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Nonclassical hydrophobic effect in membrane binding equilibria. 总被引:7,自引:0,他引:7
The enthalpy of transfer of four different amphiphilic molecules from the aqueous phase to the lipid membrane was determined by titration calorimetry. The four molecules investigated were the potential-sensitive dye 2-(p-toluidinyl)naphthalene-6-sulfonate (TNS), the membrane conductivity inducing anion tetraphenylborate (TPB), the Ca2+ channel blocker amlodipine [B?uerle, H. D., & Seelig, J. (1991) Biochemistry 30, 7203-7211], and the positively charged local anesthetic dibucaine. All four amphiphiles penetrate into the hydrophobic part of the membrane, and their binding constants, after correcting for electrostatic effects, range between 600 M-1 for dibucaine and 60,000 M-1 for tetraphenylborate. The corresponding changes in free energy were about -6 to -9 kcal/mol. Binding of the amphiphiles to membrane vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine was accompanied by exothermic heats of reaction for all four molecules. For TNS, TPB, and amlodipine, the enthalpies of transfer were almost identical and corresponded to delta H approximately -9 kcal/mol, essentially accounting for the total free energy change. Thus, the binding of these charged amphiphiles to the hydrophobic membrane was driven by enthalpy. This is in contrast to the classical hydrophobic effect, where the transfer is considered to be entropy driven. For dibucaine, the enthalpy of transfer was smaller with delta H approximately -2 kcal/mol but was still about one-third of the total free energy change. All enthalpies of transfer exhibited a distinct temperature dependence with molar heat capacities delta Cp of -30 to -100 cal mol-1K-1 for the transfer from water to the membrane.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
3.
Cantor RS 《Chemistry and physics of lipids》1999,101(1):45-56
The function of many intrinsic membrane proteins requires a conformational transition that is often strongly influenced by the molecular composition of the bilayer in which the protein is embedded. Recently, a mechanism for this shift in conformational equilibrium was suggested, in which it is argued that a shift in distribution of lateral pressures of the bilayer resulting from a change in lipid composition alters the amount of mechanical work of the protein conformational transition, if the change in the cross-sectional area profile of the protein varies with depth within the bilayer. As there is little information on the change in shape of the transmembrane region of any protein, various simple geometric models are considered. For both a generic model, and more specific models that approximate likely cooperative rearrangements of alpha-helices in bundles, it is found that the conformational equilibrium depends on the first and second integral moments of the lateral pressure distribution. In addition to revealing the possible physical underpinnings of the well-known correlation between protein activity and the 'nonlamellar' tendency of bilayer lipids, this dependence on moments of the pressure profile allows for prediction of the relative effects of different lipid compositional changes even in the absence of information on specific protein shape changes. Effects of variation in acyl chain length, degree and position of cis-unsaturation, and addition of cholesterol and small interfacially-active solutes (n-alkanols) are compared. 相似文献
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Growth hormone conformation and conformational equilibria 总被引:3,自引:0,他引:3
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While several proteins, including beta-lactamase, cytochrome c and apomyoglobin, are maximally unfolded at pH 2 by HCl in the absence of salt, the addition of anions, either from salt or acid, co-operatively induces the unfolded proteins to refold to a molten globule state, because anions bind preferentially to the compact molten globule state compared to the extended unfolded state. To study the role of the anion-dependent conformational transition at neutral pH, we synthesized a model polypeptide of 51 amino acid residues, consisting of tandem repeats of a Lys-Lys-Leu-Leu sequence and containing a turn sequence, Asn-Pro-Gly, at the center of the molecule. The model polypeptide showed no significant conformation by circular dichroism under conditions of low salt at neutral pH. However, addition of anions, either from salt or acid, induced the folding transition to an alpha-helical conformational state. The order of effectiveness of various anions in inducing the folding transition was consistent with the series of anions in inducing the molten globule of the acid-denatured protein. This suggests that the helical state of the model polypeptide is equivalent to the molten globule state. At pH values above 9, the model polypeptide also took an alpha-helical conformation, which was very similar to that induced by anions. On the basis of the chloride and pH-dependent conformational transitions, a phase diagram for the conformational states was constructed. The phase diagram was explained simply by assuming that the conformational transition is linked to the proton and the anion bindings to a limited number of amino groups and that anions bind only to the protonated groups. 相似文献
6.
