Protein transduction domains of HIV-1 and SIV TAT interact with charged lipid vesicles. Binding mechanism and thermodynamic analysis

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.     

The adsorption of Pseudomonas aeruginosa exotoxin A to phospholipid monolayers is controlled by pH and surface potential.

The interaction of Pseudomonas aeruginosa exotoxin A (ETA) with lipid monolayers was studied by measuring the variation in surface pressure. ETA adsorbs to the monolayer, occupying an average area of approximately 4.6 nm2 per molecule, up to a maximum density of one molecule per 28 nm2 of lipid film, which corresponds roughly to the cross-sectional area of the toxin. This suggests that ETA molecules adsorb until they contact each other, but insert only a small portion into the lipid film. The kinetic process could be described by a Langmuir adsorption isotherm. The apparent association and dissociation rate constants were determined, as were their dependence upon toxin concentration, membrane composition, pH, and ionic strength. Two parameters were found to be paramount for this interaction: pH and surface potential of the lipid. It appears that ETA binding occurs only in a conformational state induced by low pH and is promoted by an electrostatic interaction between a positively charged region of the protein and the negative charge of acidic phospholipids. On the basis of a simple model, the salient features of ETA involved in its adsorption were derived: 1) the existence of a conformational state induced by the protonation of a group with pK 4.5 +/- 0.2; 2) a positive charge of 1.9 +/- 0.3 e.u. able to interact with the surface potential of the membrane; 3) the fraction of potential experienced by the protein in the activated state that precedes binding, approximately 80%; 4) the intrinsic adsorption and desorption rate constants, k(a)0 = (4.8 +/- 0.3) x 10(3) M(-1) s(-1) and k(d)0 = (4.4 +/- 0.4) x 10(-4) s(-1). These rate constants are independent of pH and lipid and buffer composition, and provide a dissociation constant Kd approximately 90 nM.     

Investigations of spectrin-lipid interactions using fluoresceinphosphatidylethanolamine as a membrane probe

The binding of human erythrocyte spectrin to large unilamellar vesicles (LUVET) formed by the extrusion technique has been studied using fluoresceinphosphatidylethanolamine (FPE) as a reporter of electrostatic membrane potential. Spectrin aliquots were added to a suspension of FPE-labelled LUVETs to elucidate both the type of charge involved and the dissociation constants for spectrin binding to various lipids. All binding experiments showed serial increases in FPE fluorescence intensity upon serial additions of spectrin, indicative of increasing positive charge at the membrane surface. This proves for the first time that although exhibiting an overall net negative charge, spectrin binds to lipid surfaces by presenting positive charges to the lipid surface. Binding curves were obtained from the change in fluorescence intensity upon each spectrin addition and analysed to determine dissociation constants. A K(d) of 0.14+/-0.12 microM was found for spectrin binding to FPE-labelled phosphatidylcholine/phosphatidylserine (PC/PS) LUVETs at 22 degrees C in high salt conditions. A similar K(d) of 0.17+/-0.11 microM was obtained for spectrin binding to neutral LUVETs composed of PC. However, binding was found to be much weaker for PC/PS LUVETs under low salt conditions with a K(d) of 1.22+/-0.48 microM.     

Binding of bovine factor Va to phosphatidylcholine membranes.

