Conformational studies on the gramicidin A transmembrane channel in lipid micelles and liposomes

The interaction of gramicidin A with lysolecithin micelles and with lecithin liposomes is demonstrated by circular dichroism to result in several metastable conformational states. A stable state can be obtained after extensive heating when the gramicidin A was added dry or in ethanol solution to the phospholipid dispersion but the stable state is readily obtained when gramicidin A is added in a trifluoroethanol solution. The circular dichroism of the stable conformational state is characterized by negative ellipticity below 205 nm and principally by a positive 220 nm band on which is superposed a weak 230 nm band (the latter likely arising from tryptophan side chains). The stable conformational state is considered to be that of the functional transmembrane channel primarily on the basis of extensive studies on its interaction with sodium ions.     

Conformation of gramicidin A in Triton X‐100 micelles from CD and FTIR data: a clean example of antiparallel double β5.6 helix formation

The linear peptide gramicidin A (gA) forms prototypical ion channels specific for monovalent cations and has been extensively used to study the organization and dynamics of membrane channels. This polymorphic peptide can adopt two different types of structures, the helical dimer β6.3 (‘channel state’) and the double helical structure with two intertwined monomers. The structure of gA in micelles of detergent Triton X‐100 has been studied using CD, Fourier transform infrared, and fluorescence spectroscopy. The results obtained demonstrate that only one thermodynamically stable gA structure, the antiparallel left‐handed double helix β5.6, is formed in this membrane‐mimetic environment. The position of the tryptophan fluorescence maximum at 332 nm is the same as that in phospholipid membranes. The causative factors governing the double helix formation in the micellar medium are discussed on the basis of known physicochemical properties of Triton X‐100. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Colorimetric assay of gramicidin A in the presence of surfactants and phospholipids

Gramicidin A, a hydrophobic polypeptide containing 4 tryptophan residues/molecule, may be determined quantitatively after reaction with 4-(dimethylamino)benzaldehyde, a method previously proposed for tryptophan analysis. The assay may be carried out even in the presence of various surfactants and phospholipids at high concentrations.     

Dimer-monomer conformational equilibrium of gramicidin A in 1-alkanols as studied by h.p.l.c. and fluorescence spectroscopy

High performance size-exclusion chromatograph (HPSEC) has been successfully used to characterized the dimer-monomer conformational equilibrium of gramicidin A (GA) in a series of 1-alkanols, from methanol to 1-pentanol. The chromatographic methodology proposed has allowed a rapid and accurate determination of kinetic and thermodynamic constants in the different alcohols assayed. The validity of this chromatographic approach has been tested by comparing the values of the kinetic and thermodynamic constants obtained with those reported in the literature deduced from spectroscopic techniques. Taking advantage of the chromatographic results, the differential fluorescent features of the monomer relative to the dimer have been investigated in terms of the quantum yields ratio of the individual species, as a function of solvent polarity. Finally, the possibility of applying this HPSEC approach to the study of other auto-associating polypeptides and of GA incorporated in liposomes is also considered.     

The involvement of the lipid phase transition in the plasma-induced dissolution of multilamellar phosphatidylcholine vesicles

Unsonicated liposomes prepared from dimyristoyl phosphatidylcholine were nearly completely dissolved during a 3 h incubation with rat plasma at or close to the phase-transition temperature of 24°C. At 37 or 15°C virtually no liposomal disintegration was observed even after 24 h of incubation. The liposomal solubilization, which was monitored by turbidity measurements or by determination of phospholipid sedimentability, was accompanied by the formation of a phospholipid-protein complex similar or identical to the one we previously reported to be formed from sonicated liposomes of egg phosphatidylcholine (Scherphof, G., Roerdink, F., Waite, M. and Parks, J. (1978) Biochim. Biophys. Acta 542, 296–307). Unsonicated multilamellar liposomes made of egg phosphatidylcholine were completely resistant to the dissolving potency of plasma when incubated at 37°C. Liposomes from equimolar mixtures of dimyristoyl and dipalmitoyl phosphatidylcholine were only degraded by plasma in the temperature range between 30 and 35°C at which temperature this cocrystallizing phospholipid mixture undergoes a phase transition. However, even at these temperatures the rate of dissolution of this mixture was significantly lower than of dimyristoyl phosphatidylcholine at 24°C. In the dissolving process of this mixture a slight preference for the lower-melting component was observed.The ability of cholesterol to completely abolish the susceptibility of dimyristoyl phosphatidylcholine liposomes to plasma at a 1:2 molar ratio of cholesterol to phospholipid substantiates the essential role of the phase transition in the process of liposome solubilization.When liposomes of the monotectic mixtures dimyristoyl and distearoyl phosphatidylcholine or dilauroyl and distearoyl phosphatidylcholine were incubated with plasma at temperatures in between those at which the constituent lipids undergo a phase change in the mixture, the liposomes were slowly disolved. Under those conditions a selective removal of the lipids in the liquid-crystalline phase was observed.It is concluded that for the plasma-induced dissolution of unsonicated liposomes, which is most probably achieved by interaction with (apo)lipoproteins, the presence of phase boundaries is required in much the same way as was first reported for phospholipases by Op den Kamp, J.A.F., de Gier, J. and Van Deenen, L.L.M. (1974) Biochim. Biophys. Acta 345, 253–256).     

