Probing the pH-dependent structural features of alpha-KTx12.1, a potassium channel blocker from the scorpion Tityus serrulatus

Potassium channels are widespread in living cells and are involved in many diseases. The scorpion toxin alpha-KTx(12.1) interacts with various K(+) channels, suggesting its capacity to match diverse channel pores. It is recognized that tissue injuries may affect the pH at toxins site of action, thereby modulating both protein conformation and activity. To better understand its molecular mechanism of action, we studied alpha-KTx(12.1) using pH as a tool to explore its plasticity and NMR in combination with MD calculations to detect it. The toxin solution structure consists of an alpha-helix and a triple-stranded beta-sheet stabilized by four disulfide bridges. The NMR results show, in addition, that His28 possesses an unusually low pK(a) of 5.2. The best set of protein conformers is obtained at pH 4.5, while at pH 7.0, the reduced number of NOEs resulting from a faster hydrogen exchange does not allow to reach a good structural convergence. Nonetheless, MD calculations show that the toxin structure does not vary significantly in that pH range, while conformational changes and modifications of the surface charge distribution occur when His28 is fully protonated. Moreover, essential dynamics analysis reveals variations in the toxin's coherent motions. In conclusion, His28, with its low pK(a) value, provides alpha-KTx(12.1) with the ability to preserve its active conformation over a wide pH interval, thus expanding the range of cellular conditions where the toxin can fully exhibit its activity. Overall, the results further underline the role of histidine as a natural controller of proteins' functionality.     

The amino- and carboxyl-terminal fragments of the Bacillus thuringensis Cyt1Aa toxin have differential roles in toxin oligomerization and pore formation

The Cyt toxins produced by the bacteria Bacillus thuringiensis show insecticidal activity against some insects, mainly dipteran larvae, being able to kill mosquitoes and black flies. However, they also possess a general cytolytic activity in vitro, showing hemolytic activity in red blood cells. These proteins are composed of two outer layers of α-helix hairpins wrapped around a β-sheet. With regard to their mode of action, one model proposed that the two outer layers of α-helix hairpins swing away from the β-sheet, allowing insertion of β-strands into the membrane forming a pore after toxin oligomerization. The other model suggested a detergent-like mechanism of action of the toxin on the surface of the lipid bilayer. In this work, we cloned the N- and C-terminal domains form Cyt1Aa and analyzed their effects on Cyt1Aa toxin action. The N-terminal domain shows a dominant negative phenotype inhibiting the in vitro hemolytic activity of Cyt1Aa in red blood cells and the in vivo insecticidal activity of Cyt1Aa against Aedes aegypti larvae. In addition, the N-terminal region is able to induce aggregation of the Cyt1Aa toxin in solution. Finally, the C-terminal domain composed mainly of β-strands is able to bind to the SUV liposomes, suggesting that this region of the toxin is involved in membrane interaction. Overall, our data indicate that the two isolated domains of Cyt1Aa have different roles in toxin action. The N-terminal region is involved in toxin aggregation, while the C-terminal domain is involved in the interaction of the toxin with the lipid membrane.     

A detergent-like mechanism of action of the cytolytic toxin Cyt1A from Bacillus thuringiensis var. israelensis

The cytolytic delta-endotoxin Cyt1A from Bacillus thuringiensis var. israelensis is used in commercial preparations of environmentally safe insecticides. The current hypothesis on its mode of action is that the toxin self-assembles into well-defined cation-selective channels or pores, which results in colloid-osmotic lysis of the cell. Recently, a new hypothesis has been put forward suggesting that Cyt1A rather nonspecifically aggregates on the membrane surface and acts in a detergent-like manner. To distinguish between these two hypotheses, we investigated whether in the presence of lipid Cyt1A self-assembles into stoichiometric oligomers, which are characteristic of pores or channels, or aggregates into nonstoichiometric complexes, which would support the detergent-like model. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that in the presence of lipid Cyt1A forms protein aggregates with a broad range of molecular weights, some being too large to enter the gel. Cyt1A tryptophan (Trp) fluorescence in the presence of lipid exhibited a decrease in anisotropy and quantum yield, but an unchanged lifetime, which is consistent with the presence of toxin aggregates in the membrane. Electrostatic interactions between the charged amino acid residues and the lipid headgroups are responsible for bringing the protein to the membrane surface, while hydrophobic and/or van der Waals interactions make the membrane binding irreversible. Fluorescence photobleaching recovery, a technique that measures the diffusion coefficient of fluorescently labeled particles, and epifluorescence microscopy revealed that upon addition of Cyt1A lipid vesicles were broken into smaller, faster diffusing objects. Since no change in size or morphology of the vesicles is expected when pores are formed in the osmotically equilibrated membranes, our results support the detergent-like mode of action of Cyt1A.     