The interaction of bovine factor Va with phosphatidylcholine membranes was examined using four different fluorescence techniques: 1) changes in the fluorescence anisotropy of the fluorescent membrane probe 1,6-diphenyl-1,3,5-hexatriene (DPH) to monitor the interaction of factor Va with 1,2-dimyristoyl-3-sn-phosphatidylcholine (DMPC) small unilamellar vesicles (SUVs), 2) changes in the fluorescence anisotropy of N-(lissamine rhodamine B sulfonyl) diacyl phosphati-dylethanolamine (Rh-PE) incorporated into SUVs prepared from 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (POPC), 3) changes in the fluorescence anisotropy of fluorescein-labeled factor Va (labeled in the heavy chain) upon interaction with POPC SUVs, 4) fluorescence energy transfer from fluorescein-labeled factor Va to rhodamine-labeled POPC SUVs. In the first two sets of experiments, labeled lipid vesicles were titrated with unlabeled protein, whereas, in the latter two types of experiments, labeled factor Va was titrated with vesicles. For the weak binding observed here, it was impossible from any one binding experiment to obtain precise estimates of the three parameters involved in modeling the lipid-protein interaction, namely, the dissociation constant Kd, the stoichiometry of binding i, and the saturation value of the observable Rmax from any one experiment. However, a global analysis of the four data sets involving POPC SUVs yielded a stable estimate of the binding parameters (Kd of approximately 3.0 microM and a stoichiometry of approximately 200 lipids per bound factor Va). Binding to DMPC SUVs may be of slightly higher affinity. These observations support the contention that association of factor Va with a membrane involves a significant acidic-lipid-independent interaction along with the more commonly accepted acidic-lipid-dependent component of the total binding free energy.     

Electrostatic interactions, but not the YGNGV consensus motif, govern the binding of pediocin PA-1 and its fragments to phospholipid vesicles.

The purpose of this study was to characterize in detail the binding of pediocin PA-1 and its fragments to target membranes by using tryptophan fluorescence as a probe. Based on a three-dimensional model (Y. Chen, R. Shapira, M. Eisenstein, and T. J. Montville, Appl. Environ. Microbiol. 63:524-531, 1997), four synthetic N-terminal pediocin fragments were selected to study the mechanism of the initial step by which the bacteriocin associates with membranes. Binding of pediocin PA-1 to vesicles of phosphatidylglycerol, the major component of Listeria membranes, caused an increase in the intrinsic tryptophan fluorescence intensity with a blue shift of the emission maximum. The Stern-Volmer constants for acrylamide quenching of the fluorescence of pediocin PA-1 in buffer and in the lipid vesicles were 8.83 +/- 0.42 and 3.53 +/- 0.67 M-1, respectively, suggesting that the tryptophan residues inserted into the hydrophobic core of the lipid bilayer. The synthetic pediocin fragments bound strongly to the lipid vesicles when a patch of positively charged amino acid residues (K-11 and H-12) was present but bound weakly when this patch was mutated out. Quantitative comparison of changes in tryptophan fluorescence parameters, as well as the dissociation constants for pediocin PA-1 and its fragments, revealed that the relative affinity to the lipid vesicles paralleled the net positive charge in the peptide. The relative affinity for the fragment containing the YGNGV consensus motif was 10-fold lower than that for the fragment containing the positive patch. Furthermore, changing the pH from 6.0 to 8.0 decreased binding of the fragments containing the positive patch, probably due to deprotonation of His residues. These results demonstrate that electrostatic interactions, but not the YGNGV motif, govern pediocin binding to the target membrane.     

Benzodiazepine binding studies on living cells: application of small ligands for fluorescence correlation spectroscopy

We demonstrate the applicability of fluorescence correlation spectroscopy (FCS) for receptor binding studies using low molecular weight ligands on the membranes of living nerve cells. The binding of the benzodiazepine Ro 7-1986/602 (N-des-diethyl-fluorazepam), labeled with the fluorophore Alexa 532, to the benzodiazepine receptor was analyzed quantitatively at the membrane of single rat hippocampal neurons. The values obtained for the dissociation constant Kd = (9.9 +/- 1.9) nm and the rate constant for ligand-receptor dissociation kdisS = (1.28 +/- 0.08) x 10(-3) s(-1) show that there is a specific and high affinity interaction between the dye-labeled ligand (Ro-Alexa) and the receptor site. The binding was saturated at approx. 100 nM and displacement of 10 nM Ro-Alexa, with a 1,000-fold excess of midazolam, showed a non-specific binding of 7-10%. Additionally, two populations of the benzodiazepine receptor that differed in their lateral mobility were detected in the membrane of rat neurons. The diffusion coefficients for these two populations [D(bound1) = (1.32 +/- 0.26) microm2/s; D(bound2) = (2.63 +/- 0.63) x 10(-2) microm2/s] are related to binding sites, which shows a mono-exponential decay in a time-dependent dissociation of the ligand-receptor complex.     

