Interaction between DMPC liposomes and HM-PNIPAM polymer

The interactions of hydrophobically-modified poly-(N-isopropylacrylamides) (HM-PNIPAM) and dimyristoylphosphatidylcholine (DMPC) vesicles were investigated by the effect of the polymer on the binding of a fluorescent dye, oxonol VI, to DMPC vesicles, and on its diffusion across the membrane. On mixing with the vesicles, the dye exhibits an increase in fluorescence, which occurs in a two-stage process. The process was monitored by stopped-flow fluorescence spectrophotometry. According to the dependence of the reciprocal relaxation time on vesicle concentration, the rapid stage seems to be due to the second-order binding of the dye to the lipid membrane, a process that is almost diffusion-controlled, whereas the slow process is attributed to movement of the dye within the membrane phase. The polymer did not significantly affect the rate constant of the binding step, but it slowed down slightly the dissociation process of the dye from the membrane. However, the polymer affected the second stage, causing an increase in the reciprocal of its relaxation time, which suggests that the polymer makes the vesicle membrane more fluid.     

Insertion kinetics of a denatured alpha helical membrane protein into phospholipid bilayer vesicles

Membrane protein folding has suffered from a lack of detailed kinetic studies, particularly with regard to the insertion of denatured protein into lipid bilayers. We present a detailed in vitro kinetic study of the association of a denatured, transmembrane alpha helical protein with lipid vesicles. The mechanism of folding of Escherichia coli diacylglycerol kinase from a partially denatured state in urea has been investigated. The protein associates with lipid vesicles to give a protein, vesicle complex with an apparent association constant of 2 x 10(6) M(-1) s(-1). This association rate approaches the diffusion limit of the protein, vesicle reaction. The association of the protein with lipid vesicles is followed by a slower process occurring at observed rate of 0.031 s(-1), involving insertion into the bilayer and generation of a functional oligomer of diacylglycerol kinase. Protein aggregation competes with vesicle insertion. The urea-denatured protein monomers begin to aggregate as soon as the urea is diluted. This aggregation is faster than the association of the protein with vesicles so that most protein aggregates before it inserts into a vesicle. Increasing the vesicle concentration favours insertion of protein monomers, but at high vesicle concentrations monomers are primarily in separate vesicles and do not associate to form functional oligomers. Irreversible aggregation limits the yield of functional protein, while the data also suggest that lipid vesicles can reverse another aggregation reaction, leading to the recovery of correctly folded protein.     

A stepwise mechanism for the permeation of phloretin through a lipid bilayer

The thermodynamics of interactions between phloretin and a phosphatidylcholine (PC) vesicle membrane are characterized using equilibrium spectrophotometric titration, stopped-flow, and temperature- jump techniques. Binding of phloretin to a PC vesicle membrane is diffusion limited, with an association rate constant greater than 10(8) M-1s-1, and an interfacial activation free energy of less than 2 kcal/mol. Equilibrium binding of phloretin to a vesicle membrane is characterized by a single class of high-affinity (8 micro M), noninteracting sites. Binding is enthalpy driven (delta H = -4.9 kcal/mol) at 23 degrees C. Analysis of amplitudes of kinetic processes shows that 66 +/- 3% of total phloretin binding sites are exposed at the external vesicle surface. The rate of phloretin movement between binding sites located near the external and internal interfaces is proportional to the concentration of un-ionized phloretin, with a rate constant of 5.7 X 10(4) M-1s-1 at 23 degrees C. The rate of this process is limited by a large enthalpic (9 kcal/mol) and entropic (-31 entropy units) barrier. An analysis of the concentration dependence of the rate of transmembrane movement suggests the presence of multiple intramembrane potential barriers. Permeation of phloretin through a lipid bilayer is modeled quantitatively in terms of discrete steps: binding to a membrane surface, translocation across a series of intramembrane barriers, and dissociation from the opposite membrane surface. The permeability coefficient for phloretin is calculated as 1.9 X 10(-3) cm/s on the basis of the model presented. Structure- function relationships are examined for a number of phloretin analogues.     