Gel formation and low-temperature intramolecular conformation transition of a triple-helical polysaccharide lentinan in water

The gelation behavior of the triple-helical polysaccharide lentinan fractions having different molecular weights in water at 25 degrees C were studied by using a rheometer. The analysis of concentration and molecular weight dependence of shear stress and shear viscosity showed that aqueous lentinan is a typical shear-thinning fluid, possessing potential as a viscosity control agent, and that a weak gel with entangled network structure formed. The dynamic oscillatory behavior of lentinan in the temperature range of 1-15 degrees C was also investigated by rheologic method. The storage modulus G' and complex viscosity eta* increased first with decreasing temperature, and underwent a maximum centered at 7-9 degrees C, and then decreased with further decreasing temperature. This abnormal phenomenon was ascribed to formation of rigid structure in the gel state, which was confirmed by the experimental results from micro-DSC. The micro-DSC curves showed that an endothermic peak appeared at 7-9 degrees C for lentinan in water upon heating, which was attributable to the intramolecular order-disorder structure transition similar to triple-helical polysaccharide schizophyllan. Namely, at lower temperature, the side glucose residues of lentinan (triplix II) formed a well-organized triple-helical structure (triplix I) through hydrogen-bonding with the surrounding water molecules. Moreover, this conformation transition was proved to be thermally reversible. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 852-861, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Large scale conformational transitions in β‐structural motif of gramicidin A: kinetic analysis based on CD and FT‐IR data

Gramicidin A (gA) is a polypeptide antibiotic, which forms dimeric channels specific for monovalent cations in artificial and biological membranes. It is a polymorphic molecule that adopts a unique variety of helical conformations, including antiparallel double‐stranded ↑↓β5.6 or ↑↓β7.2 helices (number of residues per turn) and a single‐stranded β6.3 helix (the ‘channel form’). The behavior of gA‐Cs⁺ complex in the micelles of TX‐100 was studied in this work. Transfer of the complex into the micelles activates a cascade of sequential conformational transitions monitored by CD and FT‐IR spectroscopy: At the first step after Cs⁺ removal, the RH ↑↓β5.6 helix is formed, which has been discussed so far only hypothetically. Kinetics of the transitions was measured, and the activation parameters were determined. The activation energies of the ↑↓β5.6 → β‐helical monomer transition in dioxane and dioxane/water solutions were also measured for comparison. The presence of water raises the transition rate constant ~10³ times but does not lead to crucial fall of the activation energy. All activation energies were found in the 20–25 kcal/mol range, i.e. much lower than would be expected for unwinding of the double helix (when 28 H‐bonds are broken simultaneously). These results can be accounted for in the light of local unfolding (or ‘cracking’) model for large scale conformational transitions developed by the P. G.Wolynes team [Miyashita O, Onuchic JN, Wolynes PG. Proc. Natl. Acad. Sci. USA 2003; 100: 12570‐12575.]. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

The contribution of the residues from the main hydrophobic core of ribonuclease A to its pressure-folding transition state

The role of hydrophobic interactions established by the residues that belong to the main hydrophobic core of ribonuclease A in its pressure-folding transition state was investigated using the Phi-value method. The folding kinetics was studied using pressure-jump techniques both in the pressurization and depressurization directions. The ratio between the folding activation volume and the reaction volume (beta p-value), which is an index of the compactness or degree of solvation of the transition state, was calculated. All the positions analyzed presented fractional Phi f-values, and the lowest were those corresponding to the most critical positions for the ribonuclease A stability. The structure of the transition state of the hydrophobic core of ribonuclease A, from the point of view of formed interactions, is a relatively, uniformly expanded form of the folded structure with a mean Phi f-value of 0.43. This places it halfway between the folded and unfolded states. On the other hand, for the variants, the average of beta p-values is 0.4, suggesting a transition state that is 40% native-like. Altogether the results suggest that the pressure-folding transition state of ribonuclease A looks like a collapsed globule with some secondary structure and a weakened hydrophobic core. A good correlation was found between the Phi f-values and the Deltabeta p-values. Although the nature of the transition state inferred from pressure-induced folding studies and the results of the protein engineering method have been reported to be consistent for other proteins, to the best of our knowledge this is the first direct comparison using a set of mutants.     

