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1.
Machaidze G  Seelig J 《Biochemistry》2003,42(43):12570-12576
Cinnamycin (Ro 09-0198) is a tetracyclic peptide antibiotic that binds specifically to phosphatidylethanolamine (PE). Formation of a complex with phosphatidylethanolamine follows a 1:1 stoichiometry. Using high-sensitivity isothermal titration calorimetry (ITC), we have measured the thermodynamic parameters of complex formation for two different PE environments, namely, PE dissolved either in octyl glucoside (OG) micelles or in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer membrane. We have compared diacyl-PE with lyso-PE and have varied the carbon chain length from 6 to 18. Binding requires both a PE headgroup and at least one fatty acyl chain. The optimum chain length for complex formation (n) is eight. Longer chains do not enhance the binding affinity; for shorter chains, the interaction is weakened. The cinnamycin-PE complex has a binding constant K(0) of approximately 10(7)-10(8) M(-1) in the POPC membrane and only approximately 10(6) M(-1) in the octyl glucoside micelle. The difference can be attributed to the nonspecific hydrophobic interaction of cinnamycin with the lipid membrane. Complex formation is enthalpy-driven in OG micelles, whereas enthalpy and entropy make equal contributions in bilayer membranes. However, for the optimum chain length (n) of eight, the binding reaction is also completely enthalpy-driven for the bilayer membrane.  相似文献   

2.
Ro09-0198 is a cyclic peptide isolated from Streptoverticillium griseoverticillatum. This peptide caused permeability increase and aggregation of liposomes containing phosphatidylethanolamine. Liposomes containing phosphatidylserine, phosphatidylinositol or cardiolipin instead of phosphatidylethanolamine were, however, not appreciably reactive with the peptide. Among the structural analogs of phosphatidylethanolamine, dialkylphosphatidylethanolamine and 1-acylglycerophosphoethanolamine incorporated into liposomes could interact with Ro09-0198 to cause a permeability increase, whereas liposomes consisting of alkylphosphoethanolamine or phosphatidyl-N-monomethylethanolamine were insensitive to the peptide. These findings indicate that a glycerol backbone and a primary amino group of phosphatidylethanolamine are necessary for interaction with Ro09-0198 to cause membrane damage. Ro09-0198 induced a selective permeability change on liposomes. Glucose and umbelliferyl phosphate were effluxed significantly, but sucrose was only slightly permeable and inulin could not be released. Consequently, the permeability increase induced by Ro09-0198 is rather specific to molecules smaller than sucrose. Line broadening of electron spin resonance signals of spin-labeled phosphatidylethanolamine was observed upon treatment of liposomes with Ro09-0198. It was suggested from these results that Ro09-0198 can alter the physical organization of phosphatidylethanolamine in membranes, thus providing a basis for changes in membrane permeability.  相似文献   

3.
Duramycin is a 19-amino-acid tetracyclic lantibiotic closely related to cinnamycin (Ro09-0198), which is known to bind phosphatidylethanolamine (PE). The lipid specificity of duramycin was not established. The present study indicates that both duramycin and cinnamycin exclusively bind to ethanolamine phospholipids (PE and ethanolamine plasmalogen). Model membrane study indicates that the binding of duramycin and cinnamycin to PE-containing liposomes is dependent on membrane curvature, i.e., the lantibiotics bind small vesicles more efficiently than large liposomes. The binding of the lantibiotics to multilamellar liposomes induces tubulation of membranes, as revealed by electron microscopy and small-angle x-ray scattering. These results suggest that both duramycin and cinnamycin promote their binding to the PE-containing membrane by deforming membrane curvature.  相似文献   

4.
Ro09-0198, a cyclic peptide isolated from culture filtrates of Streptoverticillium griseoverticillatum, induced lysis of erythrocytes. Preincubation of the peptide with phosphatidylethanolamine reduced the hemolytic activity, whereas other phospholipids present in erythrocytes in nature had no effect. A study of the structural requirements on phosphatidylethanolamine necessary for interaction with the peptide indicates that Ro09-0198 recognizes strictly a particular chemical structure of phosphatidylethanolamine: dialkylphosphoethanolamine as well as 1-acylglycerophosphoethanolamine showed the same inhibitory effect on hemolysis induced by Ro09-0198 as diacylphospatidylethanolamine, whereas phosphoethanolamine gave no inhibitory effect. Neither phosphatidyl-N-monomethylethanolamine nor alkylphosphopropanolamine had an inhibitory effect. Consequently, the hydrophobic chain is necessary for the interaction and the phosphoethanolamine moiety is exactly recognized by the peptide. Ro-09-0198-induced hemolysis was temperature-dependent and the sensitivity of hemolysis differed greatly among animal species.  相似文献   

