Polar Interior Research Articles

Site-Specific Hydration Status of an Amphipathic Peptide in AOT Reverse Micelles

Reverse micelles formed by sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in isooctane (IO) and water have long been used as a means to provide a confined aqueous environment for various applications. In particular, AOT reverse micelles have often been used as a template to mimic membrane-water interfaces. While earlier studies have shown that membrane-binding peptides can indeed be incorporated into the polar cavity of AOT reverse micelles where they mostly fold into an alpha-helical structure, the underlying interactions leading to the ordered conformation are however not well understood. Herein, we have used circular dichroism (CD) and infrared (IR) spectroscopies in conjunction with a local IR marker (i.e., the CN group of a non-natural amino acid, p-cyano-phenylalanine) and a global IR reporter (i.e., the amide I' band of the peptide backbone) to probe the conformation as well as the hydration status of an antimicrobial peptide, mastoparan x (MPx), in AOT reverse micelles of different water contents. Our results show that at, w0=6, MPx adopts an alpha-helical conformation with both the backbone and hydrophobic side chains mostly dehydrated, whereas its backbone becomes partially hydrated at w0=20. In addition, our results suggest that the amphipathic alpha-helix so formed orients itself in such a manner that its positively charged, lysine-rich, hydrophilic face points toward the negatively charged AOT head groups, while its hydrophobic face is directed toward the polar interior of the water pool. This picture is in marked contrast to that observed for the binding of MPx to phospholipid bilayers wherein the hydrophobic surface of the bound alpha-helix is buried deeper into the membrane interior.

Kinetics of the pH‐independent hydrolyses of 4‐nitrophenyl chloroformate and 4‐nitrophenyl heptafluorobutyrate in water‐acetonitrile mixtures: consequences of solvent composition and ester hydrophobicity

AbstractThe pH‐independent hydrolyses of 4‐nitrophenyl chloroformate, NPCF and 4‐nitrophenyl heptafluorobutyrate, NPFB in aqueous acetonitrile were studied spectrophotometrically from 15 to 45 °C. The binary solvent composition covers water concentrations from 2.349 to 53.207 and from 2.745 to 53.333 mol L−1 for NPCF and NPFB, respectively. For both esters, the dependence of log (kobs), the observed rate constant, on log [water] is sigmoidal. The approximate kinetic orders with respect to water were found to be 2 and 3 for NPCF and NPFB, respectively. ΔG≠ gradually decreases as a function of increasing [water], due to a complex, quasi‐mirror image compensation of ΔH≠ and ΔS≠; both parameters increase. The structures of the transition states were probed by a proton inventory study, carried out in the presence of L2O mole fractions (L = H or D) of 0.190, 0.540, 0.890 and 0.180, 0.529, 0.890, for NPCF and NPFB, respectively. Plots of observed rate constants versus the atom fraction of deuterium in the solvent curve downward. Cyclic transition state models were fitted to the kinetic data; these models contain the ester and two water molecules (NPCF) or three water molecules (NPFB). Thus, the sigmoidal dependences of log (kobs) on log [water] are not due to a change in the number of water molecules in the transition states as a function of increasing [water]. The binary solvent mixture is micro‐heterogeneous; there exists two “micro‐domains,” one consists predominantly of coordinated water molecules, the other consists mostly of acetonitrile hydrogen‐bonded to water molecules. NPCF is 232 times more soluble in water than NPFB. That is, the former ester is dissolved in the outer, more polar periphery of these micro‐domains whereas the more hydrophobic NPFB is dissolved in their inner, less polar interiors. This conclusion is corroborated by comparing the dependence on log [water] of log [kobs], and of ET, the empirical solvent polarity parameter, as measured by solvatochromic probes of increasing hydrophobicity. Copyright © 2006 John Wiley & Sons, Ltd.