Mechanistic insights into the translocation of full length HIV-1 Tat across lipid membranes

The mechanism of how full length Tat (aa 1-86) crosses artificial lipid membranes was elucidated by means of fluorescence spectroscopy and fluorescence microscopy. It was shown that full length Tat (aa 1-86) neither forms pores in large unilamellar vesicles (LUVs) nor in giant unilamellar vesicles (GUVs) composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). In contrast, an N-terminally truncated Tat protein (aa 35-86) that lacks the structurally defined proline- and cysteine-rich region as well as the highly conserved tryptophan residue at position 11 generates pores in artificial POPC-membranes, through which a water-soluble dye up to a size of 10kDa can pass. By means of fluorescence microscopy, the transfer of fluorescently labeled full length Tat across POPC-bilayers was unambiguously visualized with a concomitant accumulation of the protein in the membrane interface. However, if the dye was attached to the protein, also pore formation was induced. The size of the pores was, however smaller than the protein size, i.e. the labeled protein with a mass of 11.6kDa passed the membrane, while a fluorescent dye with a mass of 10kDa was excluded from the vesicles' interior. The results demonstrate that pore formation is not the prime mechanism by which full length Tat crosses a membrane.     

Interaction of lipoprotein lipase and apolipoprotein C-II with sonicated vesicles of 1,2-ditetradecylphosphatidylcholine: comparison of binding constants

The interaction of lipoprotein lipase (LpL) and its activator protein, apolipoprotein C-II (apoC-II), with a nonhydrolyzable phosphatidylcholine, 1,2-ditetradecyl-rac-glycero-3-phosphocholine (C14-ether-PC), was studied by fluorescence spectroscopy. A complex of 320 molecules of C14-ether-PC per LpL was isolated by density gradient ultracentrifugation in KBr. The intrinsic tryptophan fluorescence emission spectrum of LpL was shifted from 336 nm in the absence of lipid to 330 nm in the LpL-lipid complex; the shift was associated with a 40% increase in fluorescence intensity. Addition of C14-ether-PC vesicles to apoC-II caused a 2.5-fold increase in intrinsic tryptophan fluorescence and a shift in emission maximum from 340 to 317 nm. LpL and apoC-II/C14-ether-PC stoichiometries and binding constants were determined by measuring the increase in the intrinsic tryptophan fluorescence as a function of lipid and protein concentrations; for LpL the rate and magnitude of the fluorescence increases were relatively independent of temperature in the range 4-37 degrees C. A stoichiometry of 270 PC per LpL for the LpL-lipid complex compares favorably with the value obtained in the isolated complex. The dissociation constant (Kd) of the complex is 4.3 X 10(-8) M. For apoC-II, the stoichiometry of the complex is 18 PC per apoprotein, and the Kd is 3.0 X 10(-6) M. These data suggest that LpL binds more strongly than apoC-II to phosphatidylcholine interfaces.     

Interaction of aldolase A with lecithin liposomes

The aldolase A binding to the lecithin liposomes (Kd = 2.4 +/- 0.1 X 10(-3) M) has been shown by the fluorescence and tryptophan phosphorescence at the room temperature. The interaction is accompanied by an increase in the phospholipid bilayer microviscosity, and some conformational changes in the hydrophobic part of the enzyme, pronouncing themselves in Trp-147 environment rigidity, decrease. The observation of membrane viscosity vs. incubation time revealed practically instant enzyme-membrane interaction and no gradual incorporation. The accessibility of the NAD-binding domain of aldolase for NADH in the liposome presence remains unaltered.     