Effect of low pH on the conformation of Pseudomonas exotoxin A

Previously we examined factors involved in the entry mechanism of Pseudomonas exotoxin A (PTx) at the level of lipid-protein interactions (Farahbakhsh, Z. T., Baldwin, R. L., and Wisnieski, B. J. (1986) J. Biol. Chem. 261, 11404-11408). Exposure to a low pH environment appears to be an obligatory trigger of the entry pathway. In this report we describe the effect of pH upon the conformation of PTx. We have found that the intrinsic fluorescence of PTx is strongly dependent on pH, decreasing between pH 7.4 and 4.0 with a red shift in the emission lambda max. The changes are reversible and associated with the acquisition of a binding site for the fluorescent dye 1-anilino-8-naphthalenesulfonic acid (ANS). The fluorescence intensity of ANS in the presence of PTx increases with decreasing pH and is accompanied by a blue shift in emission spectra, indicative of exposure of hydrophobic surfaces. These changes are also reversible. Both the intrinsic fluorescence and ANS binding profiles show a dramatic dependence on pH, with the transitions centered on pH 5.0 and 4.5, respectively. Circular dichroism studies reveal a 9% decrease in alpha-helicity between pH 7.7 and 4. The susceptibility of toxin to trypsin cleavage is also a function of pH, increasing with decreasing pH. The pH 7.4 cleavage profile is regained when the acid-treated samples are brought back to pH 7.4. The conformational changes observed in these pH shift experiments are likely to be physiologically significant because the conditions closely resemble those that the toxin would encounter if entry into the cytoplasm of a cell involves escape from an endosomal compartment.     

Effect of pH on the conformation and stability of the plant toxin ricin

Intrinsic protein fluorescence of native plant toxin and its isolated subunits were studied. The effect of pH was studied on: conformation of ricin and its A- and R-chains; affinity to galactose of ricin and its binding B-subunit. At two pH 5.0 and 7.0, the structural stability of toxin and subunits was estimated according to denaturational action of guanidine chloride. It was demonstrated that position of maximum and the spectrum shape of fluorescence of native toxin and catalytical A-subunit insignificantly depends on pH in the range of 3-8, whereas sufficient changes of the separameters for the ricin B-chain reveal structural transition at pH 4-5. The affinity of galactose of ricin and its isolated B-chain depends on pH, the maximal binding is observed at pH 7. The structural stability of ricin and isolated chains significantly differs at pH 7.5 and 5.0, thus the structure stability of ricin and A-chain increases, and that of B-chain decreases at pH 5.0.     

Effect of pH on the conformation of diphtheria toxin and its implications for membrane penetration

The pH-triggered change in diphtheria toxin conformation and the physical properties of the toxin above and below the transition pH have been examined. Exposure to low pH (less than or equal to 5 at 23 degrees C, less than or equal to 5.3 at 37 degrees C) triggers a rapid (t1/2 less than 30 s) change in toxin conformation; the transition occurs over a narrow pH range (0.2 unit). Below the transition pH, buried tryptophans become exposed, and the toxin becomes hydrophobic, binding very tightly to detergent. Aggregation is observed at low pH, probably due to this extreme hydrophobicity. Circular dichroism and fluorescence properties show that the low-pH conformation is not extensively unfolded. Therefore, the toxin "opens" at low pH without becoming a random coil. The conformation change is partly irreversible, and the degree of irreversibility parallels the degree of aggregation. Reduction of the disulfide bonds does not increase hydrophobicity at neutral pH. Furthermore, none of the structural variants of toxin (monomer or dimer, bound to ApUp or free, and nicked between subunits or intact) are hydrophobic at neutral pH or differ in transition pH markedly. Therefore, these factors do not mimic the effect of low pH. These observations are consistent with a functional role for the pH-triggered changes during penetration of the membranes of acidic organelles. The toxin may have adapted a conformational change similar to partial denaturation for a critical role in function. The possible nature of the pH-sensitive interactions and the effects of aggregation are discussed briefly.     