Aromatic Molecules in Restricted Geometries: Pyrene Excimer Formation in an Anchored Bilayer

The galleries of an anionic clay, Mg-Al Layered Double Hydroxide (Mg-Al LDH) have been functionalized by intercalating the anionic surfactant do-decyl sulfate. Within the galleries, the alkyl chains of the surfactant adopt a bilayer structure with the sulfate headgroup anchored to the inorganic sheet. Pyrene molecules have been solubilized in the anchored bilayer by partitioning from polar solvents. The presence of pyrene molecules induces conformational disorder in the alkyl chains of the bilayer and more importantly inhibits the rotational disordering motion of the sulfate headgroup. Pyrene fluorescence indicates formation of excimers whose intensity increases with concentration of solubilized pyrene indicating that they are mobile. Pyrene solubilized in the anchored bilayer exhibits unusual phenomena; on evacuation the excimer band disappear but reappears on releasing vacuum. It is shown that this behavior arises due to the loss of water of hydration of the headgroup on evacuation and as a consequence the pyrene moves into the less polar interior of the bilayer where it is immobile and can no longer diffuse and form excimers. The motion of pyrene into the interior of the bilayer creates free space near the surfactant chain termini, which manifests in the disappearance of the methyl-rocking mode of the ordered (-tt) end-chain conformer in the Raman spectra.

Temperature-Dependent Properties of Telechelic Hydrophobically Modified Poly(N-isopropylacrylamides) in Water: Evidence from Light Scattering and Fluorescence Spectroscopy for the Formation of Stable Mesoglobules at Elevated Temperatures

The self-assembling properties of hydrophobically modified (HM) telechelic poly(N-isopropylacrylamides) (PNIPAM) were studied in aqueous solutions of concentration ranging from 0.1 to 11 g L-1 by fluorescence spectroscopy, using N-phenyl-1-naphthylamine as a probe, and by static (SLS) and dynamic (DLS) light scattering over a temperature domain encompassing their cloud point (Tcp) and coil-to-globule transition temperature (TM). The telechelic HM−PNIPAM samples bear n-octadecyl termini, and their molar mass (Mn) ranges from 12 000 to 49 000 g mol-1 with a polydispersity index lower than 1.20. In cold aqueous solution, the HM−PNIPAM samples associate in the form of flower micelles (10.8 < RH < 17.5 nm, RG/RH ≅ 1.3−1.5) consisting of ≅16−27 polymer chains, depending on their molecular weight. In solutions heated under equilibrium conditions above TM, individual flower micelles with collapsed loops associate to form stable mesoglobules (RG/RH ∼ 0.80) comprising a few hundreds chains with a more rigid and more polar interior than the hydrophobic core of hydrated flower micelles. The size of the mesoglobules increases with increasing polymer concentration (19 < RH < 115 nm), but in all cases the mesoglobule size distributions are narrower than those of the corresponding polymer micelles in cold solutions.

Inclusion of poly-aromatic hydrocarbon (PAH) molecules in a functionalized layered double hydroxide

The internal surface of an Mg-Al layered double hydroxide has been functionalized by anchoring carboxy-methyl derivatized β-cyclodextrin cavities to the gallery walls. Neutral polyaromatic hydrocarbon (PAH) molecules have been included within the functionalized solid by driving the hydrophobic aromatic molecules from a polar solvent into the less polar interior of the anchored cyclodextrin cavities by a partitioning process. The optical (absorption and emission) properties of the PAH molecules included within the functionalized Mg-Al layered double hydroxide solid are similar to that of dilute solutions of the PAH in non-polar solvents. The unique feature of these hybrid materials is that they are thermally stable over a wide temperature range with their emission properties practically unaltered.

