Charge repulsion in the conformational stability of melittin.

Electrostatic repulsion between positively charged groups has been suggested to be critical in determining the conformation of melittin. To clarify the role of repulsive forces, we prepared a series of succinylated melittins, an acetylated melittin, and a synthetic melittin mutant, with various degrees of charge repulsion. The conformation of the melittin derivatives was examined by far-UV circular dichroism under various conditions of pH and salt at 20 degrees C. The stability of the tetrameric helical state was found to be dependent on the net charge of the peptides. The charge repulsive forces destabilized the helical state of intact melittin by 600 cal/(charge.mol of tetramer). This value was close to the corresponding one (450 cal/(charge.mol)) obtained for the acidic molten globule of horse cytochrome c [Goto, Y., & Nishikiori, S. (1991) J. Mol. Biol. 222, 679-686], which has a molecular weight and a net charge comparable to those of the tetrameric melittin. Small-angle X-ray scattering of the tetrameric melittin and the molten globule of cytochrome c showed that the two states are also comparable to each other in the radius of gyration. These results suggest that the contribution of electrostatic repulsion to the conformational stability of melittin is similar to that of the molten globule.     

Anion and pH-dependent conformational transition of an amphiphilic polypeptide

While several proteins, including beta-lactamase, cytochrome c and apomyoglobin, are maximally unfolded at pH 2 by HCl in the absence of salt, the addition of anions, either from salt or acid, co-operatively induces the unfolded proteins to refold to a molten globule state, because anions bind preferentially to the compact molten globule state compared to the extended unfolded state. To study the role of the anion-dependent conformational transition at neutral pH, we synthesized a model polypeptide of 51 amino acid residues, consisting of tandem repeats of a Lys-Lys-Leu-Leu sequence and containing a turn sequence, Asn-Pro-Gly, at the center of the molecule. The model polypeptide showed no significant conformation by circular dichroism under conditions of low salt at neutral pH. However, addition of anions, either from salt or acid, induced the folding transition to an alpha-helical conformational state. The order of effectiveness of various anions in inducing the folding transition was consistent with the series of anions in inducing the molten globule of the acid-denatured protein. This suggests that the helical state of the model polypeptide is equivalent to the molten globule state. At pH values above 9, the model polypeptide also took an alpha-helical conformation, which was very similar to that induced by anions. On the basis of the chloride and pH-dependent conformational transitions, a phase diagram for the conformational states was constructed. The phase diagram was explained simply by assuming that the conformational transition is linked to the proton and the anion bindings to a limited number of amino groups and that anions bind only to the protonated groups.     

Mechanism of acid-induced folding of proteins

We have previously shown [Goto, Y., Calciano, L. J., & Fink, A. L. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 573-577] that beta-lactamase, cytochrome c, and apomyoglobin are maximally unfolded at pH 2 under conditions of low ionic strength, but a further decrease in pH, by increasing the concentration of HCl, refolds the proteins to the A state with properties similar to those of a molten globule state. To understand the mechanism of acid-induced refolding of protein structure, we studied the effects of various strong acids and their neutral salts on the acid-unfolded states of ferricytochrome c and apomyoglobin. The conformational transition of cytochrome c was monitored at 20 degrees C by using changes in the far-UV CD and in the Soret absorption at 394 nm, and that of apomyoglobin was monitored by changes in the far-UV CD. Various strong acids (i.e., sulfuric acid, perchloric acid, nitric acid, trichloroacetic acid, and trifluoroacetic acid) refolded the acid-unfolded cytochrome c and apomyoglobin to the A states as was the case with HCl. For both proteins neutral salts of these acids caused similar conformational transitions, confirming that the anions are responsible for bringing about the transition. The order of effectiveness of anions was shown to be ferricyanide greater than ferrocyanide greater than sulfate greater than thiocyanate greater than perchlorate greater than iodide greater than nitrate greater than trifluoroacetate greater than bromide greater than chloride.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermal unfolding of tetrameric melittin: comparison with the molten globule state of cytochrome c.