Evidence of the Importance of the Met115 for Bacillus thuringiensis subsp. israelensis Cyt1Aa Protein Cytolytic Activity in Escherichia coli

Cyt1Aa is a cytolytic toxin, found together with the delta-endotoxins in Bacillus thuringiensis subsp. israelensis parasporal insecticidal crystals. The latter are used as an environmental friendly insecticide against mosquitoes and black flies. Contrary to Cry delta-endotoxin, the mode of action of Cyt1Aa is not completely understood. In the absence of direct structural data, a novel mutated cyt1Aa gene was used to obtain indirect informations on Cyt1Aa conformation changes in the lipid membrane environment. A mutated cyt1Aa gene named cyt1A97 has been isolated from a B. thuringiensis israelensis strain named BUPM97. The nucleotide sequence predicted a protein of 249 amino acids residues with a calculated molecular mass of 27 kDa. Both nucleotide and amino acid sequences similarity analysis revealed that cyt1A97 presents one amino acid different from the native cyt1Aa gene. This mutation was located in the helix α C corresponding to a substitution of Met¹¹⁵ by a Thr. The heterologous expression of the cyt1A97 and another cyt1Aa-type gene called cyt1A98, not affected by such mutation used as control, was performed in Escherichia coli. It revealed that the mutated Cyt1A97 protein was over produced as inclusion bodies showing a very weak toxicity to E. coli contrarily to Cyt1A98 that stopped E. coli growth. Hence, hydrophobic residue Met at position 115 of Cyt1Aa should play a very important role for the maintenance of the structure and cytolytic functions of Cyt1Aa.     

Studies of glutamate dehydrogenase. Regulation of glutamate dehydrogenase from Candida utilis by a pH and temperature-dependent conformational transition.

Glutamate dehydrogenase from Candida utilis undergoes a reversible conformational transition between an active and an inactive state at low pH AND low temperature. This conformational transition can also be followed by fluorescence measurements. The temperature-dependent equilibrium between the active and the inactive state is characterized by a transition temperature of 10.7 degrees C and a delta H value of 148 kcal/mol (620 kJ/mol). The temperature dependence of the enzymic activity above 15 degrees C yields an activation energy of 15 kcal/mol (63 kJ/mol), a larger value than that for the beef liver enzyme (9 kcal/mol; 38 kJ/mol). In contrast to the yeast enzyme the Arrhenius plot is linear and, therefore, the beef liver enzyme is not transformed into an inactive conformation at low temperatures. Sedimentation analysis shows that the inactivation of the Candida utilis enzyme is not caused by change in the quaternary structure. The pH dependence of the conformational transition at low pH measured by fluorescence change is characterized by a pK value of 7.01 for the enzyme in the absence and of 6.89 for the enzyme in the presence of 2-oxoglutarate with a Hill coefficient of 3.4 in both cases. Similar results are found when the pH dependence of the enzymic activity is analyzed. With the beef liver enzyme the same pK value is obtained but with a Hill coefficient of 1 indicating cooperativity only in the case of the Candida utilis enzyme. The best fit of the pH dependence of the rate constants of the fluorescence changes was obtained with pK values of 7.45 and 6.45 for the active and the inactive state respectively. In this model the lowest time constant which is obtained at the pH of the equilibrium was found to be 0.05 s-1. Preincubation experiments with the substrate 2-oxoglutarate but not with the coenzyme shift the equilibrium to the active conformation. The coenzyme obviously reduces the rate constant of the conformational transition. The sedimentation coefficient (SO20, w) and the molecular weight were found to be 11.0 S and 276 000, respectively. The enzyme molecule is built up by six polypeptide chains each having a molecular weight of 47 000.     