Relative Strengths of NH··O and CH··O Hydrogen Bonds between Polypeptide Chain Segments

Correlated ab initio calculations are used to compare the energetics when the CH and NH groups of the model dipeptide CHONHCH2CONH2 are each allowed to form a H-bond with the proton acceptor O of a peptide group. When the dipeptide is in its C7 conformation, the NH..O H-bond energy is found to be 7.4 kcal/mol, as compared to only 2.8 kcal/mol for the CH..O interaction. On the other hand, the situation reverses, and the CH..O H-bond becomes stronger than NH..O, when the dipeptide adopts a C5 structure. This reversal is important as C5 is nearly equal in stability to C7 for the dipeptide, and is representative of the commonly observed beta-sheet structure in a protein. Immersing the dipeptide-peptide pair in a model solvent weakens both sorts of H-bonds, and in a fairly uniform manner. Consequently, the trends observed in the in vacuo situation retain their validity in either aqueous solution or the protein interior. Likewise, the desolvation penalty, suffered by removing a H-bonded complex from water and placing it in the less polar interior of a protein, is quite similar for the NH..O and CH..O bonds.

Empirical lipid propensities of amino acid residues in multispan alpha helical membrane proteins

Characterizing the interactions between amino acid residues and lipid molecules is important for understanding the assembly of transmembrane helices and for studying membrane protein folding. In this study we develop TMLIP (TransMembrane helix-LIPid), an empirically derived propensity of individual residue types to face lipid membrane based on statistical analysis of high-resolution structures of membrane proteins. Lipid accessibilities of amino acid residues within the transmembrane (TM) region of 29 structures of helical membrane proteins are studied with a spherical probe of radius of 1.9 A. Our results show that there are characteristic preferences for residues to face the headgroup region and the hydrocarbon core region of lipid membrane. Amino acid residues Lys, Arg, Trp, Phe, and Leu are often found exposed at the headgroup regions of the membrane, where they have high propensity to face phospholipid headgroups and glycerol backbones. In the hydrocarbon core region, the strongest preference for interacting with lipids is observed for Ile, Leu, Phe and Val. Small and polar amino acid residues are usually buried inside helical bundles and are strongly lipophobic. There is a strong correlation between various hydrophobicity scales and the propensity of a given residue to face the lipids in the hydrocarbon region of the bilayer. Our data suggest a possibly significant contribution of the lipophobic effect to the folding of membrane proteins. This study shows that membrane proteins have exceedingly apolar exteriors rather than highly polar interiors. Prediction of lipid-facing surfaces of boundary helices using TMLIP1 results in a 54% accuracy, which is significantly better than random (25% accuracy). We also compare performance of TMLIP with another lipid propensity scale, kPROT, and with several hydrophobicity scales using hydrophobic moment analysis.

Structural Consequences of Accommodation of Four Non-cognate Amino Acid Residues in the S1 Pocket of Bovine Trypsin and Chymotrypsin

Crystal structures of P1 Gly, Val, Leu and Phe bovine pancreatic trypsin inhibitor (BPTI) variants in complex with two serine proteinases, bovine trypsin and chymotrypsin, have been determined. The association constants for the four mutants with the two enzymes show that the enlargement of the volume of the P1 residue is accompanied by an increase of the binding energy, which is more pronounced for bovine chymotrypsin. Since the conformation of the P1 side-chains in the two S1 pockets is very similar, we suggest that the difference in Δ G values between the enzymes must arise from the more polar environment of the S1 site of trypsin. This results mainly from the substitutions of Met192 and Ser189 observed in chymotrypsin with Gln192 and Asp189 present in trypsin. The more polar interior of the S1 site of trypsin is reflected by a much higher order of the solvent network in the empty pocket of the enzyme, as is observed in the complexes of the two enzymes with the P1 Gly BPTI variant. The more optimal binding of the large hydrophobic P1 residues by chymotrypsin is also reflected by shrinkage of the S1 pocket upon the accommodation of the cognate residues of this enzyme. Conversely, the S1 pocket of trypsin expands upon binding of such side-chains, possibly to avoid interaction with the polar residues of the walls. Further differentiation between the two enzymes is achieved by small differences in the shape of the S1 sites, resulting in an unequal steric hindrance of some of the side-chains, as observed for the γ-branched P1 Leu variant of BPTI, which is much more favored by bovine chymotrypsin than trypsin. Analysis of the discrimination of β-branched residues by trypsin and chymotrypsin is based on the complexes with the P1 Val BPTI variant. Steric repulsion of the P1 Val residue by the walls of the S1 pocket of both enzymes prevents the P1 Val side-chain from adopting the most optimal χ1 value.