5.
Ro09-0198, a cyclic peptide isolated from culture filtrates of Streptoverticillium griseoverticillatum, induced lysis of erythrocytes. Preincubation of the peptide with phosphatidylethanolamine reduced the hemolytic activity, whereas other phospholipids present in erythrocytes in nature had no effect. A study of the structural requirements on phosphatidylethanolamine necessary for interaction with the peptide indicates that Ro09-0198 recognizes strictly a particular chemical structure of phosphatidylethanolamine: dialkylphosphoethanolamine as well as 1-acylglycerophosphoethanolamine showed the same inhibitory effect on hemolysis induced by Ro09-0198 as diacylphosphatidylethanolamine, whereas phosphoethanolamine gave no inhibitory effect. Neither phosphatidyl-N-monomethylethanolamine nor alkylphosphopropanolamine had an inhibitory effect. Consequently, the hydrophobic chain is necessary for the interaction and the phosphoethanolamine moiety is exactly recognized by the peptide. Ro-09-0198-induced hemolysis was temperature-dependent and the sensitivity of hemolysis differed greatly among animal species.  相似文献   

6.
Application of the cardiolipin (CL)-specific fluorescent dye 10-N-nonyl-acridine orange has recently revealed CL-rich domains in the septal regions and at the poles of the Bacillus subtilis membrane (F. Kawai, M. Shoda, R. Harashima, Y. Sadaie, H. Hara, and K. Matsumoto, J. Bacteriol. 186:1475-1483, 2004). This finding prompted us to examine the localization of another phospholipid, phosphatidylethanolamine (PE), with the cyclic peptide probe, Ro09-0198 (Ro), that binds specifically to PE. Treatment with biotinylated Ro followed by tetramethyl rhodamine-conjugated streptavidin revealed that PE is localized in the septal membranes of vegetative cells and in the membranes of the polar septum and the engulfment membranes of sporulating cells. When the mutant cells of the strains SDB01 (psd1::neo) and SDB02 (pssA10::spc), which both lack PE, were examined under the same conditions, no fluorescence was observed. The localization of the fluorescence thus evidently reflected the localization of PE-rich domains in the septal membranes. Similar PE-rich domains were observed in the septal regions of the cells of many Bacillus species. In Escherichia coli cells, however, no PE-rich domains were found. Green fluorescent protein fusions to the enzymes that catalyze the committed steps in PE synthesis, phosphatidylserine synthase, and in CL synthesis, CL synthase and phosphatidylglycerophosphate synthase, were localized mainly in the septal membranes in B. subtilis cells. The majority of the lipid synthases were also localized in the septal membranes; this includes 1-acyl-glycerol-3-phosphate acyltransferase, CDP-diacylglycerol synthase, phosphatidylserine decarboxylase, diacylglycerol kinase, glucolipid synthase, and lysylphosphatidylglycerol synthase. These results suggest that phospholipids are produced mostly in the septal membranes and that CL and PE are kept from diffusing out to lateral ones.  相似文献   

7.
It is widely accepted that phosphatidylethanolamine (PE) is enriched in the cytosolic leaflet of the eukaryotic plasma membranes. To identify genes involved in the establishment and regulation of the asymmetric distribution of PE on the plasma membrane, we screened the deletion strain collection of the yeast Saccharomyces cerevisiae for hypersensitive mutants to the lantibiotic peptide Ro09-0198 (Ro) that specifically binds to PE on the cell surface and inhibits cellular growth. Deletion mutants of VPS51, VPS52, VPS53, and VPS54 encoding the components of Golgi-associated retrograde protein (GARP) complex, YPT6 encoding a Rab family small GTPase that functions with GARP complex, RIC1 and RGP1 encoding its guanine nucleotide exchange factor (GEF), and TLG2 encoding t-SNARE exhibited hypersensitivity to Ro. The mutants deleted for VPS51, VPS52, VPS53, and VPS54 were impaired in the uptake of fluorescently labeled PE. In addition, aberrant intracellular localization of the EGFP-tagged Dnf2p, the putative inward-directed phospholipid translocase (flippase) of the plasma membrane, was observed in the mutant defective in the GARP complex, Ypt6p, its GEF proteins, or Tlg2p. Our results suggest that the GARP complex is involved in the recycling of Dnf flippases.  相似文献   