Anionic lipid-induced conformational changes in human phagocyte flavocytochrome b precede assembly and activation of the NADPH oxidase complex

Phagocyte NADPH oxidases generate superoxide at high rates in defense against infectious agents, a process regulated by second messenger anionic lipids using incompletely understood mechanisms. We reconstituted the catalytic core of the human neutrophil NADPH oxidase, flavocytochrome b (Cyt b) in 99% phosphatidylcholine vesicles in order to correlate anionic lipid-dependent conformational changes in membrane-bound Cyt b and oxidase activity. The anionic lipid 10:0 phosphatidic acid (10:0 PA) specifically induced conformational changes in Cyt b as measured by a combination of fluorescence resonance energy transfer methods and size exclusion chromatography. The fluorescence lifetime of a complex between Cyt b and Cascade Blue-derivatized anti-p22(phox) antibody (CCB-CS9), increased after exposure to 10:PA by ～50% of the change observed when the complex is dissociated, indicating a structural rearrangement of p22(phox) and/or the Cyt b heme prosthetic groups. Half of the quenching relaxation occurred at 10:0 PA concentrations permissive to less than 10% full NADPH oxidase activity, but saturated near the saturation in activity in a matched cell-free oxidase assay. We conclude that anionic lipids modulate the conformation of Cyt b in the membrane and suggest they may serve to modulate the structure of Cyt b as a control mechanism for the NADPH oxidase.     

The effect of high pH upon diphtheria toxin conformation and model membrane association: role of partial unfolding

Penetration of membranes by diphtheria toxin in vivo is at least partially triggered by a low pH-induced conformational change occurring within the lumen of an acidic organelle. In order to gain insight into the nature of this change the behavior of the toxin at high pH was characterized and compared to that previously determined at low pH. We find that near pH 10.5 a major conformational change occurs. This change is accompanied by a marked decrease in fluorescence intensity, a red shift in fluorescence emission maximum, and increased susceptibility of protein fluorescence to acrylamide quenching. Differential scanning calorimetry shows that the high pH conformational change involves a cooperative endothermic unfolding transition. These changes at high pH are very similar to those induced by low pH, supporting the conclusion that the changes at low pH also involve a denaturation-like process. In addition, at high pH the toxin gains the ability to bind to model membranes, again similar to its behavior at low pH. On the basis of these studies we conclude that exposure of hydrophobic sequences due to partial unfolding is one dominating component in inducing hydrophobic behavior at both high and low pH, but that at low pH Asp/Glu protonation also contributes to hydrophobicity.     

Topography of the hydrophilic helices of membrane-inserted diphtheria toxin T domain: TH1-TH3 as a hydrophilic tether

After low pH-triggered membrane insertion, the T domain of diphtheria toxin helps translocate the catalytic domain of the toxin across membranes. In this study, the hydrophilic N-terminal helices of the T domain (TH1-TH3) were studied. The conformation triggered by exposure to low pH and changes in topography upon membrane insertion were studied. These experiments involved bimane or BODIPY labeling of single Cys introduced at various positions, followed by the measurement of bimane emission wavelength, bimane exposure to fluorescence quenchers, and antibody binding to BODIPY groups. Upon exposure of the T domain in solution to low pH, it was found that the hydrophobic face of TH1, which is buried in the native state at neutral pH, became exposed to solution. When the T domain was added externally to lipid vesicles at low pH, the hydrophobic face of TH1 became buried within the lipid bilayer. Helices TH2 and TH3 also inserted into the bilayer after exposure to low pH. However, in contrast to helices TH5-TH9, overall TH1-TH3 insertion was shallow and there was no significant change in TH1-TH3 insertion depth when the T domain switched from the shallowly inserting (P) to deeply inserting (TM) conformation. Binding of streptavidin to biotinylated Cys residues was used to investigate whether solution-exposed residues of membrane-inserted T domain were exposed on the external or internal surface of the bilayer. These experiments showed that when the T domain is externally added to vesicles, the entire TH1-TH3 segment remains on the cis (outer) side of the bilayer. The results of this study suggest that membrane-inserted TH1-TH3 form autonomous segments that neither deeply penetrate the bilayer nor interact tightly with the translocation-promoting structure formed by the hydrophobic TH5-TH9 subdomain. Instead, TH1-TH3 may aid translocation by acting as an A-chain-attached flexible tether.     