Cesium cholate: determination of X-ray crystal structure indicates participation of the ring hydroxyl groups in metal binding.

The crystal structure of cesium cholate, C(24)H(36)(OH)(3) COOCs has been determined with three-dimensional X-ray diffractometer data. It crystallized in the monoclinic space group P2(1) with unit-cell dimensions a = 11.543(5) A, b = 8.614(3) A, and c = 12.662(5) A, beta(deg) = 107.95(2), V = 1197.7 A(3) and Z = 2. The atomic parameters were refined to a final r = 0.0269 and R(omega) = 0.0280 for 2342 observed reflections. Each Cs(+) is coordinated to 7 oxygen atoms from 5 different cholate anions with Cs-O distances ranging from 2.957(4) A to 3.678(5) A. In this crystal, 5 cholates are coordinated with 1 Cs(+), and 5 Cs(+) are coordinated with 1 cholate anion. Carboxyl and all the 3 ring hydroxyl groups of cholate anion participate in binding to Cs(+) simultaneously, and there is no water molecule coordinated with the Cs(+). The pattern of successive rows arranged with polar (p) and non-polar (n) faces in apposition leads to the formation of a sandwich sheet structure with polar and non-polar channels. The Cs ions lie within the polar interior of the sandwich. The H-bond network is reorganized in forming cesium cholate from cholic acid. All the oxygen atoms in cholate anion are involved in H-bonding reciprocally or with water molecules to form an extensive 3-dimensional network of H-bonds. Compared with cholic acid and other similar type of steroids, the coordination structure and H-bonding of Cs cholate crystal are distinct.

Apolar and Polar Solvation Thermodynamics Related to the Protein Unfolding Process

Thermodynamics related to hydrated water upon protein unfolding is studied over a broad temperature range (5–125°C). The hydration effect arising from the apolar interior is modeled as an increased number of hydrogen bonds between water molecules compared with bulk water. The corresponding contribution from the polar interior is modeled as a two-step process. First, the polar interior breaks hydrogen bonds in bulk water upon unfolding. Second, due to strong bonds between the polar surface and the nearest water molecules, we assume quantization using a simplified two-state picture. The heat capacity change upon hydration is compared with model compound data evaluated previously for 20 different proteins. We obtain good correspondence with the data for both the apolar and the polar interior. We note that the effective coupling constants for both models have small variations among the proteins we have investigated.

Unexpected Interactions between Sol−Gel Silica Glass and Guest Molecules. Extraction of Aromatic Hydrocarbons into Polar Silica from Hydrophobic Solvents