Binding of phospholipids to beta-Lactoglobulin and their transfer to lipid bilayers

The bovine milk lipocalin, beta-Lactoglobulin (beta-LG), has been associated with the binding and transport of small hydrophobic and amphiphilic compounds, whereby it is proposed to increase their bioavailability. We have studied the binding of the fluorescent phospholipid-derivative, NBD-didecanoylphosphatidylethanolamine (NBD-diC10PE) to beta-LG by following the increase in amphiphile fluorescence upon binding to the protein using established methods. The equilibrium association constant, KB, was (1.2+/-0.2)x10(6) M(-1) at 25 degrees C, pH 7.4 and I=0.15 M. Dependence of KB on pH and on the monomer-dimer equilibrium of beta-LG gave insight on the nature of the binding site which is proposed to be the hydrophobic calyx formed by the beta-barrel in the protein. The monomer-dimer equilibrium of beta-LG was re-assessed using fluorescence anisotropy of Tryptophan. The equilibrium constant for dimerization, KD, was (7.0+/-1.5)x10(5) M(-1) at 25 degrees C, pH 7.4, and 0.15 M ionic strength. The exchange of NBD-diC10PE between beta-LG and POPC lipid bilayers was followed by the change in NBD fluorescence. beta-LG was shown to be a catalyst of phospholipid exchange between lipid bilayers, the mechanism possibly involving adsorption of the protein at the bilayer surface.     

Binding of oligoarginine to membrane lipids and heparan sulfate: structural and thermodynamic characterization of a cell-penetrating peptide

Cell-penetrating peptides (CPPs) comprise a group of arginine-rich oligopeptides that are able to deliver exogenous cargo into cells. A first step in the internalization of CPPs is their binding to the cell surface, a reaction likely to involve membrane phospholipids and/or heparan sulfate proteoglycans (HSPGs). The present work characterizes the interaction of R(9), one of the most efficient CPPs, with either heparan sulfate (HS) or lipid vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG). Isothermal titration calorimetry shows that R(9) binds to HS with high affinity. Assuming that HS has n independent and equivalent binding sites for R(9), we find an association constant of 3.1 x 10(6) M(-1) at 28 degrees C. At this temperature, the reaction enthalpy is DeltaH(degrees)pep = - 5.5 kcal/mol and approximately 7 R(9) molecules bind per HS chain, which is equivalent to approximately 0.95 cationic/anionic charge ratio. Delta decreases in magnitude upon an increase in temperature, and the reaction becomes entropy-driven at higher temperatures (>or=37 degrees C). The positive heat-capacity change entailed by this reaction (DeltaC(degrees)P = +167 cal mol(-1) K(-1)) indicates the loss of polar residues on R(9)-HS binding, suggesting that hydrophobic forces play no major role on binding. Calorimetric analysis of the interaction of R(9) with POPC/POPG (75:25) vesicles reveals an association constant of 8.2 x 10(4) M(-1) at 28 degrees C. Using a surface partition equilibrium model to correct for electrostatic effects, we find an intrinsic partition constant of approximately 900 M(-1), a value that is also confirmed by electrophoretic mobility measurements. This corresponds to an electrostatic contribution of approximately 33% to the total free energy of binding. Deuterium nuclear magnetic resonance (NMR) shows no change in the headgroup conformation of POPC and POPG, suggesting that binding takes place at some distance from the plane of the polar groups. (31)P NMR indicates that the lipid bilayer remains intact upon R(9) binding. The fact that R(9) binds with greater affinity to HS than to anionic lipid vesicles makes the former molecule a more likely target in binding this CPP to the cell surface.     

Kinetics of phloretin binding to phosphatidylcholine vesicle membranes

The submillisecond kinetics for phloretin binding to unilamellar phosphatidylcholine (PC) vesicles was investigated using the temperature-jump technique. Spectrophotometric studies of the equilibrium binding performed at 328 nm demonstrated that phloretin binds to a single set of independent, equivalent sites on the vesicle with a dissociation constant of 8.0 microM and a lipid/site ratio of 4.0. The temperature of the phloretin-vesicle solution was jumped by 4 degrees C within 4 microseconds producing a monoexponential, concentration-dependent relaxation process with time constants in the 30--200-microseconds time range. An analysis of the concentration dependence of relaxation time constants at pH 7.30 and 24 degrees C yielded a binding rate constant of 2.7 X 10(8) M-1 s-1 and an unbinding constant of 2,900 s-1; approximately 66 percent of total binding sites are exposed at the outer vesicle surface. The value of the binding rate constant and three additional observations suggest that the binding kinetics are diffusion limited. The phloretin analogue, naringenin, which has a diffusion coefficient similar to phloretin yet a dissociation constant equal to 24 microM, bound to PC vesicle with the same rate constant as phloretin did. In addition, the phloretin-PC system was studied in buffers made one to six times more viscous than water by addition of sucrose or glycerol to the differ. The equilibrium affinity for phloretin binding to PC vesicles is independent of viscosity, yet the binding rate constant decreases with the expected dependence (kappa binding alpha 1/viscosity) for diffusion-limited processes. Thus, the binding rate constant is not altered by differences in binding affinity, yet depends upon the diffusion coefficient in buffer. Finally, studies of the pH dependence of the binding rate constant showed a dependence (kappa binding alpha [1 + 10pH-pK]) consistent with the diffusion-limited binding of a weak acid.     