Dielectric response of hen egg white lysozyme is measured in the far infrared (5-65 cm-1, 0.15-1.95 THz, 0.6-8.1 meV) as a function of hydration. The frequency range is associated with collective vibrational modes of protein tertiary structure. The observed frequency dependence of the absorbance is broad and glass-like. For the entire frequency range, there is a slight increase in both the absorbance and index of refraction with increasing hydration for <0.27 h (mass of H2O per unit mass protein). At 0.27 h, the absorbance and index begin to increase more rapidly. This transition corresponds to the point where the first hydration shell is filled. The abrupt increase in dielectric response cannot be fully accounted for by the additional contribution to the dielectric response due to bulk water, suggesting that the protein has not yet achieved its fully hydrated state. The broad, glass-like response suggests that at low hydrations, the low frequency conformational hen egg white lysozyme dynamics can be described by a dielectric relaxation model where the protein relaxes to different local minima in the conformational energy landscape. However, the low frequency complex permittivity does not allow for a pure relaxational mechanism. The data can best be modeled with a single low frequency resonance (nu approximately 120 GHz=4 cm-1) and a single Debye relaxation process (tau approximately .03-.04 ps). Terahertz dielectric response is currently being considered as a possible biosensing technique and the results demonstrate the required hydration control necessary for reliable biosensor applications. 相似文献
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A mechanism proposed for lactose/H(+) symport by the lactose permease of Escherichia coli indicates that lactose permease is protonated prior to ligand binding. Moreover, in the ground state, the symported H(+) is shared between His322 (helix X) and Glu269 (helix VIII), while Glu325 (helix X) is charge-paired with Arg302 (helix IX). Substrate binding at the outer surface between helices IV (Glu126) and V (Arg144, Cys148) induces a conformational change that leads to transfer of the H(+) to Glu325 and reorientation of the binding site to the inner surface. After release of substrate, Glu325 is deprotonated on the inside due to re-juxtapositioning with Arg302. The conservative mutation Glu269-->Asp causes a 50-100-fold decrease in substrate binding affinity and markedly reduced active lactose transport, as well as decreased rates of equilibrium exchange and efflux. Gly-scanning mutagenesis of helix VIII was employed systematically with mutant Glu269-->Asp in an attempt to rescue function, and two mutants with increased activity are identified and characterized. Mutant Thr266-->Gly/Met267-->Gly/Glu269-->Asp binds ligand with increased affinity and catalyzes active lactose transport with a marked increase in rate; however, little improvement in efflux or equilibrium exchange is observed. In contrast, mutant Gly262-->Ala/Glu269-->Asp exhibits no improvement in ligand binding but a small increase in the rate of active transport; however, an increase in the steady-state level of accumulation, as well as efflux and equilibrium exchange is observed. Remarkably, when the two sets of mutations are combined, all translocation reactions are rescued to levels approximating those of wild-type permease. The findings support the contention that Glu269 plays a pivotal role in the mechanism of lactose/H(+) symport. Moreover, the results suggest that the two classes of mutants rescue activity by altering the equilibrium between outwardly and inwardly facing conformations of the permease such that impaired protonation and/or H(+) transfer is enhanced from one side of the membrane or the other. When the two sets of mutants are combined, the equilibrium between outwardly and inwardly facing conformations and thus protonation and H(+) transfer are restored. 相似文献
9.
Positively charged hydrophobic spin labels have been synthesized which respond to transmembrane potentials in sonicated liposomes. Electron paramagnetic resonance spectroscopy is used to show that the distribution of these probes between aqueous and membrane phases changes as a function of transmembrane potential. When liposomes are made more inside-negative, the fraction of membrane associated probe increases while the fraction of probe in the aqueous phase decreases. The results are in quantitative agreement with a simple equilibrium thermodynamic theory which allows estimation of absolute transmembrane potentials in phospholipid vesicles. 相似文献
10.