8.
In the early stages of apoptosis, phosphatidylserine (PS) is translocated from the inner side of the plasma membrane to the outer layer, which allows phagocytes to recognize and engulf the apoptotic cells. In this study we have analyzed the cell surface exposure of phosphatidylethanolamine (PE) in apoptotic CTLL-2 cells, a cytotoxic T cell line, using a tetracyclic polypeptide of 19 amino acids (Ro09-0198) which specifically recognizes the structure of PE and forms a tight equimolar complex with the phospholipid. Fluorescence microscopic analysis showed that the peptide, conjugated with fluorescence-labeled streptavidin (FL-SA-Ro), bound effectively to the cell surface of cells undergoing apoptosis in response to withdrawal of interleukin-2 from the culture media, but not to nonapoptotic cells. The binding of FL-SA-Ro to apoptotic cells was not uniform and the intense staining was observed on surface blebs of apoptotic cells. The FL-SA-Ro binding was inhibited specifically by liposomes containing PE, suggesting that PE is mainly exposed on the surface blebs of apoptotic cells. The specific binding of FL-SA-Ro to the apoptotic cells was also confirmed using a fluorescence-activated cell sorter and the time-dependent cell surface exposure of PE correlated well with the exposure of PS, as detected by the binding of annexin V. This study provides the first direct evidence that PE as well as PS is exposed on the cell surface during the early stages of apoptosis, resulting in the total loss of asymmetric distribution of aminophospholipids in the plasma membrane bilayer.  相似文献   

9.
There is evidence that membranes of rod outer segment (ROS) disks are a high-affinity Ca(2+) binding site. We were interested to see if the high occurrence of sixfold unsaturated docosahexaenoic acid in ROS lipids influences Ca(2+)-membrane interaction. Ca(2+) binding to polyunsaturated model membranes that mimic the lipid composition of ROS was studied by microelectrophoresis and (2)H NMR. Ca(2+) association constants of polyunsaturated membranes were found to be a factor of approximately 2 smaller than constants of monounsaturated membranes. Furthermore, strength of Ca(2+) binding to monounsaturated membranes increased with the addition of cholesterol, while binding to polyunsaturated lipids was unaffected. The data suggest that the lipid phosphate groups of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) in PC/PE/PS (4:4:1, mol/mol) are primary targets for Ca(2+). Negatively charged serine in PS controls Ca (2+) binding by lowering the electric surface potential and elevating cation concentration at the membrane/water interface. The influence of hydrocarbon chain unsaturation on Ca(2+) binding is secondary compared to membrane PS content. Order parameter analysis of individual lipids in the mixture revealed that Ca(2+) ions did not trigger lateral phase separation of lipid species as long as all lipids remained liquid-crystalline. However, depending on temperature and hydrocarbon chain unsaturation, the lipid with the highest chain melting temperature converted to the gel state, as observed for the monounsaturated phosphatidylethanolamine (PE) in PC/PE/PS (4:4:1, mol/mol) at 25 degrees C.  相似文献   

10.
Constituents of platelet membranes regulate the activity of the prothrombinase complex. We demonstrate that membranes containing phosphatidylcholine and phosphatidylethanolamine (PE) bind factor Va with high affinity (K(d) = ~10 nm) in the absence of phosphatidylserine (PS). These membranes support formation of a 60-70% functional prothrombinase complex at saturating factor Va concentrations. Although reduced interfacial packing does contribute to factor Va binding in the absence of PS, it does not correlate with the enhanced activity of the Xa-Va complex assembled on PE-containing membranes. Instead, specific protein-PE interactions appear to contribute to the effects of PE. In support of this, soluble C6PE binds to recombinant factor Va(2) (K(d) = ~6.5 μm) and to factor Xa (K(d) = ~91 μm). C6PE and C6PS binding sites of factor Xa are specific, distinct, and linked, because binding of one lipid enhances the binding and activity effects of the other. C6PE triggers assembly (K(d)(app) = ~40 nm) of a partially active prothrombinase complex between factor Xa and factor Va(2), compared with K(d)(app) for C6PS ~2 nm. These findings provide new insights into the possible synergistic roles of platelet PE and PS in regulating thrombin formation, particularly when exposed membrane PS may be limiting.  相似文献   