Investigations of the effects of ethanol on warfarin binding to human serum albumin

Ethanol effects on warfarin binding to human serum albumin (HSA) have been studied by equilibrium dialysis and fluorescence methods at pH 7.4 in phosphate-buffered saline at 37 degrees C. In the presence of various amounts of ethanol fluorescence intensity of bound warfarin decreased significantly but this intensity reduction was not solely from displacement of bound warfarin from HSA. By comparing fluorescence and equilibrium dialysis data we concluded that fluorescence intensity reduction of warfarin was mainly the result of changes in the surrounding environment of the warfarin binding site by ethanol interaction with HSA and that displacement of bound warfarin was not significant compared to the fluorescence intensity changes. The dissociation constant of warfarin binding to HSA decreased with an increasing amount of ethanol. From the changes in fluorescence intensity upon warfarin binding to HSA with the presence of ethanol ranging from 0 to 5.0% the following dissociation constants (Kd) were determined: 0% ethanol 5.39 +/- 0.2 microM, 0.1% ethanol 5.86 +/- 0.1 microM, 0.3% ethanol 5.83 +/- 0.2 microM, 0.5% ethanol 6.76 +/- 0.1 microM, 1% ethanol 7.01 +/- 0.1 microM, 3% ethanol 9.9 +/- 0.7 microM, 5% ethanol 13.01 +/- 0.1 microM. From the equilibrium dialysis with the same ranges of ethanol presence the following Kd values were obtained: 0% ethanol 6. 62 +/- 1.6 microM, 0.1% ethanol 6.81 +/- 1.1 microM, 0.3% ethanol 8. 26 +/- 2.5 microM, 0.5% ethanol 8.86 +/- 1.9 microM, 1% ethanol 11. 01 +/- 4.2 microM, 3% ethanol 20.75 +/- 2.4 microM, 5% ethanol 21.67 +/- 2.2 microM. The results suggest that warfarin bound to HSA was displaced by ethanol. These data indicate that ethanol influence on warfarin binding to HSA may alter the pharmacokinetics of warfarin.     

Cucurbitacin delta 23-reductase from the fruit of Cucurbita maxima var. Green Hubbard. Physicochemical and fluorescence properties and enzyme-ligand interactions.

Cucurbitacin delta 23-reductase from Cucurbita maxima var. Green Hubbard fruit displays an apparent Mr of 32,000, a Stokes radius of 263 nm and a diffusion coefficient of 8.93 X 10(-7) cm2 X s-1. The enzyme appears to possess a homogeneous dimeric quaternary structure with a subunit Mr of 15,000. Two tryptophan and fourteen tyrosine residues per dimer were found. Emission spectral properties of the enzyme and fluorescence quenching by iodide indicate the tryptophan residues to be buried within the protein molecule. In the pH range 5-7, where no conformational changes were detected, protonation of a sterically related ionizable group with a pK of approx. 6.0 markedly influenced the fluorescence of the tryptophan residues. Protein fluorescence quenching was employed to determine the dissociation constants for binding of NADPH (Kd 17 microM), NADP+ (Kd 30 microM) and elaterinide (Kd 227 microM). Fluorescence energy transfer between the tryptophan residues and enzyme-bound NADPH was observed.     

Soluble Glycosaminoglycans Inhibit the Interaction of TAT−PTD with Lipid Vesicles

Several models have been proposed for translocation of cell-penetrating peptides across membranes, but no general consensus on the mechanism of this process has emerged. It was hypothesized that heparan sulfate on the cell surface may play a role. We used fluorescence spectroscopy to study the effect of three soluble glycosaminoglycans—heparan sulfate, low-molecular-weight heparin, and dermatan sulfate—on the interaction of the fluorescently labeled peptide TAT−PTD with negatively charged small unilamellar vesicles. We found that the presence of glycosaminoglycans results in an order-of-magnitude increase in the apparent dissociation constant K _d of the electrostatic component of the peptide/membrane interaction (from 0.13 to 2.6 mM). Thus, rather than aiding in the peptide’s penetration, soluble glycosaminoglycans competitively decrease TAT−PTD’s binding to the membrane, presumably by neutralizing its charge, and thereby attenuating electrostatic forces involved in the interaction. Our results, however, do not exclude a possible role of membrane-anchored glycosaminoglycans in the endocytotic transduction of CPPs across the cell membrane.     

