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Biophysical Characterization (biophysical + characterization)
Selected AbstractsStructural and Biophysical Characterization of XIAP BIR3 G306E Mutant: Insights in Protein Dynamics and Application for Fragment-Based Drug DesignCHEMICAL BIOLOGY & DRUG DESIGN, Issue 3 2009Cathy D. Moore Previous reports describe modulators of X-linked inhibitor of apoptosis (XIAP),caspase interaction designed from the AVPI N-terminal peptide sequence of second mitochondria-derived activator of caspase. A fragment-based drug design strategy was initiated to identify therapeutic non-peptidomimetic antagonists of X-linked inhibitor of apoptosis protein,protein interactions. Fragments that bind to the AVPI binding site of BIR3 (bacculoviral inhibitory repeat) were identified, and to further localize the fragment binding within the AVPI binding site, a point mutation was designed which alters the dynamics of flexible loops and blocks PI region of the binding cleft, thus enabling definition of weakly bound small molecules in the AV portion of the binding cleft. Nuclear magnetic resonance analysis confirmed the G306E mutation stabilizes the AV pocket. Biophysical characterization of the mutant confirms conformation change within the PI sub-pocket as evidenced by a significant diminishment in binding affinity of AVPI mimetics, yet the binding affinity of the smaller AV mimetics is maintained or slightly improved in the mutant compared with wild-type. Additional data from non-covalent mass spectrometry analysis shows enhanced binding of AV mimetics to the G306E mutant over the wild-type. The presented data outline a protein engineering strategy that allowed mapping of AV-replacements with better sensitivity and precision. [source] Biophysical characterization of synthetic rhamnolipidsFEBS JOURNAL, Issue 22 2006Jörg Howe Synthetic rhamnolipids, derived from a natural diacylated glycolipid, RL-2,214, produced by Burkholderia (Pseudomonas) plantarii, were analyzed biophysically. Changes in the chemical structures comprised variations in the length, the stereochemistry and numbers of the lipid chains, numbers of rhamnoses, and the occurrence of charged or neutral groups. As relevant biophysical parameters, the gel (,) to liquid crystalline (,) phase behavior of the acyl chains of the rhamnoses, their three-dimensional supramolecular aggregate structure, and the ability of the compounds to intercalate into phospholipid liposomes in the absence and presence of lipopolysaccharide-binding protein were monitored. Their biological activities were examined as the ability to induce cytokines in human mononuclear cells and to induce chemiluminescence in monocytes. Depending on the particular chemical structures, the physicochemical parameters as well as the biological test systems show large variations. This relates to the acyl chain fluidity, aggregate structure, and intercalation ability, as well as the bioactivity. Most importantly, the data extend our conformational concept of endotoxicity, based on the intercalation of naturally originating amphiphilic virulence factors into membranes from immune cells. This ,endotoxin conformation', produced by amphiphilic molecules with a hydrophilic charged backbone and apolar hydrophobic moiety, and adopting inverted cubic aggregate structures, causes high mechanical stress in target immune cells on integral proteins, eventually leading to cell activation. Furthermore, biologically inactive rhamnolipids with lamellar aggregate structures antagonize the endotoxin-induced activity in a way similar to lipid A-derived antagonists. [source] Biophysical characterization of the interaction of Limulus polyphemus endotoxin neutralizing protein with lipopolysaccharideFEBS JOURNAL, Issue 10 2004Jörg Andrä Endotoxin-neutralizing protein (ENP) of the horseshoe crab is one of the most potent neutralizers of endotoxins [bacterial lipopolysaccharide (LPS)]. Here, we report on the interaction of LPS with recombinant ENP using a variety of physical and biological techniques. In biological assays (Limulus amebocyte lysate and tumour necrosis factor-, induction in human mononuclear cells), ENP causes a strong reduction of the immunostimulatory ability of LPS in a dose-dependent manner. Concomitantly, the accessible negative surface charges of LPS and lipid A (zeta potential) are neutralized and even converted into positive values. The gel to liquid crystalline phase transitions of LPS and lipid A shift to higher temperatures indicative of a rigidification of the acyl chains, however, the only slight enhancement of the transition enthalpy indicates that the hydrophobic moiety is not strongly disturbed. The aggregate structure of lipid A is converted from a cubic into a multilamellar phase upon ENP binding, whereas the secondary structure of ENP does not change due to the interaction with LPS. ENP contains a hydrophobic binding site to which the dye 1-anilino-8-sulfonic acid binds at a Kd of 19 µm, which is displaced by LPS. Because