Properties of a solute may differ greatly between a free solution and that solution confined in pores of a sol−gel glass. We studied the entry of various aromatic organic compounds from solution into the monolith of sol−gel silica immersed in this solution. Partitioning of the solute is quantified by the uptake coefficient, the ratio of its concentrations in the glass and in the surrounding solution at equilibrium. The dependence of this coefficient on the solvent gives insight into possible interactions between the solute and the silica matrix. We report the uptake of 31 compounds altogether: 18 halogen derivatives of benzene; 5 condensed (fused) aromatics; and stilbene and three substituted derivatives of it, each in both cis and trans configurations. When the solvent is hexane, the uptake coefficients are as follows: 1.0−1.9 for the halobenzenes; 3.0−4.6 for the hydrocarbons; and 3.3−4.9 for the stilbenes. When the solvent is carbon tetrachloride or dichloromethane, the uptake coefficients become 0.82−1.39 for the hydrocarbons and 0.90−1.25 for the stilbenes. The excessive uptake of organic compounds from hexane is unexpected, for it amounts to extraction of nonpolar or slightly polar solutes from a nonpolar solvent into a polar interior of silica glass. The solute−silica interactions responsible for this extraction are not of the van der Waals type. Our findings are consistent with hydrogen bonding between the aromatic π system in the solutes and the hydroxyl groups on the silica surface. Hexane cannot interact with this surface but dichloromethane and carbon tetrachloride can: they shield the hydroxyl groups from the organic solvents and thus suppress the hydrogen bonding. This explanation is supported by the emission spectra of the aromatic compound pyrene when it is dissolved in acetonitrile, dichloromethane, cyclohexyl chloride, and hexane and when it is taken up by monoliths of sol−gel silica from these four solutions. The relative intensities of the emission bands designated III and I change greatly when pyrene is taken up from hexane but remain unchanged when it is taken up from the other three solvents. Evidently, hexane does not, whereas the other three solvents do, line the silica surface and shield it from approach by pyrene molecules. Even though solute molecules are much smaller than the pores in the sol−gel glass, diffusion of these molecules into the monolith may result in an uneven partitioning at equilibrium. This fact must be taken into consideration in the design of biosensors, immobilized catalysts, and other composite materials because their function depends on the entry of analytes, substrates, and other chemicals into the glass matrix.

Photoisomerization of indolinespirobenzopyran in anionic clay matrices of layered double hydroxides

Sulfonated indolinespirobenzopyran (SP–SO3–) was intercalated into the Mg/Al or Zn/Al layered double hydroxides (LDHs) in the presence of toluene-p-sulfonic acid (PTS). The new intercalates exhibited reversible photoisomerization between SP–SO3– and merocyanine (MC). The presence of PTS was critical for the reversible photoisomerization. MC was very stable and the rate constant of decolorization was <9 × 10–7S–1. SP interacted with non-polar portions between the layers although MC interacted with the polar interior surface of the LDH.

A novel strategy for the formulation of medicinal aerosols

The delivery of medicinally active compounds to the respiratory tract requires that the natural aerodynamic filtration capacity of the lung is overcome, and therefore poses a major challenge to pharmaceutical formulation. Metered dose inhalers (M Dl's) formulated as suspensions of a micronised drug in a chlorofluorocarbon (CFC) blend represent a major device for the delivery of locally active drugs used in the treatment of respiratory disorders. Despite the identification and optimisation ol the major parameters which influence the performance of MDI's such as particle size, vapour pressure, surfactant concentration (Poll et al, 1969; Moren, 1978; Newman et al, 1982), their use in practise results in extensive extrapulmonary deposition with frequently <10% of the metered dose actually reaching the targeted site. Whilst poor coordination by the patient between the commencement of inspiration and actuation of the device contributes to their poor efficacy, most of the aerosol output immediately after actuation is, in fact, aerodynamically unfavourable for pulmonary deposition (Moren and Anderson, 1981 ). The design of more elaborate actuators to encourage efficient breakup and/or collection of non-respirable aerosols droplets (Byron et al, 1989), though logical, is largely restricted by the presence of micronised drug particles within the formulation. Homogeneous systems, where the drug is dissolved in the propellant blend offers potential advantages both in formulation and in actuator and valve design which, prospectively, could lead to an improved therapeutic efficacy of this dosage form. Amphiphatic molecules in apolar media may associate into reversed or inverted micellar solutions (Luisi et al, 1988) which are homogeneous and optically transparent and the ability of such systems to solubilise quantities of water has been well docu me nted (Arkin and Singleterry, 1949; Keh and Valeur, 1981 ). The result of incorporating water into such systems is the production of a water-pool and the formation of swollen micelles, which could serve as centres for the solubilisation of drugs of various physico-chemical characteristics. We have investigated the aggregation of a soya lecithin (SPC) in a model CFC, trichlorotrifluoroethane (P113), using an iodine solubilisation method and determined the potential to solubilise water within their polar interior. Spectroscopic and viscometric techniques were employed to determine aggregation number of SPC solutions and to study the effect of solubilised water (expressed as the molar ratio of water:surfactant, R) on this and the size and shape of the reversed micelles. Drug solubility studies for a model hydrophilic compound were performed over a range of values of R and in separate experiments, the influence of added water on the micellar core polarity was investigated using a fluorescence probe. Finally, pressurised packs containing a range of surfactant concentrations and solubilised water have been formulated and the aerosols produced from those units following activation through a number of various orifices characterised.