The effect of pH on the conformation and stability of the structure of plant toxin-ricin

The effect of pH on the conformation of ricin and its A- and B-chains has been studied by measuring their intrinsic fluorescence. At pH 5.0 and 7.5, the structural stability of toxin and subunits was estimated according to the denaturing action of guanidine hydrochloride. It was demonstrated that the fluorescence of native toxin and catalytic A-subunit does not depend significantly on pH in the range pH 3-8, whereas ricin B-chain undergoes a structural transition at pH less than 5.0. The structural stability of ricin and isolated chains differs significantly at pH 7.5 and 5.0; the structural stability of ricin and the A-chain increases, whereas that of the B-chain decreases.     

Role of receptor interaction in the mode of action of insecticidal Cry and Cyt toxins produced by Bacillus thuringiensis

Cry toxins from Bacillus thuringiensis are used for insect control. Their primary action is to lyse midgut epithelial cells. In this review we will summarize recent findings on the Cry toxin-receptor interaction and the role of receptor recognition in their mode of action. Cry toxins interact sequentially with multiple receptors. In lepidopteran insects, Cry1A monomeric toxins interact with the first receptor and this interaction triggers oligomerization of the toxins. The oligomer then interacts with second receptor inducing insertion into membrane microdomains and larval death. In the case of mosquitocidal toxins, Cry and Cyt toxins play a part. These toxins have a synergistic effect and Cyt1Aa overcomes Cry toxin resistance. Recently, it was proposed that Cyt1Aa synergizes or suppresses resistance to Cry toxins by functioning as a membrane-bound receptor for Cry toxin.     

Ion-conducting channels produced by botulinum toxin in planar lipid membranes

The interaction of botulinum neurotoxin (Botx) with planar lipid membranes was studied by measuring the ability of the toxin to form ion-conducting channels. Channel formation was pH dependent. At physiological pH, Botx formed no channels, whereas at pH 6.6, the toxin formed channels with a unit conductance of 12 pS in 0.1 M NaCl. The rate of channel formation increased with decreasing pH, reaching a maximum at pH 6.1, and then decreased at lower values of pH. The channels, once formed, were permanent entities in the membrane throughout the course of an experiment and fluctuated between an open and a closed state. The rate of channel formation depended upon the square of the toxin concentration, suggesting an aggregation step is involved in channel formation. The data were consistent with the hypothesis that Botx enters cells through endocytosis, followed by its release into the cytoplasm at low pH.     

Study of the correlation of secondary structure of beta-amyloid peptide (Abeta40) with the hydrophobic exposure under different conditions

Abeta is the core protein of extracellular plaque of Alzheimer's disease, and its neurotoxicity is relative to its conformation. In the current work, the effects of various factors, such as pH, ionic strength and lipid membranes, on the secondary structure of Abeta were studied by circular dichroism. In addition, we detected the exposure of hydrophobic sites of Abeta under different conditions using ANS fluorescence. The results showed that the hydrophobic exposure of the protein was correlated with the content of 3betasheet conformation in the phospholipid-containing environment. The beta-sheet content and hydrophobic exposure of Abeta both increased when reacted with pure PC vesicles, while no beta-sheet content and very low hydrophobic exposure were detected after reaction with 30% cholesterol containing PC vesicles. Since beta-sheet conformation is considered as the toxic conformation of Afbeta such correlation may be important for the pathology of AD.     