11.
The constitution and configuration of Ro 09-0198 (cinnamycin) have been determined in DMSO. Further investigations in aqueous solution, in SDS micelles and in a lipid bilayer have been done to study the influence of different environments on the conformation of the peptide. It turned out that in spite of the polycyclic structure of the molecule, the conformation is drastically changed going from water to SDS micelles. Ro 09-0198 orients itself in lipid bilayers as expected from its amphiphilic structure. According to a nuclear Overhauser effect spectroscopy experiment under magic angle spinning (MAS) conditions, the molecule is incorporated into the membrane with its hydrophobic part inside the bilayer.  相似文献   

12.
By employing diphenylhexatriene steady-state fluorescence anisotropy, pyrenedecanoic acid excimer formation, and high sensitivity scanning calorimetry we have demonstrated that the liposomes containing phosphatidylethanolamine (PE) and various mole fractions of ganglioside GD1a had a gel-liquid crystalline phase transition between 15 and 25 degrees C. Calorimetric measurements indicated that these phase transitions were broad and centered between 17 and 21 degrees C. The enthalpy change of the transition was linearly dependent on the ganglioside concentration up to 10.0 mol% and plateaued between 11.4-16.2 mol%. The high enthalpy change (37 kcal/mol of GD1a added into the PE bilayer) indicates the existence of PE-GD1a complex structure in the liposomal membrane. It is proposed that semi-fluid domains containing six PE and one ganglioside molecule are present in the PE-GD1a membranes at temperatures above gel-liquid crystalline phase transition. The Sendai virus induced leakage of PE-GD1a liposomes has been investigated by using an entrapped, self-quenching fluorescent dye, calcein. The leakage rate was dependent on the mole fraction of ganglioside GD1a and was maximal at 6.3 mol%. Arrhenius plots of the leakage rates showed breaks in the 20-25 degrees C temperature range, which correspond to the gel-liquid crystalline phase transition of the target liposomes. These data suggest that the rate of Sendai virus-induced leakage can be regulated via fluidity modulation by changing the PE to GD1a ratio at constant temperatures.  相似文献   

13.
T Nowak  M J Lee 《Biochemistry》1977,16(7):1343-1350
The formation of multiple ligand complexes with muscle pyruvate kinase was measured in terms of dissociation constants and the standard free energies of formation were calculated. The binding of Mn2+ to the enzyme (KA = 55 +/- 5 X 10(-6) M; deltaF degrees = -5.75 +/- 0.05 kcal/mol) and to the enzyme saturated with phosphoenolpyruvate (conditional free energy) KA' = 0.8 +/- 0.4 X 10(-6) M; deltaF degrees = -8.22 +/- 0.34 kcal/mol) has been measured under identical conditions giving a free energy of coupling, delta(deltaF degrees) = -2.47 +/- 0.34 kcal/mol. Such a large negative free energy of coupling is diagnostic of a strong positively cooperative effect in ligand binding. The binding of the substrate phosphoenolpyruvate to free enzyme and the enzyme-Mn2+ complex was, by necessity, measured by different methods. The free energy of phosphoenolpyruvate binding to free enzyme (KS = 1.58 +/- 0.10 X 10(-4)M; deltaF degrees = -5.13 +/- 0.04 kcal/mol) and to the enzyme-Mn2+ complex (K3 = 0.75 +/- 0.10 X 10(-6)M; deltaF degrees = -8.26 +/- 0.07 kcal/mol) also gives a large negative free energy of coupling, delta(deltaF degrees) = -3.16 +/- 0.08 kcal/mol. Such a large negative value confirms reciprocal binding effects between the divalent cation and the substrate phosphoenolpyruvate. The binding of Mn2+ to the enzyme-ADP complex was also investigated and a free energy of coupling, delta(deltaF degrees) = -0.08 +/- 0.08 kcal/mol, was measured, indicative of little or no cooperativity in binding. The free energy of coupling with Mn2+ and pyruvate was measured as -1.52 +/- 0.14 kcal/mol, showing a significant amount of cooperativity in ligand binding but a substantially smaller effect than that observed for phosphoenolpyruvate binding. The magnitude of the coupling free energy may be related to the role of the divalent cation in the formation of the enzyme-substrate complexes. In the absence of the activating monovalent cation, the coupling free energies for phosphoenolpyruvate and pyruvate binding decrease by 40-60% and 25%, respectively, substantiating a role for the monovalent cation in the formation of enzyme-substrate complexes with phosphoenolpyruvate and with pyruvate.  相似文献   