Fluorescence studies of the interactions of serum albumin and rat alpha1-fetoprotein with aflatoxin B1. Specificity and binding parameters.

The interaction of bovine serum albumin and rat alpha1-fetoprotein with aflatoxin B1 has been followed by the fluorescence quenching of the protein in presence of the ligand. The binding parameters (n, number of sites and Kd, dissociation constant) have been determined for the bovine serum albumin-alflatoxin B1 system: n = 3.5 and Kd = 3.1 +/- 0.5 . 10(-5) M and for the alpha-fetoprotein-aflatoxin system: n = 4 and Kd = 3.7 +/-0.5 . 10(-5) M. The competition of anilino-naphthalene-sulfonate and aflatoxin B1 for the same hydrophobic sites on bovine serum albumin has been demonstrated. The fluorescence quenching of various proteins (lysozymes, egg-albumin, gamma-globulin) by aflatoxin B1 have shown that there is not a strict specificity of aflatoxin towards proteins.     

Revised Role of Glycosaminoglycans in TAT Protein Transduction Domain-mediated Cellular Transduction

Cellular uptake of the human immunodeficiency virus TAT protein transduction domain (PTD), or cell-penetrating peptide, has previously been surmised to occur in a manner dependent on the presence of heparan sulfate proteoglycans that are expressed ubiquitously on the cell surface. These acidic polysaccharides form a large pool of negative charge on the cell surface that TAT PTD binds avidly. Additionally, sulfated glycans have been proposed to aid in the interaction of TAT PTD and other arginine-rich PTDs with the cell membrane, perhaps aiding their translocation across the membrane. Surprisingly, however, TAT PTD-mediated induction of macropinocytosis and cellular transduction occurs in the absence of heparan sulfate and sialic acid. Using labeled TAT PTD peptides and fusion proteins, in addition to TAT PTD-Cre recombination-based phenotypic assays, we show that transduction occurs efficiently in mutant Chinese hamster ovary cell lines deficient in glycosaminoglycans and sialic acids. Similar results were obtained in cells where glycans were enzymatically removed. In contrast, enzymatic removal of proteins from the cell surface completely ablated TAT PTD-mediated transduction. Our findings support the hypothesis that acidic glycans form a pool of charge that TAT PTD binds on the cell surface, but this binding is independent of the PTD-mediated transduction mechanism and the induction of macropinocytotic uptake by TAT PTD.     

Tissue factor and its extracellular soluble domain: the relationship between intermolecular association with factor VIIa and enzymatic activity of the complex.

We find that the isolated, extracellular domain of tissue factor (TF1-218; sTF) exhibits only 4% of the activity of wild-type transmembrane TF (TF1-263) in an assay that measures the conversion of factor X to Xa by the TF:VIIa complex. Further, the activity of sTF is manifest only when vesicles consisting of phosphatidylserine and phosphatidylcholine (30/70 w/w) are present. To determine whether the decreased activity results from weakened affinity of sTF for VIIa, we studied their interaction using equilibrium ultracentrifugation, fluorescence anisotropy, and an activity titration. Ultracentrifugation of the sTF:VIIa complex established a stoichiometry of 1:1 and an upper limit of 1 nM for the equilibrium dissociation constant (Kd). This value is in agreement with titrations of dansyl-D-Phe-L-Phe-Arg chloromethyl ketone active site labeled VIIa (DF-VIIa) with sTF using dansyl fluorescence anisotropy as the observable. Pressure dissociation experiments were used to obtain quantitative values for the binding interaction. These experiments indicate that the Kd for the interaction of sTF with DF-VIIa is 0.59 nM (25 degrees C). This value may be compared to a Kd of 7.3 pM obtained by the same method for the interaction of DF-VIIa with TF1-263 reconstituted into phosphatidylcholine vesicles. The molar volume change of association was found to be 63 and 117 mL mol-1 for the interaction of DF-VIIa with sTF and TF1-263, respectively. These binding data show that the sTF:VIIa complex is quantitatively and qualitatively different from the complex formed by TF1-263 and VIIa.     