Conotoxins as sensors of local pH and electrostatic potential in the outer vestibule of the sodium channel

We examined the block of voltage-dependent rat skeletal muscle sodium channels by derivatives of mu-conotoxin GIIIA (muCTX) having either histidine, glutamate, or alanine residues substituted for arginine-13. Toxin binding and dissociation were observed as current fluctuations from single, batrachotoxin-treated sodium channels in planar lipid bilayers. R13X derivatives of muCTX only partially block the single-channel current, enabling us to directly monitor properties of both muCTX-bound and -unbound states under different conditions. The fractional residual current through the bound channel changes with pH according to a single-site titration curve for toxin derivatives R13E and R13H, reflecting the effect of changing the charge on residue 13, in the bound state. Experiments with R13A provided a control reflecting the effects of titration of all residues on toxin and channel other than toxin residue 13. The apparent pKs for the titration of residual conductance are shifted 2-3 pH units positive from the nominal pK values for histidine and glutamate, respectively, and from the values for these specific residues, determined in the toxin molecule in free solution by NMR measurements. Toxin affinity also changes dramatically as a function of pH, almost entirely due to changes in the association rate constant, kon. Interpreted electrostatically, our results suggest that, even in the presence of the bound cationic toxin, the channel vestibule strongly favors cation entry with an equivalent local electrostatic potential more negative than -100 mV at the level of the "outer charged ring" formed by channel residues E403, E758, D1241, and D1532. Association rates are apparently limited at a transition state where the pK of toxin residue 13 is closer to the solution value than in the bound state. The action of these unique peptides can thus be used to sense the local environment in the ligand--receptor complex during individual molecular transitions and defined conformational states.     

Tetracaine-membrane interactions: effects of lipid composition and phase on drug partitioning, location, and ionization

Interactions of the local anesthetic tetracaine with unilamellar vesicles made of dimyristoyl or dipalmitoyl phosphatidylcholine (DMPC or DPPC), the latter without or with cholesterol, were examined by following changes in the drug's fluorescent properties. Tetracaine's location within the membrane (as indicated by the equivalent dielectric constant around the aromatic fluorophore), its membrane:buffer partition coefficients for protonated and base forms, and its apparent pK(a) when adsorbed to the membrane were determined by measuring, respectively, the saturating blue shifts of fluorescence emission at high lipid:tetracaine, the corresponding increases in fluorescence intensity at this lower wavelength with increasing lipid, and the dependence of fluorescence intensity of membrane-bound tetracaine (TTC) on solution pH. Results show that partition coefficients were greater for liquid-crystalline than solid-gel phase membranes, whether the phase was set by temperature or lipid composition, and were decreased by cholesterol; neutral TTC partitioned into membranes more strongly than the protonated species (TTCH(+)). Tetracaine's location in the membrane placed the drug's tertiary amine near the phosphate of the headgroup, its ester bond in the region of the lipids' ester bonds, and associated dipole field and the aromatic moiety near fatty acyl carbons 2-5; importantly, this location was unaffected by cholesterol and was the same for neutral and protonated tetracaine, showing that the dipole-dipole and hydrophobic interactions are the critical determinants of tetracaine's location. Tetracaine's effective pK(a) was reduced by 0.3-0.4 pH units from the solution pK(a) upon adsorption to these neutral bilayers, regardless of physical state or composition. We propose that the partitioning of tetracaine into solid-gel membranes is determined primarily by its steric accommodation between lipids, whereas in the liquid-crystalline membrane, in which the distance between lipid molecules is larger and steric hindrance is less important, hydrophobic and ionic interactions between tetracaine and lipid molecules predominate.     

Phosphorylation alters the pH-dependent active state equilibrium of rhodopsin by modulating the membrane surface potential.

Phosphorylation reduces the lifetime and activity of activated G protein-coupled receptors, yet paradoxically shifts the metarhodopsin I-II (MI-MII) equilibrium (K(eq)) of light-activated rhodopsin toward MII, the conformation that activates G protein. In this report, we show that phosphorylation increases the apparent pK for MII formation in proportion to phosphorylation stoichiometry. Decreasing ionic strength enhances this effect. Gouy-Chapman theory shows that the change in pK is quantitatively explained by the membrane surface potential, which becomes more negative with increasing phosphorylation stoichiometry and decreasing ionic strength. This lowers the membrane surface pH compared to the bulk pH, increasing K(eq) and the rate of MII formation (k(1)) while decreasing the back rate constant (k(-)(1)) of the MI-MII relaxation. MII formation has been observed to depend on bulk pH with a fractional stoichiometry of 0.6-0.7 H(+)/MII. We find that the apparent fractional H(+) dependence is an artifact of altering the membrane surface charge during a titration, resulting in a fractional change in membrane surface pH compared to bulk pH. Gouy-Chapman calculations of membrane pH at various phosphorylation levels and ionic strengths suggest MII formation behavior consistent with titration of a single H(+) binding site with 1:1 stoichiometry and an intrinsic pK of 6.3 at 0.5 degrees C. We show evidence that suggests this same site has an intrinsic pK of 5.0 prior to light activation and its protonation before activation greatly enhances the rate of MII formation.     