Involvement of disulfide bonds and histidine 172 in a unique beta-sheet to alpha-helix transition of alpha 1-acid glycoprotein at the biomembrane interface

Human alpha(1)-acid glycoprotein (AGP), which is comprised of 183 amino acid residues and 5 carbohydrate chains, is a major plasma protein that binds to basic and neutral drugs as well as to steroid hormones. It has a beta-sheet-rich structure in aqueous solution. Our previous findings suggest that AGP forms an alpha-helix structure through an interaction with biomembranes. We report herein on a study of the mechanism of alpha-helix formation in AGP using various modified AGPs. The disulfide reduced AGP (R-AGP) was extensively unfolded, whereas asialylated AGP (A-AGP) maintained the native structure. Intriguingly, reduced and asialylated AGP (RA-AGP) increased the alpha-helix content as observed in the presence of biomembrane models, and showed a significant decrease in ligand binding capacity. This suggests that AGP has an innate tendency to form an alpha-helix structure, and disulfide bonds are a key factor in the conformational transition between the beta-sheet and alpha-helix structures. However, RA-AGP with all histidine residues chemically modified (HRA-AGP) was found to lose the intrinsic ability to form an alpha-helix structure. Furthermore, disulfide reduction of the H172A mutant expressed in Pichia pastoris also caused a similar loss of folding ability. The present results indicate that disulfide bonds and the C-terminal region, including H172 of AGP, play important roles in alpha-helix formation in the interaction of the protein with biomembranes.     

Polyelectrolytic aspects of the thermodynamics of conformational transition: κ‐carrageenan in formamide

A recently published method for the determination of the enthalpy and entropy changes of nonionic origin upon conformational transition of linear biopolyelectrolytes in solution [J. C. Benegas, A. Cesàro, R. Rizzo and S. Paoletti (1998) Biopolymers, Vol. 45, pp. 203–216] has been extended from the case of aqueous salt solutions to that of the organic solvent formamide (FA). The calculation have been applied to the case of the intramolecular transition of the K⁺ salt form of the sulfated polysaccharide κ‐carrageenan. The method proved to be effective in providing the desired data in FA, as it has been previously been successful for the water cases. The comparison between the predicted enthalpy change of transition and the microcalorimetric experimental one turned out to be excellent, thereby ensuring on the validity of the approach. © 1999 John Wiley & Sons, Inc. Biopoly 49: 127–130, 1999     

Modulating repeat protein stability: the effect of individual helix stability on the collective behavior of the ensemble

Repeat proteins are tandem arrays of a small structural motif, in which tertiary structure is stabilized by interactions within a repeat and between neighboring repeats. Several studies have shown that this modular structure is manifest in modular thermodynamic properties. Specifically, the global stability of a repeat protein can be described by simple linear models, considering only two parameters: the stability of the individual repeated units (H) and the coupling interaction between the units (J). If the repeat units are identical, single values of H and J, together with the number of repeated units, is sufficient to completely describe the thermodynamic behavior of any protein within a series. In this work, we demonstrate how the global stability of a repeat protein can be changed, in a predictable fashion, by modifying only the H parameter. Taking a previously characterized series of consensus tetratricopeptide repeats (TPR) (CTPRa) proteins, we introduced mutations into the basic repeating unit, such that the stability of the individual repeat unit was increased, but its interaction with neighboring units was unchanged. In other words, we increased H but kept J constant. We demonstrated that the denaturation curves for a series of such repeat proteins can be fit and additional curves can be predicted by the one-dimensional Ising model in which only H has changed from the original fit for the CTPRa series. Our results show that we can significantly increase the stability of a repeat protein by rationally increasing the stability of the units (H), whereas the interaction between repeats (J) remains unchanged.     