Macroscopic models for studies of electrostatic interactions in proteins: limitations and applicability.

The validity of macroscopic models for calculations of electrostatic energies in proteins is examined. The Tanford-Kirkwood (TK) model is extended to include the self energy of the ionized groups. It is shown that ionized groups cannot exist inside nonpolar regions of proteins and argued that the experimental finding of ions inside proteins proves that the corresponding local environment is polar. The modified TK model (MTK model), which adjusts charge-charge interactions by the corresponding solvent accessibilities, is found to be inconsistent with the TK model, on which it is based. The MTK model corresponds to a polar interior whereas the TK model assumes a nonpolar interior. It is shown that models that assume a high dielectric constant for proteins give reasonable results for interactions between charged groups at equilibrium. It is then explained why, in contradiction to common belief, protein interiors are polar around charged groups. It is argued that in focusing on charge-charge interactions one overlooks the key contribution of the protein dipoles in determining the self energy of charges in the interior of proteins.

Structures of membrane proteins

The possible conformations of integral membrane proteins are restricted by the nature of their environment. In order to satisfy the requirement of maximum hydrogen bonding, those portions of the polypeptide chain which are in contact with lipid hydrocarbon must be organized into regions of regular secondary structure. As possible models of the intramembranous regions of integral membrane proteins, three types of regular structures are discussed. Two, the alpha helix and the beta-pleated sheet, are regularly occurring structural features of soluble proteins. The third is a newly proposed class of conformations called beta helices. These helices have unique features which make them particularly well-suited to the lipid bilayer environment. The central segment of the membrane-spanning protein glycophorin can be arranged into a beta helix with a hydrophobic exterior and a polar interior containing charged amino-acid side chains. Such structures could function as transmembrane ion channels. A model of the activation process based on a hypothetical equilibrium between alpha and beta helical forms of a transmembrane protein is presented. The model can accurately reproduce the kinetics and voltage dependence of the channels in nerve.

Co-oligopeptides of glycine and aromatic amino acids with variable distance between the aromatic residues IV. Spectroscopic properties of tryptophan-containing peptides in a cyclohexane phase

l-Tryptophan, l-tryptophanylglycine, glycyl- l-tryptophan, glycyl- l-tryptophanylglycine and glycyl- l-tryptophanylglycylglycyl- l-tryptophanylglycine have been transferred from an aqueous solution (generally 0.1 M NaOH) to cyclohexane, using the quaternary ammonium salt trioctylmethyl ammonium chloride (NR 4 +Cl −, soluble in cyclohexane but not in water) as the transporting agent. The spectroscopic properties of l-tryptophan and tryptophan-containing peptides have been studied in the cyclohexane phase. With respect to the aqueous solutions, ultraviolet absorption spectra are characterized by a considerable red shift of the absorption maxima and by a hypochromicity of up to 10%. Fluorescence spectra generally show emission maxima which are characteristic of polar environments, accompanied by a significant enhancement of the quantum yield. CD spectra have also been investigated for all peptides and compared with those for aqueous systems reported in preceding publications. All these spectral changes cannot be attributed solely to the cyclohexane solvent effect. It is suggested that these anomalous spectral properties of the tryptophan-containing compounds in the cyclohexane-NR 4 + solution are due to the influence the electrostatic field of the ion pair has on the indole chromophore. The possible implications of this finding for the spectroscopic properties of aromatic residues buried in the polar interior of proteins are discussed.