Spontaneous phosphatidylcholine transfer by collision between vesicles at high lipid concentration

The transfer kinetics of [3H]-1-palmitoyl-2-oleoylphosphatidylcholine ([3H]POPC) and 1-palmitoyl-2-(pyrenyldecanoyl)phosphatidylcholine (PyrPC) from POPC small unilamellar vesicles were examined at 37 degrees C with lipid concentrations ranging from 0.1 to 40 mM. The rate of [3H]POPC transfer was determined by analyzing the movement of this lipid from charged donor to neutral acceptor vesicles. The rate of decay of the ratio of the intensity of pyrene excimer fluorescence to that from the pyrene monomer (E/M) upon addition of an unlabeled vesicle population to a population containing PyrPC was used to evaluate PyrPC transfer. For both lipids, the kinetic data are best described by a model which assumes that transfer occurs by vesicle collisions as well as by desorption from the bilayer. For [3H]POPC, the off-rate constant is 0.014 h-1 while the collisional rate constant is 0.0016 mM-1 h-1. PyrPC has an off-rate constant of 0.023 h-1 and a collisional constant of 0.0015 mM-1 h-1. These numbers were calculated by assuming the rate of interbilayer transfer to be negligible relative to that of intervesicular transfer. The large transfer fluxes in the high vesicle concentration range where the collisional process dominates suggest that spontaneous transfer may be of importance in membrane biogenesis.     

Membrane binding kinetics of protein kinase C betaII mediated by the C2 domain.

Conventional isoforms of protein kinase C (PKC) are activated when their two membrane-targeting modules, the C1 and C2 domains, bind the second messengers diacylglycerol (DG) and Ca2+, respectively. This study investigates the mechanism of Ca2+-induced binding of PKC betaII to anionic membranes mediated by the C2 domain. Stopped-flow fluorescence spectroscopy reveals that Ca2+-induced binding of the isolated C2 domain to anionic vesicles proceeds via at least two steps: (1) rapid binding of two or more Ca2+ ions to the free domain with relatively low affinity and (2) diffusion-controlled association of the Ca2+-occupied domain with vesicles. Ca2+ increases the affinity of the C2 domain for anionic membranes by both decreasing the dissociation rate constant (k(off)) and increasing the association rate constant (k(on)) for membrane binding. For binding to vesicles containing 40 mol % anionic lipid in the presence of 200 microM Ca2+, k(off) and k(on) are 8.9 s(-1) and 1.2 x 10(10) M(-1) x s(-1), respectively. The k(off) value increases to 150 s(-1) when free Ca2+ levels are rapidly reduced, decreasing the average lifetime of the membrane-bound C2 domain (tau = k(off)(-1)) from 110 ms in the presence of Ca2+ to 6.7 ms when Ca2+ is rapidly removed. Experiments addressing the role of electrostatic interactions reveal that they stabilize either the initial C2 domain-membrane encounter complex or the high-affinity membrane-bound complex. Specifically, lowering the phosphatidylserine mole fraction or including MgCl2 in the binding reaction decreases the affinity of the C2 domain for anionic vesicles by both reducing k(on) and increasing k(off) measured in the presence of 200 microM Ca2+. These species do not affect the k(off) value when Ca2+ is rapidly removed. Studies with PKC betaII reveal that Ca2+-induced binding to membranes by the full-length protein proceeds minimally via two kinetically resolvable steps: (1) a rapid bimolecular association of the enzyme with vesicles near the diffusion-controlled limit and, most likely, (2) subsequent conformational changes of the membrane-bound enzyme. As is the case for the C2 domain, k(off) for full-length PKC betaII increases when Ca2+ is rapidly removed, reducing tau from 11 s in the presence of Ca2+ to 48 ms in its absence. Thus, both the C2 domain and the slow conformational change prolong the lifetime of the PKC betaII-membrane ternary complex in the presence of Ca2+, with rapid membrane release triggered by removal of Ca2+. These results provide a molecular basis for cofactor regulation of PKC whereby the C2 domain searches three-dimensional space at the diffusion-controlled limit to target PKC to relatively common anionic phospholipids, whereupon a two-dimensional search is initiated by the C1 domain for the more rare, membrane-partitioned DG.     