Whereas melittin at micromolar concentrations is unfolded under conditions of low salt at neutral pH, it transforms to a tetrameric alpha-helical structure under several conditions, such as high peptide concentration, high anion concentration, or alkaline pH. The anion- and pH-dependent stabilization of the tetrameric structure is similar to that of the molten globule state of several acid-denatured proteins, suggesting that tetrameric melittin might be a state similar to the molten globule state. To test this possibility, we studied the thermal unfolding of tetrameric melittin using far-UV CD and differential scanning calorimetry. The latter technique revealed a broad but distinct heat absorption peak. The heat absorption curves were consistent with the unfolding transition observed by CD and were explainable by a 2-state transition mechanism between the tetrameric alpha-helical state and the monomeric unfolded state. From the peptide or salt-concentration dependence of unfolding, the heat capacity change upon unfolding was estimated to be 5 kJ (mol of tetramer)-1 K-1 at 30 degrees C and decreased with increasing temperature. The observed change in heat capacity was much smaller than that predicted from the crystallographic structure (9.2 kJ (mol of tetramer)-1 K-1), suggesting that the hydrophobic residues of tetrameric melittin in solution are exposed in comparison with the crystallographic structure. However, the results also indicate that the structure is more ordered than that of a typical molten globule state. We consider that the conformation is intermediate between the molten globule state and the native state of globular proteins.     

Reduction of the buried intrachain disulfide bond of the constant fragment of the immunoglobulin light chain: global unfolding under physiological conditions

The constant (CL) fragment of the immunoglobulin light chain contains only one intrachain disulfide bond buried in the interior of the molecule. The kinetics of reduction with dithiothreitol of the disulfide bond were studied at various concentrations of guanidine hydrochloride at pH 8.0 and 25 degrees C. It was found that the disulfide bond is reduced even in the absence of guanidine hydrochloride. The results of the reduction kinetics were compared with those of the unfolding and refolding kinetics of the CL fragment previously reported [Goto, Y., & Hamaguchi, K. (1982) J. Mol. Biol. 156, 891-910]. It was shown that the reduction of the disulfide bond proceeds through a species with a conformation very similar to that of the fully unfolded one and that the CL fragment undergoes global unfolding transition even in water.     

Fusion of phospholipid vesicles induced by an amphiphilic model peptide: close correlation between fusogenicity and hydrophobicity of the peptide in an alpha-helix.

A model peptide with 51 amino acid residues consisting of tandem repeats of a Lys-Lys-Leu-Leu sequence and a turn sequence of Asn-Pro-Gly at the center of the molecule has a random conformation at neutral pH but adopts an amphiphilic alpha-helical form in the presence of various salts or nucleotides [Goto, Y., & Aimoto, S. (1991) J. Mol. Biol. 218, 387-396; Goto, Y., Okamura, N., & Aimoto, S. (1991) J. Biochem. (Tokyo) 109, 746-750]. The interaction of this model peptide with liposome membranes and the resulting alpha-helical conformational transition and membrane fusion as well as the effect of the nucleotide ATP on these events were examined at neutral pH. The peptide associated stoichiometrically with liposome membranes composed of phosphatidylserine (PS) and phosphatidylcholine (PC) in a molar ratio of 2:1, resulting in formation of an amphiphilic alpha-helix and induction of fusion of the liposomes. However, the final fusion level was not correlated with the amount of binding or the helix content and was found to increase on an increase in hydrophobicity of the peptide in the alpha-helical form by neutralization of its positive charges by the negative charges of PS. In contrast, in the presence of ATP, the peptide bound completely to the PS/PC membranes at a lower concentration of liposome and concomitantly induced membrane fusion, indicating that ATP cooperates with PS to neutralize the charges of the peptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR studies on the monomer–tetramer transition of melittin in an aqueous solution at high and low temperatures

Melittin, a peptide of 26 amino acid residues, has been used as a model peptide for protein folding and unfolding, and extensive research has been done into its structure and conformational stability. Circular dichroism (CD) studies have demonstrated that melittin in an aqueous solution undergoes a transition from a helical tetramer to a random coil monomer not only by heating but also by cooling from room temperature (i.e., heat- and cold-denaturation, respectively). The heat-denaturation has been also examined by nuclear magnetic resonance (NMR) experiments, however, no NMR data have been presented on the cold-denaturation. In this paper, using proton ((1)H) NMR spectroscopy, we show that melittin undergoes conformational transitions from the monomer to the tetramer to the monomer by elevating temperature from 2 to 70 °C. Only melittin including a trans proline peptide bond participates in the transitions, whereas melittin including a cis proline one does not. The tetramer has maximum conformation stability at around 20 °C, and cooperativity of the heat-denaturation is extremely low.     