14.
Ro09-0198 (Ro) is a tetracyclic peptide antibiotic that binds specifically to phosphatidylethanolamine (PE) and causes cytolysis. To investigate the molecular basis of transbilayer movement of PE in biological membranes, we have isolated a series of budding yeast mutants that are hypersensitive to the Ro peptide. One of the most sensitive mutants, designated ros3 (Ro-sensitive 3), showed no significant change in the cellular phospholipid composition or in the sensitivity to amphotericin B, a sterol-binding polyene macrolide antibiotic. These results suggest that the mutation of ros3 affects the PE organization on the plasma membrane, rather than PE synthesis or overall organization of the membrane structures. By functional complementation screening, we identified the gene ROS3 affected in the mutant, and we showed that the hypersensitive phenotype was caused by the defective expression of the ROS3 gene product, Ros3p, an evolutionarily conserved protein with two putative transmembrane domains. Disruption of the ROS3 gene resulted in a marked decrease in the internalization of fluorescence-labeled analogs of PE and phosphatidylcholine, whereas the uptake of fluorescence-labeled phosphatidylserine and endocytic markers was not affected. Neither expression levels nor activities of ATP-binding cassette transporters of the ros3Delta cells differed from those of wild type cells, suggesting that Ros3p is not related to the multidrug resistance activities. Immunochemical analyses of the structure and subcellular localization showed that Ros3p was a glycosylated membrane protein localized in both the plasma membrane and the endoplasmic reticulum, and that a part of Ros3p was associated with the detergent-insoluble glycolipid-enriched complexes. These results indicate that Ros3p is a membrane glycoprotein that plays an important role in the phospholipid translocation across the plasma membrane.  相似文献   

15.
Ziegler A  Blatter XL  Seelig A  Seelig J 《Biochemistry》2003,42(30):9185-9194
Cell-penetrating peptides (CPPs) traverse cell membranes of cultured cells very efficiently by a mechanism not yet identified. Recent theories for the translocation suggest either the binding of the CPPs to extracellular glycosaminoglycans or the formation of inverted micelles with negatively charged lipids. In the present study, the binding of the protein transduction domains (PTD) of human (HIV-1) and simian immunodeficiency virus (SIV) TAT peptide (amino acid residues 47-57, electric charge z(p) = +8) to membranes containing various proportions of negatively charged lipid (POPG) is characterized. Monolayer expansion measurements demonstrate that TAT-PTD insertion between lipids requires loosely packed monolayer films. For densely packed monolayers (pi > 29 mN/m) and lipid bilayers, no insertion is possible, and binding occurs via electrostatic adsorption to the membrane surface. Light scattering experiments show an aggregation of anionic lipid vesicles when the electric surface charge is neutralized by TAT-PTD, the observed stoichiometry being close to the theoretical value of 1:8. Membrane binding was quantitated with isothermal titration calorimetry and three further methods. The reaction enthalpy is Delta H degrees approximately equal to -1.5 kcal/mol peptide and is almost temperature-independent with Delta C(p) degrees approximately 0 kcal/(mol K), indicating equal contributions of polar and hydrophobic interactions to the reaction heat capacity. The binding of TAT-PTD to the anionic membrane is described by an electrostatic attraction/chemical partition model. The electrostatic attraction energy, calculated with the Gouy-Chapman theory, accounts for approximately 80% of the binding energy. The overall binding constant, K(app), is approximately 10(3)-10(4) M(-1). The intrinsic binding constant (K(p)), corrected for electrostatic effects and describing the partitioning of the peptide between the lipid-water interface and the membrane, is small and is K(p) approximately 1-10 M(-1). Deuterium and phosphorus-31 nuclear magnetic resonance demonstrate that the lipid bilayer remains intact upon TAT-PTD binding. The NMR data provide no evidence for nonbilayer structures and also not for domain formation. This is further supported by the absence of dye efflux from single-walled lipid vesicles. The electrostatic interaction between TAT-PTD and anionic phosphatidylglycerol is strong enough to induce a change in the headgroup conformation of the anionic lipid, indicating a short-lived but distinct correlation between the TAT-PTD and the anionic lipids on the membrane outside. TAT-PTD has a much lower affinity for lipid membranes than for glycosaminoglycans, making the latter interaction a more probable pathway for CPP binding to biological membranes.  相似文献   