A stopped-flow kinetic study of the interaction of potential-sensitive oxonol dyes with lipid vesicles

The interaction of the dyes oxonol V and oxonol VI with unilamellar dioleoylphosphatidylcholine vesicles was investigated using a fluorescence stopped-flow technique. On mixing with the vesicles, both dyes exhibit an increase in their fluorescence, which occurs in two phases. According to the dependence of the reciprocal relaxation time on vesicle concentration, the rapid phase appears to be due to a second-order binding of the dye to the lipid membrane, which is very close to being diffusion-controlled. The slow phase is almost independent of vesicle concentration, and it is suggested that this may be due to a change in dye conformation or position within the membrane, possibly diffusion across the membrane to the internal monolayer. The response times of the dyes to a rapid jump in the membrane potential has also been investigated. Oxonol VI was found to respond to the potential change in less than 1 s, whereas oxonol required several minutes. This has been attributed to lower mobility of oxonol V within the lipid membrane.     

Self-association of the polyene antibiotic nystatin in dipalmitoylphosphatidylcholine vesicles: a time-resolved fluorescence study.

The interaction between Nystatin and small unilamellar vesicles of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, both in gel (T = 21 degrees C) and in liquid-crystalline (T = 45 degrees C) phases, was studied by steady-state and time-resolved fluorescence measurements by taking advantage of the intrinsic tetraene fluorophore present in this antibiotic. It was shown that Nystatin aggregates in aqueous solution with a critical concentration of 3 microM. The enhancement in the fluorescence intensity of the antibiotic was applied to study the membrane binding of Nystatin, and it was shown that the antibiotic had an almost fivefold higher partition coefficient for the vesicles in a gel (P = (1.4 +/- 0.1) x 10(3)) than in a liquid-crystalline phase (P = (2.9 +/- 0.1) x 10(2)). Moreover, a time-resolved fluorescence study was used to examine Nystatin aggregation in the membrane. The emission decay kinetics of Nystatin was described by three and two exponentials in the lipid membrane at 21 degrees C and 45 degrees C, respectively. Nystatin mean fluorescence lifetime is concentration-dependent in gel phase lipids, increasing steeply from 11 to 33 ns at an antibiotic concentration of 5-6 microM, but the fluorescence decay parameters of Nystatin were unvarying with the antibiotic concentration in fluid lipids. These results provide evidence for the formation of strongly fluorescent antibiotic aggregates in gel-phase membrane, an interpretation that is at variance with a previous study. However, no antibiotic self-association was detected in a liquid-crystalline lipid bilayer within the antibiotic concentration range studied (0-14 microM).     

The interaction of a synthetic mitochondrial signal peptide with lipid membranes is independent of transbilayer potential.

We have used fluorescence measurements and assays of vesicle disruption (contents leakage) to monitor the interaction between lipid vesicles and a synthetic peptide corresponding to the N-terminal 27 amino acids of rat mitochondrial pre-ornithine carbamyltransferase (pOCT). This peptide and two fluorescent derivatives bind reversibly to vesicles composed of neutral and anionic phospholipids with increasing affinity as the proportion of anionic lipids in the vesicles increases. The affinity of the peptide for lipid vesicles is unaffected by the presence of a transbilayer potential (inside negative) of at least -80 mV across the vesicle membranes. Our results support the proposal that the signal sequence of pOCT may promote an initial association of the precursor protein with mitochondrial membranes prior to binding to a specific receptor. However, we find no evidence that the pOCT signal sequence can subsequently undergo transfer into or across the lipid bilayer, even in the presence of a transmembrane potential of the magnitude previously found to support the import of precursor proteins into mitochondria.     