lipopolysaccharide-binding protein (LBP) is not able to bind to LPS when ENP and LPS are preincubated, tight binding of ENP to LPS can be deduced with a Kd in the low nonomolar range. Importantly, ENP is able to incorporate by itself into target phospholipid liposomes, and is also able to mediate the intercalation of LPS into the liposomes thus acting as a transport protein in a manner similar to LBP. Thus, LPS,ENP complexes might enter target membranes of immunocompetent cells, but are not able to activate due to the ability of ENP to change LPS aggregates from an active into an inactive form. [source] Biophysical characterization of the interaction of high-density lipoprotein (HDL) with endotoxinsFEBS JOURNAL, Issue 23 2002Klaus Brandenburg The interaction of bacterial endotoxins [lipopolysaccharide (LPS) and the ,endotoxic principle' lipid A], with high-density lipoprotein (HDL) from serum was investigated with a variety of physical techniques and biological assays. HDL exhibited an increase in the gel to liquid crystalline phase transition temperature Tc and a rigidification of the acyl chains of the endotoxins as measured by Fourier-transform infrared spectroscopy and differential scanning calorimetry. The functional groups of the endotoxins interacting with HDL are the phosphates and the diglucosamine backbone. The finding of phosphates as target groups is in accordance to measurements of the electrophoretic mobility showing that the zeta potential decreases from ,50 to ,60 mV to ,20 mV at binding saturation. The importance of the sugar backbone as further target structure is in accordance with the remaining negative potential and competition experiments with polymyxin B (PMB) and phase transition data of the system PMB/dephosphorylated LPS. Furthermore, endotoxin binding to HDL influences the secondary structure of the latter manifesting in a change from a mixed ,-helical/,-sheet structure to a predominantly ,-helical structure. The aggregate structure of the lipid A moiety of the endotoxins as determined by small-angle X-ray scattering shows a change of a unilamellar/inverted cubic into a multilamellar structure in the presence of HDL. Fluorescence resonance energy transfer data indicate an intercalation of pure HDL, and of [LPS],[HDL] complexes into phospholipid liposomes. Furthermore, HDL may enhance the lipopolysaccharide-binding protein-induced intercalation of LPS into phospholipid liposomes. Parallel to these observations, the LPS-induced cytokine production of human mononuclear cells and the reactivity in the Limulus test are strongly reduced by the addition of HDL. These data allow to develop a model of the [endotoxin]/[HDL] interaction. [source] Biophysical characterization of polymeric and liposomal gene delivery systems using empirical phase diagramsJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 10 2006Marika Ruponen Abstract A major problem with the pharmaceutical use of nonviral gene delivery systems arises from their limited characterization due to their size and heterogeneity. In this study, we provide a more intuitive view of their structure and behavior employing an empirically based phase diagram approach. Complexes formed between plasmid DNA and four cationic carriers (a monovalent lipid, the same monovalent lipid combined with a helper lipid, polylysine, and a branched form of polyethyleneimine), at both positive and negative nitrogen/phosphorous ratios, are characterized employing dynamic light scattering, circular dichroism, and extrinsic dye fluorescence as methods sensitive to various aspects of the structure of the complexes. These measurements were performed as a function of pH and ionic strength to perturb the electrostatic contacts that are key to complex formation. Using a multidimensional eigenvalue approach, the data are presented in the form of a colored, five dimensional diagram. The resultant eight empirical phase diagrams display three to five variably resolved phases. In contrast, the phase diagram of the plasmid alone showed only two to three such phases. Each state is assigned to a particular form of the complex in terms of their size, extent of collapse and conformation of the associated DNA component. The utility of this approach is then briefly discussed. © 2006 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 95:2101,2114, 2006 [source] A preliminary crystallographic study of recombinant NicX, an Fe2+ -dependent 2,5-dihydroxypyridine dioxygenase from Pseudomonas putida KT2440ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2010José Ignacio Jiménez NicX from Pseudomonas putida KT2440 is an Fe2+ -dependent dioxygenase that is involved in the aerobic degradation of nicotinic acid. The enzyme converts 2,5-dihydroxypyridine to N -formylmaleamic acid when overexpressed in Escherichia coli. Biophysical characterization of NicX by analytical gel-filtration chromatography revealed that it behaves as an oligomeric assembly in solution, with an apparent molecular weight that is consistent with a hexameric