Two-dimensional 1H NMR study of d(br5C-G)3 in the Z-form. Self association and flexibility of the left-handed double helix

The Z conformation of the auto complementary hexanucleoside pentaphosphate d(br5C-G)3 in 1 M NaClO4 solution has been investigated by using 2D NMR techniques. NOESY experiments performed at different temperatures show that the oligonucleotide exhibits end-to-end associations at room temperature. The conformation of the hexanucleotide molecules is very similar to that found in the crystal which was described by Chevrier et al. (J. Mol. Biol., 1986, 188, 707-719) as a Z-I form. When the temperature is increased the aggregates are dissociated and a conformational change is observed which is interpreted as a Z-I in equilibrium Z-II transition.     

Irreversible Changes Occur in Chromatin Structure Upon Dissociation Of Histone H1

Abstract

The role of histone H1 in the actual interactions bringing about chromatin folding is investigated by studying the reversibility of its dissociation. HI was dissociated by increase of the NaCl concentration and reassociated by dialysis, without removal from the dialysis bag. To scrutinize the fidelity of this stoichiometric form of chromatin reconstitution, we use circular dichroism, nuclease digestion, thermal denaturation and the sensitive electric birefringence method. No alteration of the repeat length and no nucleosomal sliding are observed upon the reassociation procedure. However, under all the different conditions investigated, the original value of the positive electric birefringence is never recovered, indicating an irreversible change of structure. CD and melting profiles confirm that DNA-protein interactions are modified, and orientational relaxation time measurements indicate that these structural perturbations affect the salt- induced transition of poly nucleosomal fibers. The striking conclusion of these studies is that variations of ionic concentration are sufficient to induce irreversible structural alterations affecting the higher-order folding of chromatin. It is of interest that the only sample which exhibits behavior upon reassociation comparable to that of native chromatin is the one which experienced the fastest salt transitions. We suggest that these conformational changes arise from the unbinding to DNA of certain basic tails of histone(s), and that a competition for DNA binding locations exists upon the reassociation. These results are then additional arguments (Mazen, A., Hacques, M.F. and Marion, C., J. Mol. Biol. 194, 741–745 (1987)), to suggest that dissociation of H1 might modify a direct interaction between basic tails of core histones and H1.     

Influence of NaCl and sorbitol on the stability of conformations of cytochrome c

Influence of ionic (NaCl) and non-ionic (sorbitol) additives on structural transitions of cytochrome c was investigated by circular dichroism, optical and EPR spectroscopy. Transformations of cytochrome c, induced by the acidification of solution and temperature perturbation, were monitored in the heme pocket together with changes in the secondary structure. NaCl and sorbitol exhibited antagonistic effect on the acid-induced transition of the protein. Sorbitol enhanced the stability of native conformation while NaCl destabilized this state. The midpoints of acid-induced transitions in the axial coordination of heme as well as in the secondary structure occurred nearly at the same pH values. However, temperature-induced transitions in the unfolding of the secondary structure were almost coincidental with the cleavage of Met80–Fe bond only in the sorbitol solutions. In the salt solution the Met80–Fe bond was markedly more labile than the secondary structure.     