Temperature-jump and voltage-jump experiments at planar lipid membranes support an aggregational (micellar) model of the gramicidin A ion channel

Summary The kinetics of formation and dissociation of channels formed by gramicidin A and two analogues in planar lipid membranes was studied using a laser temperature-jump technique developed earlier [Brock, W., Stark, G., Jordan, P.C. (1981),Biophys. Chem. 13:329–348]. The time course of the electric current was found to agree with a single exponential term plus a linear drift. In case of gramicidin A the relaxation time was identical to that reported for V-jump experiments [Bamberg, E., Läuger, P. (1973),J. Membrane Biol. 11:177–194], which were interpreted on the basis of a dimerization reaction. The same results were obtained for gramicidin A and for chemically dimerized malonyl-bis-desformylgramicidin. It is therefore suggested that the dimerization represents a parallel association of two dimers to a tetramer. There is evidence that the tetramer, contrary to the presently favored dimer hypothesis, is the smallest conductance unit of an active gramicidin channel. An additional V-jump-induced relaxation process of considerably larger time constant is interpreted as a further aggregation of gramicidin dimers.Abbreviations GA gramicidin A - OPG O-pyromellitylgramicidin A - MBDG malonyl-bis-desformylgramicidin     

The compact and expanded denatured conformations of apomyoglobin in the methanol-water solvent

We have performed a detailed study of methanol-induced conformational transitions of horse heart apomyoglobin (apoMb) to investigate the existence of the compact and expanded denatured states. A combination of far- and near-ultraviolet circular dichroism, NMR spectroscopy, and small-angle X-ray scattering (SAXS) was used, allowing a phase diagram to be constructed as a function of pH and the methanol concentration. The phase diagram contains four conformational states, the native (N), acid-denatured (U(A)), compact denatured (I(M)), and expanded helical denatured (H) states, and indicates that the compact denatured state (I(M)) is stable under relatively mild denaturing conditions, whereas the expanded denatured states (U(A) and H) are realized under extreme conditions of pH (strong electric repulsion) or alcohol concentration (weak hydrophobic interaction). The results of this study, together with many previous studies in the literature, indicate the general existence of the compact denatured states not only in the salt-pH plane but also in the alcohol-pH plane. Furthermore, to determine the general feature of the H conformation we used several proteins including ubiquitin, ribonuclease A, alpha-lactalbumin, beta-lactoglobulin, and Streptomyces subtilisin inhibitor (SSI) in addition to apoMb. SAXS studies of these proteins in 60% methanol showed that the H states of these all proteins have expanded and nonglobular conformations. The qualitative agreement of the experimental data with computer-simulated Kratky profiles also supports this structural feature of the H state.     

Orientational constraints as three-dimensional structural constraints from chemical shift anisotropy: the polypeptide backbone of gramicidin A in a lipid bilayer.

Chemical shifts observed from samples that are uniformly aligned with respect to the magnetic field can be used as very high-resolution structural constraints. This constraint takes the form of an orientational constraint rather than the more familiar distance constraint. The accuracy of these constraints is dependent upon the quality of the tensor characterization. Both tensor element magnitudes and tensor orientations with respect to the molecular frame need to be considered. Here these constraints have been used to evaluate models for the channel conformation of gramicidin A. Of the three models used, the one experimentally derived model of gramicidin in sodium dodecyl sulfate micelles fits the data least well.     

Two peptide fragments G55-I72 and K97-A109 from staphylococcal nuclease exhibit different behaviors in conformational preferences for helix formation

Two synthetic peptides, SNasealpha1 and SNasealpha2, corresponding to residues G55-I72 and K97-A109, respectively, of staphylococcal nuclease (SNase), are adopted for detecting the role of helix alpha1 (E57-A69) and helix alpha2 (M98-Q106) in the initiation of folding of SNase. The helix-forming tendencies of the two SNase peptide fragments are investigated using circular dichroism (CD) and two-dimensional (2D) nuclear magnetic resonance (NMR) methods in water and 40% trifluoroethanol (TFE) solutions. The coil-helix conformational transitions of the two peptides in the TFE-H2O mixture are different from each other. SNasealpha1 adopts a low population of localized helical conformation in water, and shows a gradual transition to helical conformation with increasing concentrations of TFE. SNasealpha2 is essentially unstructured in water, but undergoes a cooperative transition to a predominantly helical conformation at high TFE concentrations. Using the NMR data obtained in the presence of 40% TFE, an ensemble of alpha-helical structures has been calculated for both peptides in the absence of tertiary interactions. Analysis of all the experimental data available indicates that formation of ordered alpha-helical structures in the segments E57-A69 and M98-Q106 of SNase may require nonlocal interactions through transient contact with hydrophobic residues in other parts of the protein to stabilize the helical conformations in the folding. The folding of helix alpha1 is supposed to be effective in initiating protein folding. The formation of helix alpha2 depends strongly on the hydrophobic environment created in the protein folding, and is more important in the stabilization of the tertiary conformation of SNase.     