Enthalpy-driven apolipoprotein A-I and lipid bilayer interaction indicating protein penetration upon lipid binding

The interaction of lipid-free apolipoprotein A-I (apoA-I) with small unilamellar vesicles (SUVs) of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) with and without free cholesterol (FC) was studied by isothermal titration calorimetry and circular dichroism spectroscopy. Parameters reported are the affinity constant (K(a)), the number of protein molecules bound per vesicle (n), enthalpy change (DeltaH degrees), entropy change (DeltaS degrees ), and the heat capacity change (DeltaC(p) degrees). The binding process of apoA-I to SUVs of POPC plus 0-20% (mole) FC was exothermic between 15 and 37 degrees C studied, accompanied by a small negative entropy change, making enthalpy the main driving force of the interaction. The presence of cholesterol in the vesicles increased the binding affinity and the alpha-helix content of apoA-I but lowered the number of apoA-I bound per vesicle and the enthalpy and entropy changes per bound apoA-I. Binding affinity and stoichiometry were essentially invariant of temperature for binding to SUVs of POPC/FC at a molar ratio of 6/1 at (2.8-4) x 10(6) M(-1) and 2.4 apoA-I molecules bound per vesicle or 1.4 x 10(2) phospholipids per bound apoA-I. A plot of DeltaH degrees against temperature displayed a linear behavior, from which the DeltaC(p) degrees per mole of bound apoA-I was calculated to be -2.73 kcal/(mol x K). These results suggested that binding of apoA-I to POPC vesicles is characterized by nonclassical hydrophobic interactions, with alpha-helix formation as the main driving force for the binding to cholesterol-containing vesicles. In addition, comparison to literature data on peptides suggested a cooperativity of the helices in apoA-I in lipid interaction.     

Modulation of membrane fusion by membrane fluidity: temperature dependence of divalent cation induced fusion of phosphatidylserine vesicles

We have investigated the temperature dependence of the fusion of phospholipid vesicles composed of pure bovine brain phosphatidylserine (PS) induced by Ca2+ or Mg2+. Aggregation of the vesicles was monitored by 90 degrees light-scattering measurements, fusion by the terbium/dipicolinic acid assay for mixing of internal aqueous volumes, and release of vesicle contents by carboxyfluorescein fluorescence. Membrane fluidity was determined by diphenylhexatriene fluorescence polarization measurements. Small unilamellar vesicles (SUV, diameter 250 A) or large unilamellar vesicles (LUV, diameter 1000 A) were used, and the measurements were done in 0.1 M NaCl at pH 7.4. The following results were obtained: (1) At temperatures (0-5 degrees C) below the phase transition temperature (Tc) of the lipid, LUV (PS) show very little fusion in the presence of Ca2+, although vesicle aggregation is rapid and extensive. With increasing temperature, the initial rate of fusion increases dramatically. Leakage of contents at the higher temperatures remains limited initially, but subsequently complete release occurs as a result of collapse of the internal aqueous space of the fusion products. (2) SUV (PS) are still in the fluid state down to 0 degree C, due to the effect of bilayer curvature, and fuse rapidly in the entire temperature range from 0 to 35 degrees C in the presence of Ca2+. The initial rate of leakage is low relative to the rate of fusion. At higher temperatures (15 degrees C and above), subsequent collapse of the vesicles' internal space causes complete release.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR study of volatile anesthetic binding to nicotinic acetylcholine receptors

New lines of evidence suggest that volatile anesthetics interact specifically with proteins. Direct binding analysis, however, has been largely limited to soluble proteins. In this study, specific interaction was investigated between isoflurane, a clinically important volatile anesthetic, and membrane-bound nicotinic acetylcholine receptors (nAChRs) from Torpedo electroplax, using (19)F nuclear magnetic resonance spectroscopy and gas chromatography. The receptors were reconstituted into 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid vesicles. After correcting for nonspecific partitioning into the lipid, the equilibrium dissociation constant, K(d), of isoflurane binding to nAChR at 15 degrees C was found to be 0.36 +/- 0.03 mM. This value is within the clinically relevant concentration range of the agent. Based on the receptor concentrations in the vesicle suspension assayed by the bicinchoninic acid method and the fraction of bound isoflurane, X(b), determined by gas chromatography, an estimate of an average of 9-10 specifically bound isoflurane molecules can be made for each receptor, or two for each subunit. Upon binding, the transverse relaxation time constant (T(2)) of (19)F resonance of isoflurane is decreased by nearly three orders of magnitude, indicating a dramatic reduction in the mobility of specifically bound isoflurane. Kinetic analysis reveals that the off rate of binding, k(-1), is 1.7 x 10(4) s(-1). The on rate, k(+1), can thus be calculated to be approximately 4.8 x 10(7) M(-1) s(-1), suggesting a nearly diffusion-limited association. This is in contrast to anesthetic binding to a soluble protein, bovine serum albumin (BSA), where k(+1) and k(-1) are at least an order of magnitude slower. It is concluded that the presence of lipids may be critical for the correct evaluation of binding kinetics between volatile anesthetics and neuronal receptors.     