A two-process model describes the hydrogen exchange behavior of cytochrome c in the molten globule state with various extents of acetylation

Acetylation of Lys residues of horse cytochrome c steadily stabilizes the molten globule state in 18 mM HCl as more Lys residues are acetylated [Goto and Nishikiori (1991) J. Mol. Biol. 222, 679-686]. The dynamic features of the molten globule state were characterized by hydrogen/deuterium exchange of amide protons, monitored by mass spectrometry as each deuteration increased the protein mass by 1 Da. Electrospray mass spectrometry enabled us to monitor simultaneously the exchange kinetics of more than seven species with a different number of acetyl groups. One to four Lys residue-acetylated cytochrome c showed almost no protection of the amide protons from rapid exchange. The transition from the unprotected to the protected state occurred between five and eight Lys residue-acetylated species. For species with more than nine acetylated Lys residues, the exchange kinetics were independent of the extent of acetylation, and 26 amide protons were protected at 60 min of exchange, indicating the formation of a rigid hydrophobic core with hydrogen-bonded secondary structures. The apparent transition to the protected state required a higher degree of acetylation than the conformational transition measured by circular dichroism, which had a midpoint at about four acetylated residues. This difference in the transitions suggested a two-process model in which the exchange occurs either from the protected folded state or from the unprotected unfolded state through global unfolding. On the basis of a two-process model and with the reported values of the exchange and stability parameters, we simulated the exchange kinetics of a series of acetylated cytochrome c species. The simulated kinetics reproduced the observed kinetics well, indicating validity of this model for hydrogen exchange of the molten globule state.     

Acid- and base-induced conformational transitions of equinatoxin II

We have investigated the acid- and base-induced conformational transitions of equinatoxin II (EqTxII), a pore-forming protein, by a combination of CD-spectroscopy, ultrasonic velocimetry, high precision densimetry, viscometry, gel electrophoresis, and hemolytic activity assays. Between pH 7 and 2, EqTxII does not exhibit any significant structural changes. Below pH 2, EqTxII undergoes a native-to-partially unfolded transition with a concomitant loss of its rigid tertiary structure and the formation of a non-native secondary structure containing additional alpha-helix. The acid-induced denatured state of EqTxII exhibits a higher intrinsic viscosity and a lower adiabatic compressibility than the native state. Above 50 degrees C, the acid-induced denatured state of EqTxII reversibly denatures to a more unfolded state as judged by the far UV CD spectrum of the protein. At alkaline pH, EqTxII undergoes two base-induced conformational transitions. The first transition occurs between pH 7 and 10 and results in a partial disruption of tertiary structure, while the secondary structure remains largely preserved. The second transition occurs between pH II and 13 and results in the complete loss of tertiary structure and the formation of a non-native, more alpha-helical secondary structure. The acid- and base-induced partially unfolded states of EqTxII form water-soluble oligomers at low salt, while at high salt (> 350 mM NaCl), the acid-induced denatured state precipitates. The hemolytic activity assay shows that the acid- and base-induced denatured states of EqTxII exhibit significantly reduced activity compared to the native state.     

Acid titrations of poly(dG-dC).poly(dG-dC) in aqueous solution and in a w/o microemulsion

The model polynucleotide poly(dG-dC).poly(dG-dC) (polyGC) was titrated with a strong acid (HCl) in aqueous unbuffered solutions and in the quaternary w/o microemulsion CTAB/n-pentanol/n-hexane/water. The titrations, performed at several concentrations of NaCl in the range 0.005 to 0.600 M, were followed by recording the modifications of the electronic absorption and of the CD spectra (210< or = lambda < or =350 nm) upon addition of the acid. In solution, the polynucleotide undergoes two acid-induced transitions, neither of which corresponds to denaturation of the duplex to single coil. The first transition leads to the Hoogsteen type synG.C+ duplex, while the second leads to the C+.C duplex. The initial B-form of polyGC was recovered by back-titration with NaOH. The apparent pKa values were obtained for both steps of the titration, at all salt concentrations. A reasonably linear dependence of pKa1 and pKa2 from p[NaCl] was obtained, with both pKa values decreasing with increasing ionic strength. In microemulsion, at salt concentrations < or = 0.300 M, an acid-induced transition was observed, matching the first conformational transition recorded also in solution. However, further addition of acid led to denaturation of the protonated duplex. Renaturation of polyGC was obtained by back-titration with NaOH. At salt concentrations > 0.300 M, polyGC is present as a mixture of B-form and psi- aggregates, that slowly separate from the microemulsion. The acid titration induces at first a conformational transition similar to the one observed at low salt or in solution, then denaturation occurs, which is however preceded by the appearance of a transient conformation, that has been tentatively classified as a left-handed Z double helix.     