Conformation and mode of organization of amphiphilic membrane components: a conformational analysis. 总被引:2,自引:0,他引:2
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The insulin receptor is an integral membrane glycoprotein (Mr approximately 300,000) composed of two alpha-subunits (Mr approximately 130,000) and two beta-subunits (Mr approximately 95,000) linked by disulphide bonds. This oligomeric structure divides the receptor into two functional domains such that alpha-subunits bind insulin and beta-subunits possess tyrosine kinase activity. The amino acid sequence deduced from cDNA of the single polypeptide chain precursor of human placental insulin receptor revealed that alpha- and beta-subunits consist of 735 and 620 residues, respectively. The alpha-subunit is hydrophilic, disulphide-bonded, glycosylated and probably extracellular. The beta-subunit consists of a short extracellular region which links the alpha-subunit through disulphide bridges, a hydrophobic transmembrane region and a longer cytoplasmic region which is structurally homologous with other tyrosine kinases like the src oncogene product and EGF receptor kinases. The cellular function of insulin receptors is dual: transmembrane signalling and endocytosis of hormone. The binding of insulin to its receptor on the cell membrane induces transfer of signal from extracellular to cytoplasmic receptor domains leading to activation of cell metabolism and growth. In addition, hormone-receptor complexes are internalized leading to intracellular proteolysis of insulin, whereas receptors are recycled to the membrane. These phenomena are kinetically well-characterized, but their molecular mechanisms remain obscure. Insulin receptor in different tissues and animal species are homologous in their structure and function, but show also significant differences regarding size of alpha-subunits, binding kinetics, insulin specificity and receptor-mediated degradation. We suggest that this heterogeneity of receptors may be linked to the diversity in insulin effects on metabolism and growth in various cell types. The purified insulin receptor phosphorylates its own beta-subunit and exogenous protein and peptide substrates on tyrosine residues, a reaction which is insulin-sensitive, Mn2+-dependent and specific for ATP. Tyrosine phosphorylation of the beta-subunit activates receptor kinase activity, and dephosphorylation with alkaline phosphatase deactivates the kinase. In intact cells or impure receptor preparations, a serine kinase is also activated by insulin. The cellular role of two kinase activities associated with the insulin receptor is not known, but we propose that the tyrosine- and serine-specific kinases mediate insulin actions on metabolism and growth either through dual-signalling or sequential pathways.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
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Nanotubules formed by highly hydrophobic amphiphilic alpha-helical peptides and natural phospholipids
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Furuya T Kiyota T Lee S Inoue T Sugihara G Logvinova A Goldsmith P Ellerby HM 《Biophysical journal》2003,84(3):1950-1959
We previously reported that the 18-mer amphiphilic alpha-helical peptide, Hel 13-5, consisting of 13 hydrophobic residues and five hydrophilic amino acid residues, can induce neutral liposomes (egg yolk phosphatidylcholine) to adopt long nanotubular structures and that the interaction of specific peptides with specific phospholipid mixtures induces the formation of membrane structures resembling cellular organelles such as the Golgi apparatus. In the present study we focused our attention on the effects of peptide sequence and chain length on the nanotubule formation occurring in mixture systems of Hel 13-5 and various neutral and acidic lipid species by means of turbidity measurements, dynamic light scattering measurements, and electron microscopy. We designed and synthesized two sets of Hel 13-5 related peptides: 1) Five peptides to examine the role of hydrophobic or hydrophilic residues in amphiphilic alpha-helical structures, and 2) Six peptides to examine the role of peptide length, having even number residues from 12 to 24. Conformational, solution, and morphological studies showed that the amphiphilic alpha-helical structure and the peptide chain length (especially 18 amino acid residues) are critical determinants of very long tubular structures. A mixture of alpha-helix and beta-structures determines the tubular shapes and assemblies. However, we found that the charged Lys residues comprising the hydrophilic regions of amphiphilic structures can be replaced by Arg or Glu residues without a loss of tubular structures. This suggests that the mechanism of microtubule formation does not involve the charge interaction. The immersion of the hydrophobic part of the amphiphilic peptides into liposomes initially forms elliptic-like structures due to the fusion of small liposomes, which is followed by a transformation into tubular structures of various sizes and shapes. 相似文献