Binding of peptides with basic residues to membranes containing acidic phospholipids.

There are clusters of basic amino acids on many cytoplasmic proteins that bind transiently to membranes (e.g., protein kinase C) as well as on the cytoplasmic domain of many intrinsic membrane proteins (e.g., glycophorin). To explore the possibility that these basic residues bind electrostatically to monovalent acidic lipids, we studied the binding of the peptides Lysn and Argn (n = 1-5) to bilayer membranes containing phosphatidylserine (PS) or phosphatidylglycerol (PG). We made electrophoretic mobility measurements using multilamellar vesicles, fluorescence and equilibrium binding measurements using large unilamellar vesicles, and surface potential measurements using monolayers. None of the peptides bound to vesicles formed from the zwitterionic lipid phosphatidylcholine (PC) but all bound to vesicles formed from PC/PS or PC/PG mixtures. None of the peptides exhibited specificity between PS and PG. Each lysine residue that was added to Lys2 decreased by one order of magnitude the concentration of peptide required to reverse the charge on the vesicle; equivalently it increased by one order of magnitude the binding affinity of the peptides for the PS vesicles. The simplest explanation is that each added lysine binds independently to a separate PS with a microscopic association constant of 10 M-1 or a free energy of approximately 1.4 kcal/mol. Similar, but not identical, results were obtained with the Argn peptides. A simple theoretical model combines the Gouy-Chapman theory (which accounts for the nonspecific electrostatic accumulation of the peptides in the aqueous diffuse double layer adjacent to the membrane) with mass action equations (which account for the binding of the peptides to greater than 1 PS). This model can account qualitatively for the dependence of binding on both the number of basic residues in the peptides and the mole fraction of PS in the membrane.     

Equilibrium and kinetics of the allosteric transition of GroEL studied by solution X-ray scattering and fluorescence spectroscopy

We have studied the ATP-induced allosteric structural transition of GroEL using small angle X-ray scattering and fluorescence spectroscopy in combination with a stopped-flow technique. With X-ray scattering one can clearly distinguish the three allosteric states of GroEL, and the kinetics of the transition of GroEL induced by 85 microM ATP have been observed directly by stopped-flow X-ray scattering for the first time. The rate constant has been found to be 3-5s(-1) at 5 degrees C, indicating that this process corresponds to the second phase of the ATP-induced kinetics of tryptophan-inserted GroEL measured by stopped-flow fluorescence. Based on the ATP concentration dependence of the fluorescence kinetics, we conclude that the first phase represents bimolecular non-cooperative binding of ATP to GroEL with a bimolecular rate constant of 5.8 x 10(5)M(-1)s(-1) at 25 degrees C. Considering the electrostatic repulsion between negatively charged GroEL (-18 of the net charge per monomer at pH 7.5) and ATP, the rate constant is consistent with a diffusion-controlled bimolecular process. The ATP-induced fluorescence kinetics (the first and second phases) at various ATP concentrations (< 400 microM) occur before ATP hydrolysis by GroEL takes place and are well explained by a kinetic allosteric model, which is a combination of the conventional transition state theory and the Monod-Wyman-Changeux model, and we have successfully evaluated the equilibrium and kinetic parameters of the allosteric transition, including the binding constant of ATP in the transition state of GroEL.     

Multi-method global analysis of thermodynamics and kinetics in reconstitution of monellin

Accurate and precise determinations of thermodynamic parameters of binding are important steps toward understanding many biological mechanisms. Here, a multi-method approach to binding analysis is applied and a detailed error analysis is introduced. Using this approach, the binding thermodynamics and kinetics of the reconstitution of the protein monellin have been quantitatively determined in detail by simultaneous analysis of data collected with fluorescence spectroscopy, surface plasmon resonance and isothermal titration calorimetry at 25 degrees C, pH 7.0 and 150 mM NaCl. Monellin is an intensely sweet protein composed of two peptide chains that form a single globular domain. The kinetics of the reconstitution reaction are slow, with an association rate constant, k(on) of 8.8 x 10(3) M(-1) s(-1) and a dissociation rate constant, k(off) of 3.1 x 10(-4) s(-1). The equilibrium constant K(A) is 2.8 x 10(7) M(-1) corresponding to a standard free energy of association, DeltaG degrees , of -42.5 kJ/mol. The enthalpic component, DeltaH degrees , is -18.7 kJ/mol and the entropic contribution, DeltaS degrees , is 79.8 J mol(-1) K(-1) (-TDeltaS degrees = -23.8 kJ/mol). The association of monellin is therefore a bimolecular intra-protein association whose energetics are slightly dominated by entropic factors.     