Adenine nucleotides affect the binding of 3-phosphoglycerate to pig muscle 3-phosphoglycerate kinase

Pig muscle 3-phosphoglycerate kinase was complexed with 1-anilino-8-naphthalenesulfonate (ANS) in order to monitor the binding of substrates to the enzyme. The enzyme-dye interaction did not influence the enzymic activity under the experimental conditions used. By measuring the substrate-dependent change in the fluorescence emission of ANS molecules tightly bound to the enzyme (Kd less than or equal to 0.05 mM), fluorimetric titrations were carried out in 0.1 M Tris/HCl buffer pH 7.5, containing 5 mM mercaptoethanol, at 20 degrees C. The dissociation constants obtained for the separate bindings of 3-phosphoglycerate, MgATP, 1,3-bisphosphoglycerate and MgADP were 0.03 +/- 0.01 mM, 0.15 +/- 0.10 mM, 0.00005 +/- 0.00001 mM and 0.15 +/- 0.10 mM respectively. binding of 3-phosphoglycerate is weakened when MgATP is also bound to the enzyme: the dissociation constant of 3-phosphoglycerate in this ternary complex (0.25 +/- 0.08 mM) is comparable to its Km value (0.38 +/- 0.10 mM). The same weakening can be observed in the non-productive ternary complexes where MgATP is replaced by MgADP (Kd = 0.20 +/- 0.10 mM) or AMP (Kd = 0.12 +/- 0.05 mM), whereas adenosine has no such effect. This indicates the importance of the negatively charged phosphate(s) of nucleotides in influencing the binding of 3-phosphoglycerate. In contrast to 3-phosphoglycerate, the binding of the substrate analogue, glycerol 3-phosphate is practically not affected by the presence of MgATP: the dissociation constant to the free enzyme (0.40 +/- 0.10 mM) is comparable to its inhibitory constant (0.70 +/- 0.20 mM). This finding and the similarity of the dissociation constant of glycerol 3-phosphate binding (0.40 +/- 0.10 mM) and the Km value of 3-phosphoglycerate (0.38 +/- 0.10 mM) suggest that, during the enzymic reaction, binding of 3-phosphoglycerate occurs probably without involvement of the carboxyl group.     

Interaction of fluorescence labeled single-stranded DNA with hexameric DNA-helicase RepA: a photon and fluorescence correlation spectroscopy study

Fluorescence correlation spectroscopy (FCS) was used to characterize the interaction of fluorescence labeled single-stranded DNA (ssDNA) with hexameric RepA DNA-helicase (hRepA) encoded by plasmid RSF1010. The apparent dissociation constants, Kd(app), for the equilibrium binding of 12mer, 30mer, and 45mer ssDNA 5'-labeled with BFL to hRepA dimer in the presence of 0.5 mM ATPgammaS at pH 5.8 and 25 degrees C were determined to be 0.58 +/- 0.12, 0.52 +/- 0.07, and 1.66 +/- 0.32 microM, respectively. Binding curves are compatible with one binding site for ssDNA present on hRepA dimer, with no indication of cooperativity. At pH 7.6 in the presence of ATPgammaS and at pH 5.8 in the absence of ATPgammaS, complex formation between ssDNA and hRepA was too weak for measuring complete binding curves by FCS. Under these conditions, the dissociation constant, Kd(app), is in the range between 10 and 250 microM. The kinetics of complex formation at pH 5.8 are faster than the time resolution (approximately 10-20 s) of FCS experiments under pseudo-first-order conditions, with respect to BFL-ssDNA. Photon correlation spectroscopy (PCS) experiments yielded, within the experimental error range, the same values for the apparent hydrodynamic radii, R(h), of hRepA dimer and its complex with ssDNA as determined by FCS (R(h) = 6.6 +/- 1 nm). hRepA starts to aggregate under acidic conditions (相似文献   