Effect of water potential on sol-gel transition and intermolecular interaction of gelatin near the transition temperature

The sol-gel transition of gelatin, measured by thermal analysis and viscosity measurement, was analyzed in terms of the change in hydration state of polymer molecules. A new thermodynamic model was proposed in which the effect of water potential is explicitly taken into account for the evaluation of the free energy change in the sol-gel transition process. Because of the large number of water molecules involved and the small free energy change in the transition process, the contribution of water activity, a(W), was proved to be not negligible in the sol-gel transition process in solutions containing such low-molecular cosolutes as sugars, glycerol, urea, and formamide. The gel-stabilization effect of sugars and glycerol was linear with a(W), which seemed consistent with the contribution of water potential in the proposed model. The different stabilization effect among sugars and glycerol was explained by the difference in solvent ordering, which affects hydrophobic interaction among protein molecules. The gel-destabilization effect of urea and formamide could be explained only by the direct binding of them to protein molecules through hydrogen bonding. On the contrary, the polymer-polymer interaction, measured by the viscosity analysis, in polyethyleneglycol and dextran solutions was not sensitive to the change in a(W), suggesting that no substantial change in hydration state with a(W) occurred in these polymer solutions.     

分子动力学模拟尿素对水溶液中蛋白质构象转变的影响

采用分子动力学方法和全原子模型研究尿素和水分子对模型蛋白S-肽链结构转化的影响。模拟结果显示S-肽链的变性速率常数k值随着尿素浓度的增加而先降低后升高,在尿素浓度为2.9 mol/L时达到最低值。模拟了不同尿素浓度下尿素-肽链、水-肽链以及肽链分子氢键的形成状况。结果表明:尿素浓度较低时,尿素分子与S-肽链的极性氨基酸侧链形成氢键,但不破坏其分子内的骨架氢键,尿素在S-肽链水化层外形成限制性空间,增强了S-肽链的稳定性。随着尿素的升高,尿素分子进入S-肽链内部并与其内部氨基酸残基形成氢键,导致S-肽链的骨架氢键丧失,S-肽链发生去折叠。上述模拟结果与文献报道的实验结果一致,从分子水平上揭示了尿素对蛋白质分子结构变化的影响机制,对于研究和发展蛋白质折叠及稳定化技术具有指导意义。     

Temperature dependence of single channel currents and the peptide libration mechanism for ion transport through the gramicidin A transmembrane channel

Summary A study of the temperature dependence of gramicidin A conductance of K⁺ in diphytanoyllecithin/n-decane membranes shows the plot of In (single channel conductance) as a function of reciprocal temperature to be nonlinear for the most probable set of conductance, states. These results are considered in terms of a series of barriers, of the dynamics of channel conformation,vis-a-vis the peptide libration mechanism, and of the effect of lipid viscosity on side chain motions again as affecting the energetics of peptide libration.     

Protein conformational transitions coupled to binding in molecular recognition of unstructured proteins: deciphering the effect of intermolecular interactions on computational structure prediction of the p27Kip1 protein bound to the cyclin A-cyclin-dependent kinase 2 complex

The relationship between folding mechanism coupled to binding and structure prediction of the tertiary complexes is studied for the p27(Kip) (1) protein which has an intrinsically disordered unbound form and undergoes a functional folding transition during complex formation with the phosphorylated cyclin A-cyclin-dependent kinase 2 (Cdk2) binary complex. Hierarchy of p27(Kip1) structural loss determined in our earlier studies from temperature-induced Monte Carlo simulations and subsequent characterization of the transition state ensemble (TSE) for the folding reaction have shown that simultaneous ordering of the p27(Kip1) native intermolecular interface for the beta-hairpin and beta-strand secondary structure elements is critical for nucleating a rapid kinetic transition to the native tertiary complex. In the present study, we investigate the effect of forming specific intermolecular interactions on structure prediction of the p27(Kip1) tertiary complex. By constraining different secondary structure elements of p27(Kip1) in their native bound conformations and conducting multiple simulated annealing simulations, we analyze differences in the success rate of predicting the native structure of p27(Kip1) in the tertiary complex. In accordance with the nucleation-condensation mechanism, we have found that further stabilization of the native intermolecular interface for the beta-hairpin and beta-strand elements of p27(Kip1), that become ordered in the TSE, but are hardly populated in the unbound state, results in a consistent acquisition of the native bound structure. Conversely, the excessive stablization of the local secondary structure elements, which are rarely detected in the TSE, has a detrimental effect on convergence to the native bound structure.