16.
OCP1 and OCP2, the most abundant proteins in the cochlea, are putative subunits of an SCF E3 ubiquitin ligase. Previous work has demonstrated that they form a heterodimeric complex. The thermodynamic details of that interaction are herein examined by isothermal titration calorimetry. At 25 degrees C, addition of OCP1 to OCP2 yields an apparent association constant of 4.0 x 10(7) M(-1). Enthalpically-driven (DeltaH=-35.9 kcal/mol) and entropically unfavorable (-TDeltaS=25.5 kcal/mol), the reaction is evidently unaccompanied by protonation/deprotonation events. DeltaH is strongly dependent on temperature, with DeltaC(p)=-1.31 kcal mol(-1) K(-1). Addition of OCP2 to OCP1 produces a slightly less favorable DeltaH, presumably due to the requirement for dissociation of the OCP2 homodimer prior to OCP1 binding. The thermodynamic signature for OCP1/OCP2 complex formation is inconsistent with a rigid-body association and suggests that the reaction is accompanied by a substantial degree of folding.  相似文献   

17.
Apical membrane vesicles were prepared from bovine tracheal epithelial cells. These membranes were enriched in alkaline phosphatase specific activity 35-fold compared to cellular homogenates. Steady-state fluorescence polarization studies of these membranes, using three fluorophores, demonstrated that they possessed a relatively low fluidity. Studies using the probe 1,6-diphenyl-1,3,5-hexatriene detected thermotropic transitions at 25.7 +/- 0.4 and 26.8 +/- 0.6 degrees C in these membranes and their liposomes, respectively. Analysis of the composition of these membranes revealed a fatty acyl saturation index of 0.59 +/- 0.02, a protein/lipid ratio (w/w) of 0.60 +/- 0.06, a cholesterol/phospholipid ratio (mol/mol) of 0.83 +/- 0.11, and a sphingomyelin/lecithin ratio (mol/mol) of 0.64 +/- 0.10. Membrane vesicles were osmotically active when studied by a stopped-flow nephelometric technique. Arrhenius plots of rates of osmotic water efflux demonstrated break points at approximately 28 and 18 degrees C, with activation energies of 16.7 +/- 0.2 kcal mol-1 from 35 to 28 degrees C, 8.3 +/- 0.5 kcal mol-1 from 28 to 18 degrees C, and approximately 3.0 kcal mol-1 below 18 degrees C. Treatment of membrane vesicles with benzyl alcohol, a known fluidizer, decreased lipid order (increased fluidity) and increased the rate of osmotic water efflux. The present results suggest that water crosses tracheal epithelial cell apical membranes by solubility-diffusion across the lipid domain and that increases in fluidity correlate with increases in the water permeability of these membranes.  相似文献   

18.
The mechanism of recognition of proteins and peptides by antibodies and the factors determining binding affinity and specificity are mediated by essentially the same features. However, additional effects of the usually unfolded and flexible solution structure of peptide ligands have to be considered. In an earlier study we designed and optimized six peptides (pepI to pepVI) mimicking the discontinuous binding site of interleukin-10 for the anti-interleukin-10 monoclonal antibody (mab) CB/RS/1. Three of them were selected for analysis of their solution conformation by circular dichroism measurements. The peptides differ in the content of alpha-helices and in the inducibility of helical secondary structures by trifluoroethanol. These properties, however, do not correlate with the binding affinity. PepVI, a 32-mer cyclic epitope mimic, has the highest affinity to mab CB/RS/1 identified to date. CD difference spectroscopy suggests an increase of the alpha-helix content of pepVI with complex formation. Binding of pepVI to mab CB/RS/1 is characterized by a large negative, favorable binding enthalpy and a smaller unfavorable loss of entropy (DeltaH degrees = -16.4 kcal x mol(-1), TDeltaS degrees = -6.9 kcal x mol(-1)) resulting in DeltaG degrees = -9.5 kcal x mol(-1) at 25 degrees C as determined by isothermal titration calorimetry. Binding of pepVI is enthalpically driven over the entire temperature range studied (10-35 degrees C). Complex formation is not accompanied by proton uptake or release. A negative heat capacity change DeltaC(p) of -0.354 kcal x mol(-1) x K(-1) was determined from the temperature dependence of DeltaH degrees. The selection of protein mimics with the observed thermodynamic properties is promoted by the applied identification and iterative optimization procedure.  相似文献   