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The chemical synthesis and X-ray crystal structure analysis of a model peptide incorporating a conformationally flexible beta-Ala residue: Boc-beta-Ala-Pda, 1 (C23H46N2O3: molecular weight = 398.62) have been described. The peptide crystallized in the crystal system triclinic with space group P21: a = 5.116(3) A, b = 5.6770(10) A, c = 21.744(5) A; alpha = 87.45 degrees, beta = 86.87 degrees, gamma = 90.0 degrees; Z = 1. An attractive feature of the crystal molecular structure of 1 is the induction of a reasonably extended backbone conformation of the beta-Ala moiety, i.e. the torsion angles phi approximately -115 degrees, mu approximately 173 degrees and psi approximately 122 degrees, correspond to skew-, trans and skew+ conformation, respectively, by an unbranched hydrophobic alkyl chain, Pda, which prefers an all-anti orientation (theta1 approximately -153 degrees, theta2 approximately ellipsis theta14 approximately +/-178 degrees ). The observation is remarkable because, systematic conformational investigations of short linear beta-Ala peptides of the type Boc-beta-Ala-Xaa-OCH3 (Xaa = Aib or Acc6) have shown that the chemical and stereochemical characters of the neighboring moieties may be critical in dictating the overall folded and/or unfolded conformational features of the beta-Ala residue. The overall conformation of 1 is typical of a 'bar'. It appears convincing that, in addition to a number of hydrophobic contacts between the parallel arranged molecules, an array of conventional N-HellipsisO=C intermolecular H-bonding interactions stabilize the crystal molecular structure. Moreover, the resulting 14-membered pseudo-ring motif, generated by the amide-amide interactions between the adjacent molecules, is completely devoid of nonconventional C-HellipsisO interaction. The potentials of the conformational adaptation of the beta-Ala residue, to influence and stabilize different structural characteristics have been highlighted. 相似文献
14.
Dennison SR Hauss T Dante S Brandenburg K Harris F Phoenix DA 《Biophysical chemistry》2006,119(2):115-120
The replacement of hydrogen with deuterium is frequently used in conjunction with neutron diffraction to investigate peptide-membrane interaction. This isotopic substitution in an amino acid residue radically changes the neutron scatter pattern of the peptide, thereby allowing its localisation within the bilayer with the aid of derived Fourier maps. Nonetheless, this technique relies on the generally held assumption that normal and isotopically enriched protein species do not differ significantly in structure or biological activity. Recently, this assumption has been questioned and here, diffraction data from studies on a membrane interactive peptide clearly challenge the reliability of this assumption. 相似文献
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An amphiphilic peptide with a 3-arginine stretch and a 6-valine stretch was evaluated as a gene carrier. The short amphiphilic peptide, R3V6, not only formed micelles in aqueous solution, but was also able to deliver plasmid DNA (pDNA) into cells without toxicity. In this research, various amphiphilic peptides were synthesized with a 3-arginine stretch and a 6-valine, -alanine, -leucine, or -phenylalanine stretch. In vitro transfection assays in human embryonic kidney 293 cells showed that R3V6 and R3L6 peptides had higher transfection efficiencies than R3A6, R3F6, and poly-L-lysine (PLL). Since the peptide micelles had hydrophobic cores, a hydrophobic anti-cancer drug, bis-chloronitrosourea (BCNU),was able to be loaded into the cores of the micelles. The incorporation of the hydrophobic drug into the cores of the peptide micelles may stabilize the micelle structure and increase the transfection efficiency. The in vitro transfection assay with BCNU-loaded R3V6 (R3V6-BCNU) or R3L6 (R3L6-BCNU) showed that the BCNU-loaded peptide micelles had a higher transfection efficiency than the peptide micelles without BCNU. R3V6-BCNU and R3L6-BCNU had the highest transfection at a 0.8:1 weight ratio (BCNU:R3V6) and a 1.2:1 weight ratio (BCNU:R3L6), respectively. Furthermore, compared to simple diffusion, a more efficient delivery of the drug into cells may be facilitated by endocytosis of the micelles. R3L6-BCNU and R3V6-BCNU had higher cell toxicity to cells than BCNU alone. Therefore, the R3V6- and R3L6-BCNU may be useful for drug and gene combination cancer therapy. 相似文献
17.