Phase diagram for acidic conformational states of apomyoglobin

The conformation of apomyoglobin under acidic conditions depends on both pH and salt conditions. Using KCl and HCl, a phase diagram for the pH and ionic strength-dependent conformational states was constructed based on the change in the far-ultraviolet circular dichroism. Acid-denatured apomyoglobin consists of two conformational states, the largely unfolded state (UA) and the relatively compact, acidic intermediate state (A). The UA state is stable below pH 3 and at low ionic strength. High ionic strength favors the A state. Because the minimal ionic strength increases significantly with a decrease in pH below 2, the UA state is limited to only a small region between pH 1 and 3.     

Mechanism of anion-cation selectivity of amphotericin B channels

Summary Zero current potential and conductance of ionic channels formed by polyene antibiotic amphotericin B in a lipid bilayer were studied in various electrolyte solutions. Nonpermeant magnesium and sulphate ions were used to independently vary the concentration of monovalent anions and cations as well as to maintain the high ionic strength of the two solutions separated by the membrane. Under certain conditions the channels select very strongly for anions over cations. They are permeable to small inorganic anions. However, in the absence of these anions the channels are practically impermeable to any cation. In the presence of a permeant anion the contribution of monovalent cations to channel conductance grows with an increase in the anion concentration. The ratio of cation-to-anion permeability coefficients is independent of the membrane potential and cation concentration, but it does depend linearly on the sum of concentrations of a permeant anion in the two solutions. These results are accounted for on the assumption that a cation can enter only an anion-occupied channel to form an ionic pair at the center of the channel. The cation is also assumed to slip past the anion and then to leave the channel for the opposite solution. This model with only few parameters can quantitatively describe the concentration dependences of conductance and zero current potential under various conditions.     

Nucleosomes undergo slow spontaneous gaping

In eukaryotes, DNA is packaged into a basic unit, the nucleosome which consists of 147 bp of DNA wrapped around a histone octamer composed of two copies each of the histones H2A, H2B, H3 and H4. Nucleosome structures are diverse not only by histone variants, histone modifications, histone composition but also through accommodating different conformational states such as DNA breathing and dimer splitting. Variation in nucleosome structures allows it to perform a variety of cellular functions. Here, we identified a novel spontaneous conformational switching of nucleosomes under physiological conditions using single-molecule FRET. Using FRET probes placed at various positions on the nucleosomal DNA to monitor conformation of the nucleosome over a long period of time (30–60 min) at various ionic conditions, we identified conformational changes we refer to as nucleosome gaping. Gaping transitions are distinct from nucleosome breathing, sliding or tightening. Gaping modes switch along the direction normal to the DNA plane through about 5–10 angstroms and at minutes (1–10 min) time scale. This conformational transition, which has not been observed previously, may be potentially important for enzymatic reactions/transactions on nucleosomal substrate and the formation of multiple compression forms of chromatin fibers.     

Concentration-dependent effects of anions on the anaerobic oxidation of hemoglobin and myoglobin

The redox potentials of hemoglobin and myoglobin and the shapes of their anaerobic oxidation curves are sensitive indicators of globin alterations surrounding the active site. This report documents concentration-dependent effects of anions on the ease of anaerobic oxidation of representative hemoglobins and myoglobins. Hemoglobin (Hb) oxidation curves reflect the cooperative transition from the T state of deoxyHb to the more readily oxidized R-like conformation of metHb. Shifts in the oxidation curves for Hb A(0) as Cl(-) concentrations are increased to 0.2 m at pH 7.1 indicate preferential anion binding to the T state and destabilization of the R-like state of metHb, leading to reduced cooperativity in the oxidation process. A dramatic reversal of trend occurs above 0.2 m Cl(-) as anions bind to lower affinity sites and shift the conformational equilibrium toward the R state. This pattern has been observed for various hemoglobins with a variety of small anions. Steric rather than electronic effects are invoked to explain the fact that no comparable reversal of oxygen affinity is observed under identical conditions. Evidence is presented to show that increases in hydrophilicity in the distal heme pocket can decrease oxygen affinity via steric hindrance effects while increasing the ease of anaerobic oxidation.     