Calcium binding of troponin C. I. A potentiometric titration study.

Structural changes of troponin C on calcium binding were studied by hydrogen ion titration, circular dichroism, and fluorescence measurements. The potentiometric titration curves in the carboxyl region are shifted towards lower pH with calcium binding. The intrinsic pK of the carboxyl groups at the calcium binding sites decreases by 0.8 pK unit on calcium binding; on the other hand, magnesium ions have little effect on the intrinsic pK of the carboxyl groups. The intrinsic pK of the imidazole group is not affected by calcium binding. The value of w, an electrostatic interaction factor, is identical for calcium-free and calcium-bound troponin C and is about half of the value calculated assuming a compact sphere. The results of difference titration on the calcium binding indicate that the pH of troponin C solution increases on addition of CaCl2 up to 2 mol of Ca2+ per mol of troponin C and then decreases on further addition of CaCl2. The pH increase is depressed in the presence of MgCl2, in the low pH region, or at high ionic strength. The pH increase is also observed on addition of MgCl2. The ellipticity at 222 nm was measured under the same conditions as the difference titration measurements, and the relation between the pH change and the conformational change of troponin C on calcium binding is discussed based on the results obtained. The number of calcium binding sites and the binding constants estimated by analysis of these difference titration curves were in agreement with the results of Potter and Gergely (22). No magnesium binding site was observed. The tyrosine fluorescence measurements indicated that the binding site near tyrosine-109 is one of the high affinity sites.     

pH-dependent fusion of phosphatidylcholine small vesicles. Induction by a synthetic amphipathic peptide

A synthetic, amphipathic 30-amino acid peptide with the major repeat unit Glu-Ala-Leu-Ala (GALA) was designed to mimic the behavior of the fusogenic sequences of viral fusion proteins. GALA is a water-soluble peptide with an aperiodic conformation at neutral pH and becomes an amphipathic alpha-helix as the pH is lowered to 5.0 where it interacts with bilayers. Fluorescence energy transfer measurements indicated that GALA induced lipid mixing between phosphatidylcholine small unilamellar vesicles but not large unilamellar vesicles. This lipid mixing occurred only at pH 5.0 and not at neutral pH. Concomitant with lipid mixing, the vesicles increased in diameter from 500 to 750 to 1000 A as measured by dynamic light scattering and internal volume determination. GALA induced leakage of small molecules (Mr 450) at pH 5.0 was too rapid to permit detection of contents mixing. However, retention of larger molecules (Mr 4100) under the same conditions suggests that vesicle fusion is occurring. For a 100/1 lipid/peptide ratio all vesicles fused just once, whereas for a 50/1 ratio higher order fusion products formed. A mass action model gives good simulation of the kinetics of increase in fluorescence intensity and yields rate constants of aggregation and fusion. As the lipid to peptide ratio decreases from 100/1 to 50/1 both rate constants of aggregation and fusion increase, indicating that GALA is a genuine inducer of vesicle fusion. The presence of divalent cations which can alter GALAs conformation at pH 7.5 had little effect on its lipid mixing activity. GALA was modified by altering the sequence while keeping the amino acid composition constant or by shortening the sequence. These peptides did not have any lipid mixing activity nor did they induce an increase in vesicle size. Together, these results indicate that fusion of phosphatidylcholine small unilamellar vesicles induced by GALA requires both a peptide length greater than 16 amino acids as well as a defined topology of the hydrophobic residues.