Binding of a fluorescent dansylcadaverine-substance P analogue to negatively charged phospholipid membranes

We have investigated the binding of a new dansylcadaverine derivative of substance P (DNC-SP) with negatively charged small unilamellar vesicles composed of a mixture of phosphatidylcholine (PC) and either phosphatidylglycerol (PG) or phosphatidylserine (PS) using fluorescence spectroscopic techniques. The changes in fluorescence properties were used to obtain association isotherms at variable membrane negative charges and at different ionic strengths. The experimental association isotherms were analyzed using two binding approaches: (i) the Langmuir adsorption isotherm and the partition equilibrium model, that neglect the activity coefficients; and (ii) the partition equilibrium model combined with the Gouy-Chapman formalism that considers electrostatic effects. A consistent quantitative analysis of each DNC-SP binding curve at different lipid composition was achieved by means of the Gouy-Chapman approach using a peptide effective interfacial charge (v) value of (0.95 +/- 0.02), which is lower than the physical charge of the peptide. For PC/PG membranes, the partition equilibrium constant were 7.8 x 10(3) M(-1) (9/1, mol/mol) and 6.9 x 10(3) M(-1) (7/3, mol/mol), whereas for PC/PS membranes an average value of 6.8 x 10(3) M(-1) was estimated. These partition equilibrium constants were similar to those obtained for the interaction of DNC-SP with neutral PC membranes (4.9 x 10(3) M(-1)), as theoretically expected. We demonstrate that the v parameter is a determinant factor to obtain a unique value of the binding constant independently of the surface charge density of the vesicles. Also, the potential of fluorescent dansylated SP analogue in studies involving interactions with cell membranes is discussed.     

Isothermal titration calorimetric studies on the binding of a family 6 carbohydrate-binding module of Clostridium thermocellum xynA with xlylooligosaccharides

The family 6 carbohydrate-binding module (CBM) of Clostridium thermocellum XynA was expressed, and the binding equilibria of the CBM with xylooligosaccharides (degree of polymerization DP = 2-8) were observed by isothermal titration calorimetry (ITC) at pH 8. The association constant, Ka, increased with increasing DP from 5 x 10(3) M(-1) (DP = 2) to approximately 5 x 10(5) M(-1) (DP = 5-8) at 20 degrees C. The Ka values at 60 degrees C were about 1/10 of those at 20 degrees C. The binding was found to be an enthalpy-driven reaction. The DP dependence of the thermodynamic parameters of the binding reaction suggested the size of the ligand-binding site to be 5 xylose units long.     

Phospholipid-binding properties of bovine factor V and factor Va.

Factor V and factor Va binding to single bilayer phospholipid vesicles was investigated by light-scattering intensity measurements. This technique allows the measurement of free and phospholipid-bound protein concentrations from which equilibrium constants can be obtained. As controls, the Ca2+-dependent phospholipid binding of prothrombin and factor X were also studied. The average values obtained for the dissociation constants (Kd) and lipid to protein ratio at saturation, moles/mole (n), for prothrombin (Kd = 2.3 X 10(-6) M, n = 104) and factor X (Kd = 2.5 X 10(-6) M, n = 46) binding to vesicles containing 25% Folch fraction III and 75% phosphatidylcholine in the presence of 2 mM Ca2+ were in agreement with those reported in the literature. The average factor V and factor Va values for the dissociation constants and lipid to protein ratio at saturation (moles/mole) were Kd = 7.2 X 10(-8) M and n = 270 for factor V and Kd = 4.4 X 10(-7) M and n = 76 for factor Va. In contrast to prothrombin and factor X, factor V and factor Va demonstrated Ca2+-independent lipid binding. In addition, the number of factor V and factor Va molecules bound per vesicle was found to be dependent both on the phosphatidylserine content of the vesicle and the ionic strength of the buffer.