Physicochemical studies of the protein-lipid interactions in melittin-containing micelles

Complexes of melittin with detergents and phospholipids have been characterized by fluorescence, circular dichroism, ultracentrifugation, quasi-elastic light scattering and 1H nuclear magnetic resonance (NMR) experiments. By ultracentrifugation and quasi-elastic light-scattering measurements it is shown that melittin forms stoichiometrically well-defined complexes with dodecylphosphocholine micelles consisting of one melittin molecule and approximately forty detergent molecules. Evidence from fluorescence, circular dichroism and 1H nuclear magnetic resonance experiments indicates that the conformation of melittin bound to micelles of various detergents or of diheptanoyl phosphatidylcholine is largely independent of the type of lipid and furthermore appears to be quite closely related to the conformation of melittin bound to phosphatidylcholine bilayers. 1H NMR is used to investigate the conformation of micelle-bound melittin in more detail and to compare certain aspects of the melittin conformation in the micelles with the spatial structures of monomeric and self-aggregated tetrameric melittin in aqueous solution. The experience gained with this system demonstrates that high resolution NMR of complexes of membrane proteins with micelles provides a viable method for conformational studies of membrane proteins.     

The acid-induced folded state of Sac7d is the native state

Sac7d unfolds at low pH in the absence of salt, with the greatest extent of unfolding obtained at pH 2. We have previously shown that the acid unfolded protein is induced to refold by decreasing the pH to 0 or by addition of salt (McCrary BS, Bedell J. Edmondson SP, Shriver JW, 1998, J Mol Biol 276:203-224). Both near-ultraviolet circular dichroism spectra and ANS fluorescence enhancements indicate that the acid- and salt-induced folded states have a native fold and are not molten globular. 1H,15N heteronuclear single quantum coherence NMR spectra confirm that the native, acid-, and salt-induced folded states are essentially identical. The most significant differences in amide 1H and 15N chemical shifts are attributed to hydrogen bonding to titrating carboxyl side chains and through-bond inductive effects. The 1H NMR chemical shifts of protons affected by ring currents in the hydrophobic core of the acid- and salt-induced folded states are identical to those observed in the native. The radius of gyration of the acid-induced folded state at pH 0 is shown to be identical to that of the native state at pH 7 by small angle X-ray scattering. We conclude that acid-induced collapse of Sac7d does not lead to a molten globule but proceeds directly to the native state. The folding of Sac7d as a function of pH and anion concentration is summarized with a phase diagram that is similar to those observed for other proteins that undergo acid-induced folding except that the A-state is encompassed by the native state. These results demonstrate that formation of a molten globule is not a general property of proteins that are refolded by acid.     

The temperature dependence of human erythrocyte transport of phosphate, phosphite and hypophosphite

The temperature dependence of the erythrocyte anion transport protein (Band 3 or AE1) mediated influx of three nonspherical substrates, the divalent anions phosphate and phosphite, and the monovalent hypophoshite, were determined. Phase transitions were found in the temperature dependence of the influxes of all three anions. The 95% confidence limits for the transition temperatures were: 34.6-38.1 degrees C, 7.4-9.1 degrees C and 6.7-9.7 degrees C for phosphate, phosphite and hypophosphite, respectively, while the critical influx rates at the transitions were 29-50, 64-102 and 26-58 ions/s per carrier, respectively. That the critical rates rather than the transition temperatures are of similar magnitude indicates that the transitions are related to transport mechanisms rather than to thermal protein conformational changes. These critical rates are two orders of magnitude lower than those reported for the self-exchange of Cl- and Br- (Brahm, J. (1977) J. Gen. Physiol. 70, 283-306). The critical rate of monovalent hypophosphite is similar to that of divalent phosphate and phosphite, but not to that of Cl- indicating that this effect is mediated by the structure of the substrate rather than by its charge. The disparity in the rates rc at which phase transitions occur in AE1-mediated transport of spherical and nonspherical anions indicates a difference in the interaction between the two classes of anions and the protein.