species. NicX was crystallized by the hanging-drop vapour-diffusion method at 291,K. Diffraction data were collected to a resolution of 2.0,Å at the ESRF. The crystals most probably belong to the orthorhombic space group C222 or C2221. The estimated Matthews coefficient was 2.4,Å3,Da,1, corresponding to 50% solvent content, which is consistent with the presence of three protein molecules in the asymmetric unit. Analysis of the crystal data together with chromatographic results supports NicX being a hexameric assembly composed of two cyclic trimers. Currently, crystallization of recombinant selenomethionine-containing NicX is in progress. [source] Structural and Biophysical Characterization of XIAP BIR3 G306E Mutant: Insights in Protein Dynamics and Application for Fragment-Based Drug DesignCHEMICAL BIOLOGY & DRUG DESIGN, Issue 3 2009Cathy D. Moore Previous reports describe modulators of X-linked inhibitor of apoptosis (XIAP),caspase interaction designed from the AVPI N-terminal peptide sequence of second mitochondria-derived activator of caspase. A fragment-based drug design strategy was initiated to identify therapeutic non-peptidomimetic antagonists of X-linked inhibitor of apoptosis protein,protein interactions. Fragments that bind to the AVPI binding site of BIR3 (bacculoviral inhibitory repeat) were identified, and to further localize the fragment binding within the AVPI binding site, a point mutation was designed which alters the dynamics of flexible loops and blocks PI region of the binding cleft, thus enabling definition of weakly bound small molecules in the AV portion of the binding cleft. Nuclear magnetic resonance analysis confirmed the G306E mutation stabilizes the AV pocket. Biophysical characterization of the mutant confirms conformation change within the PI sub-pocket as evidenced by a significant diminishment in binding affinity of AVPI mimetics, yet the binding affinity of the smaller AV mimetics is maintained or slightly improved in the mutant compared with wild-type. Additional data from non-covalent mass spectrometry analysis shows enhanced binding of AV mimetics to the G306E mutant over the wild-type. The presented data outline a protein engineering strategy that allowed mapping of AV-replacements with better sensitivity and precision. [source] Membrane permeability and antimicrobial kinetics of cecropin P1 against Escherichia coli,JOURNAL OF PEPTIDE SCIENCE, Issue 6 2009Steven Arcidiacono Abstract The interaction of cecropin P1 (CP1) with Escherichiacoli was investigated to gain insight into the time-dependent antimicrobial action. Biophysical characterizations of CP1 with whole bacterial cells were performed using both fluorescent and colorimetric assays to investigate the role of membrane permeability and lipopolysaccharide (LPS) binding in lytic behavior. The kinetics of CP1 growth inhibition assays indicated a minimal inhibitory concentration (MIC) of 3 µM. Bactericidal kinetics at the MIC indicated rapid killing of E.coli (<30 min). Membrane permeability studies illustrated permeation as a time-dependent event. Maximum permeability at the MIC occurred within 30 min, which correlates to the bactericidal action. Further investigation showed that the immediate permeabilizing action of CP1 is concentration-dependent, which correlates to the concentration-dependent nature of the inhibition assays. At the MIC and above, the immediate permeability was significant enough that the cells could not recover and exhibit growth. Below the MIC, immediate permeability was evident, but the level was insufficient to inhibit growth. Dansyl polymyxin B displacement studies showed LPS binding is essentially the same at all concentrations investigated. However, it does appear that only the immediate interaction is important, because binding continued to increase over time beyond cell viability. Our studies correlated CP1 bactericidal kinetics to membrane permeability suggesting CP1 concentration-dependent killing is driven by the extent of the immediate permeabilizing action of the peptide. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd. [source] Quantitative model of cellulite: three-dimensional skin surface topography, biophysical characterization, and relationship to human perceptionINTERNATIONAL JOURNAL OF COSMETIC SCIENCE, Issue 4 2005L. K. Smalls Gynoid lipodystrophy (cellulite) is the irregular, dimpled skin surface of the thighs, abdomen, and buttocks in 85% of post-adolescent women. The distinctive surface morphology is believed to result when subcutaneous adipose tissue protrudes into the lower reticular dermis, thereby creating irregularities at the surface. The biomechanical properties of epidermal and dermal tissue may also influence severity. Cellulite-affected thigh sites were measured in 51 females with varying degrees of cellulite, in 11 non-cellulite controls, and in 10 male controls. A non-contact high-resolution three-dimensional (3D) laser surface scanner was used to quantify the skin surface morphology and determine specific