Intrinsic fluorescence of binding-site fragments of the nicotinic acetylcholine receptor: perturbations produced upon binding alpha-bungarotoxin

Synthetic peptides corresponding to sequences contained within residues 173-204 of the alpha-subunit in the nicotinic acetylcholine receptor (nAChR) of Torpedo californica bind the competitive antagonist alpha-bungarotoxin (BGTX) with relative high affinity. Since the synthetic peptide fragments of the receptor and BGTX each contain a small number of aromatic residues, intrinsic fluorescence studies were used to investigate their interaction. We examined a number of receptor-derived peptide fragments of increasing length (4-32 amino acids). Changes in the lambda max and quantum yield with increasing polypeptide chain length suggest an increase in the hydrophobicity of the tryptophan environment. When selective excitation and subtraction were used to reveal the tyrosine fluorescence of the peptides, a significant red shift in emission was observed and was found to be due to an excited-state tyrosinate. The binding of BGTX to the receptor-derived peptide fragments resulted in a large increase in fluorescence. In addition, at equilibrium, the lambda max of tryptophan fluorescence was shifted to shorter wavelengths. The. fluorescence enhancement, which was saturable with either peptide or BGTX, was used to determine the dissociation constants for the complexes. At pH 7.4, the apparent Kd for a dodecameric peptide (alpha 185-196), consisting of residues 185-196 in the alpha-subunit of the nAChR from Torpedo californica, was 1.4 microM. The Kd for an 18-mer (alpha 181-198), consisting of residues 181-198 of the Torpedo alpha-subunit, was 0.3 microM. No binding or enhanced fluorescence was observed with an irrelevant synthetic peptide of comparable composition.(ABSTRACT TRUNCATED AT 250 WORDS)     

蛋白转导域透膜作用的研究进展

HIV-TAT蛋白转导域（Protein transduction domain,,PTD）是新近发现的一种在蛋白转导过程中能高效穿过生物膜的结构域,源自人类免疫缺陷病毒Tat蛋白的一段碱性氨基酸多肽,能与多肽、蛋白质及DNA等分子连接并跨膜导入绝大部分的组织细胞或透过血脑屏障,转导效率高且对细胞无损伤。TAT-PTD与细胞膜之间的电荷作用,吸附于膜表面,依赖脂筏介导的巨胞饮作用进入细胞。TAT融合蛋白系统是一种极有价值的运载工具,在基础医学研究和临床治疗方面有着广泛的应用前景。     

Binding of a mitochondrial presequence to natural and artificial membranes: role of surface potential.

The binding of a synthetic mitochondrial presequence to large, negatively charged, unilamellar vesicles and to unenergized yeast mitochondria has been measured. The presequence, which corresponds to the amino-terminal 25 residues of the yeast cytochrome oxidase subunit IV precursor, was labeled with a fluorescent probe and used to examine the importance of the surface potentials of membranes on the interactions with the presequence. Binding of the fluorescent presequence to the membranes was determined by measuring a decrease in the fluorescence emission of the bound presequence. Binding both to the vesicles and to the mitochondria could be described as a simple partitioning of the presequence between the aqueous and lipid phases. The partitioning was found to depend on the ionic strength of the medium, and the Gouy-Chapman theory could be used to describe the partitioning at various ionic strengths. Application of the theory allowed the determination of an apparent charge on the presequence (+2.31 +/- 0.25), salt-independent apparent partition coefficients for vesicles (99 +/- 84 M-1) and for unenergized mitochondria (14.5 +/- 3.6 L g-1), and an estimated charge density for the mitochondrial outer membrane (-0.0124 +/- 0.0016 C m-2). This study shows that electrostatic effects are significant for the binding of a mitochondrial presequence both to lipid vesicles and to mitochondria, the natural target membrane of the presequence. The accumulation of positively charged presequences at the negative mitochondrial surface and the subsequent partitioning of the presequences directly into the mitochondrial outer membrane probably represent early steps in the translocation of precursor proteins into mitochondria.