19.
The thermodynamics of zinc hematoporphyrin (ZnHP) dimerization and ZnHP-membrane binding were studied. The dimerization equilibrium was determined over the temperature range 19-40 degrees C, using fluorometric techniques. The dimerization constant obtained at 37 degrees C (neutral pH in phosphate-buffered saline) is 4.6 (+/- 0.6) X 10(4) M-1. The dimerization was found to decrease with temperature over the range 19-36 degrees C, the data allowing the extraction of the following thermodynamic parameters for the temperature range 19-31 degrees C: delta G0 = -9.3 kcal/mol, delta H0 = -7.4 kcal/mol, delta S0 = -6.4 eu. For temperatures above 36 degrees C the dimerization was found to be temperature independent, giving the following parameters: delta G0 = -6.6 kcal/mol, delta H0 = 0 kcal/mol, delta S0 = 21.2 eu. On the basis of the data the case is made for the existence of two types of ZnHP dimers, differing in the location of the fifth Zn2+ ligand and in the nature of the contribution of the solvent to the dimerization. For the membrane binding, large unilamellar liposomes served to model biological membranes. The binding of ZnHP to the liposomes was found to be similar, quantitatively, to the corresponding metal-free molecule, namely, fitting a case of one type of site and giving a binding constant of 1600 +/- 160 M (neutral pH and 37 degrees C) which is independent of the length of the porphyrin-liposome.  相似文献   

20.
T Wieprecht  M Beyermann  J Seelig 《Biochemistry》1999,38(32):10377-10387
Magainins are positively charged amphiphatic peptides which permeabilize cell membranes and display antimicrobial activity. They are usually thought to bind specifically to anionic lipids, and binding studies have been performed almost exclusively with negatively charged membranes. Here we demonstrate that binding of magainins to neutral membranes, a reaction which is difficult to assess with spectroscopic means, can be followed with high accuracy using isothermal titration calorimetry. The binding mechanism can be described by a surface partition equilibrium after correcting for electrostatic repulsion by means of the Gouy-Chapman theory. Unusual thermodynamic parameters are observed for the binding process. (i) The three magainin analogues that were investigated bind to neutral membranes with large exothermic reaction enthalpies DeltaH of -15 to -18 kcal/mol (at 30 degrees C). (ii) The reaction enthalpies increase with increasing temperature, leading to a large positive heat capacity DeltaC(p) of approximately 130 cal mol(-)(1) K(-)(1) (at 25 degrees C). (iii) The Gibbs free energies of binding DeltaG are between -6.4 and -8.6 kcal/mol, resulting in a large negative binding entropy DeltaS. The binding of magainin to small unilamellar vesicles is hence an enthalpy-driven reaction. The negative DeltaH and DeltaS and the large positive DeltaC(p) contradict the conventional understanding of the hydrophobic effect. CD experiments reveal that the membrane-bound fraction of magainin is approximately 80% helical at 8 degrees C, decreasing to approximately 60% at 45 degrees C. Since the random coil --> alpha-helix transition in aqueous solution is known to be an exothermic process, the same process occurring at the membrane surface is shown to account for up to 65% of the measured reaction enthalpy. In addition to membrane-facilitated helix formation, the second main driving force for membrane binding is the insertion of the nonpolar amino acid side chains into the lipid bilayer. It also contributes a negative DeltaH and follows the pattern for the nonclassical hydrophobic effect. Addition of cholesterol drastically reduces the extent of peptide binding and reveals an enthalpy-entropy compensation mechanism. Membrane permeability was measured with a dye assay and correlated with the extent of peptide binding. The level of dye efflux is linearly related to the amount of surface-bound peptide and can be traced back to a membrane perturbation effect.  相似文献   

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