roughness values. The scans were evaluated by experts and na,ve judges (n = 62). Body composition was evaluated via dual-energy X-ray absorptiometry; dermal thickness and the dermal,subcutaneous junction were evaluated via high-resolution 3D ultrasound and surface photography under compression. Biomechanical properties were also measured. The roughness parameters Svm (mean depth of the lowest valleys) and Sdr (ratio between the roughness surface area and the area of the xy plane) were highly correlated to the expert image grades and, therefore, designated as the quantitative measures of cellulite severity. The strength of the correlations among na,ve grades, expert grades, and roughness values confirmed that the data quantitatively evaluate the human perception of cellulite. Cellulite severity was correlated to BMI, thigh circumference, percent thigh fat, architecture of the dermal,subcutaneous border (ultrasound surface area, red-band SD from compressed images), compliance, and stiffness (negative correlation). Cellulite severity was predicted by the percent fat and the area of the dermal,subcutaneous border. The biomechanical properties did not significantly contribute to the prediction. Comparison of the parameters for females and males further suggests that percent thigh fat and surface area roughness deviation are the distinguishing features of cellulite. [source] Interaction of S413 -PV cell penetrating peptide with model membranes: relevance to peptide translocation across biological membranesJOURNAL OF PEPTIDE SCIENCE, Issue 5 2007Miguel Mano Abstract Cell penetrating peptides (CPPs) have been successfully used to mediate the intracellular delivery of a wide variety of molecules of pharmacological interest both in vitro and in vivo, although the mechanisms by which the cellular uptake occurs remain unclear and controversial. Following our previous work demonstrating that the cellular uptake of the S413 -PV CPP occurs mainly through an endocytosis-independent mechanism, we performed a detailed biophysical characterization of the interaction of this peptide with model membranes. We demonstrate that the interactions of the S413 -PV peptide with membranes are essentially of electrostatic nature. As a consequence of its interaction with negatively charged model membranes, the S413 -PV peptide becomes buried into the lipid bilayer, which occurs concomitantly with significant peptide conformational changes that are consistent with the formation of a helical structure. Comparative studies using two related peptides demonstrate that the conformational changes and the extent of cell penetration are dependent on the peptide sequence, indicating that the helical structure acquired by the S413 -PV peptide is relevant for its nonendocytic uptake. Overall, our data suggest that the cellular uptake of the S413 -PV CPP is a consequence of its direct translocation through cell membranes, following conformational changes induced by peptide-membrane interactions. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd. [source] Development of a cytokine analog with enhanced stability using computational ultrahigh throughput screeningPROTEIN SCIENCE, Issue 5 2002Peizhi Luo Abstract Granulocyte-colony stimulating factor (G-CSF) is used worldwide to prevent neutropenia caused by high-dose chemotherapy. It has limited stability, strict formulation and storage requirements, and because of poor oral absorption must be administered by injection (typically daily). Thus, there is significant interest in developing analogs with improved pharmacological properties. We used our ultrahigh throughput computational screening method to improve the physicochemical characteristics of G-CSF. Improving these properties can make a molecule more robust, enhance its shelf life, or make it more amenable to alternate delivery systems and formulations. It can also affect clinically important features such as pharmacokinetics. Residues in the buried core were selected for optimization to minimize changes to the surface, thereby maintaining the active site and limiting the designed protein's potential for antigenicity. Using a structure that was homology modeled from bovine G-CSF, core designs of 25,34 residues were completed, corresponding to 1021,1028 sequences screened. The optimal sequence from each design was selected for biophysical characterization and experimental testing; each had 10,14 mutations. The designed proteins showed enhanced thermal stabilities of up to 13°C, displayed five-to 10-fold improvements in shelf life, and were biologically active in cell proliferation assays and in a neutropenic mouse model. Pharmacokinetic studies in monkeys showed that subcutaneous injection of the designed analogs results in greater systemic exposure, probably attributable to improved absorption from the subcutaneous compartment. These results show that our computational method can be used to develop improved pharmaceuticals and illustrate its utility as a powerful protein design tool. [source] | ||||||||||