Hapten-linked conformational equilibria in immunolglobulins XRPC-24 and J-539 observed by chemical relaxation. 总被引:3,自引:0,他引:3
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The interaction of oligogalactan haptens with the murine myeloma proteins XRPC-24 and J-539 has been investigated by the fluorescence temperature-jump method. The relaxation spectrum is composed of two processes, the faster representing hapten assocaition and the slower a protein isomerization. In both cases the concentration dependence of relaxation times and amplitudes was consistent with the general mechanism formulated by Lancet and Pecht (1976, Proc. Natl. Acad. Sci. U.S.A. 73:3549), in which the equilibrium between two conformations of the protein is shifted by hapten binding. The intact proteins and their Fab fragment had identical kinetic behavior, indicating that the conformational changes are located in the Fab region. Temperature dependence analysis for protein J-539 permitted the calculation of activation parameters and led to a consistent energy profile for all the elementary steps. The conformational states are separated by large activation barriers, but have similar free energies. The results suggest that hapten-induced conformational changes in immunoglobulins are more general phenomena than was previously thought. 相似文献
18.
The conformational equilibrium of a decapeptide renin inhibitor (Renin Inhibitory Peptide (RIP), NH-P-H-P-F-H-F-F-V-Y-K-CO2H) in water, methanol and trifluoroethanol has been investigated. The value of a combined spectroscopic approach was apparent, with the need to define conformational states that were mixtures of conformational forms. Similarities between this study and that of the Melanin Concentrating Hormone (MCH) core peptide (5-14) are notable [1]. In water, two beta-turn conformations and an extended form were found to be in equilibrium, with cis/trans isomerism at Pro-3. Extended conformations associated with the P(II) helix and irregular forms were more favoured in aqueous environments. In MeOH and TFE, two beta-turn conformations associated with overlapping sequences and cis/trans isomerism at Pro-3 amide bond were seen to be in equilibrium. 2D ROESY and chemical-exchange cross-peaks were detected by 1H NMR and used to build up detailed models of the interconverting beta-turn conformations of RIP. 相似文献
19.
The equilibria and dynamics of the disorder-to-order transition of the anionic polysaccharide iota-carrageenan have been studied in the presence of tetramethyl-ammonium salts. By the use of a stopped-flow polarimeter, the rate equation and temperature dependence of the observed forward rate-constant were found to accord with a co-operative dimerisation process. Activation parameters for helix nucleation were shown to be independent of the anion for solutions containing tetramethylammonium chloride and bromide, i.e., ΔH‡ = 1 ±3 kJ.mol−1, ΔS‡ = −178 ±10 J.mol−1.K−1, ΔG‡298K = 54 ±2 kJ.mol−1, and knuc,298K = 1880 ±80 dm3.mol−1.s−1. The temperature dependence of optical rotation was also shown to be independent of the anion present. 相似文献
20.
Folding proteins with a simple energy function and extensive conformational searching. 总被引:7,自引:6,他引:1
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We describe a computer algorithm for predicting the three-dimensional structures of proteins using only their amino acid sequences. The method differs from others in two ways: (1) it uses very few energy parameters, representing hydrophobic and polar interactions, and (2) it uses a new "constraint-based exhaustive" searching method, which appears to be among the fastest and most complete search methods yet available for realistic protein models. It finds a relatively small number of low-energy conformations, among which are native-like conformations, for crambin (1CRN), avian pancreatic polypeptide (1PPT), melittin (2MLT), and apamin. Thus, the lowest-energy states of very simple energy functions may predict the native structures of globular proteins. 相似文献