115, and S117 of EndoT5 are involved in the coordination of calcium ions by forming the core of the EF-like Ca2+-binding loop while the charged residues D122 and E123 of EndoT5 contribute to maintaining the loop net charge density. The results suggest that Ca2+ binding to the EndoT5 molecule could be essential for the stabilization of the long mobile loop in the catalytically active "open" conformation. The possible mechanism of Ca2+ regulation of EndoT5 activity during bacteriophage T5's life cycle through the Ca2+ concentration difference between the cytoplasm and the periplasm of the host bacteria cell has been discussed. The study reveals valuable insight into the role of calcium in the regulation of phage-induced bacterial lysis.