Micellization of conjugated chenodeoxy- and ursodeoxycholates and solubilization of cholesterol into their micelles: comparison with other four conjugated bile salts species

Micelle formations of sodium glyco- and taurochenodeoxycholate (NaGCDC and NaTCDC) and sodium glyco- and tauroursodeoxycholates (NaGUDC and NaTUDC) was studied at 308.2 K for their critical micelle concentrations at various NaCl concentrations by pyrene fluorescence probe, and the degree of counterion binding to micelle was determined using the Corrin-Harkins plots. The degree of counterion binding was found to be 0.37-0.38 for chenodeoxycholate conjugates, while the determination of the degree was quite difficult for ursodeoxycholate conjugates. The change of I1/I3 values on the fluorescence spectrum with the conjugate bile salt concentration suggested two steps for their bile salt aggregation. The first step is a commencement of smaller aggregates, the first cmc, and the second one is a starting of stable aggregates, the second cmc. The aggregation number was determined at 308.2 K and 0.15 M NaCl concentration by static light scattering: 16.3 and 11.9 for sodium NaGCDC and NaTCDC, and 7.9 and 7.1 for NaGUDC and NaTUDC, respectively. The solubilization of cholesterol into the bile salt micelles in the presence of coexisting cholesterol phase and the maximum additive concentration (MAC) of cholesterol was determined against the bile salt concentration. The standard Gibbs energy change for the solubilization was evaluated, where the micelles were regarded as a chemical species. The solubilization was stabilized in the order of NaGUDC approximately = NaTUDC < NaTC < NaGC < NaTCDC < NaGCDC < NaTDC < NaGDC, where the preceding results were taken into the order.     

Spectrophotometric determination of the critical micellar concentration of bile salts using bilirubin monoglucuronide as a micellar probe. Utility of derivative spectroscopy.

We have developed a simple biologically non-invasive method for determining the critical micellar concentration (CMC) of bile salts using pure naturally occurring bilirubin IX alpha monoglucuronide (BMG), an important bile pigment present in virtually all mammalian biles. This methodology employs visible absorbance spectroscopy of BMG in bile salts over a range of bile salt concentrations that include the reported CMC. Using 100 microM-BMG in 0.4 M-imidazole buffer at pH 7.8, we calculated that the CMC for sodium taurochenodeoxycholate is between 2.5 and 3.0 mM based on: (1) an abrupt change in lambda max. in this concentration range, (2) a precipitous decrease in the amplitude of the absorbance shoulder at 450 nm, (3) a sudden decrease in the second derivative absorbance of BMG at 400 nm and an increase in absorbance at 470 nm, (4) a sharp change in the 4th derivative absorbance at 375 and 395 nm. In contrast, sodium taurocholate, a bile salt that reportedly does not have a CMC but continuously self-associates over a wide concentration range, exhibited none of these changes. The use of derivative spectroscopy enhances the ability to detect the CMC changes and also indicates the number of BMG species in solution and their relative energy states.     

Micelle formation of sodium deoxycholate and sodium ursodeoxycholate (part 1)

Micellization of sodium deoxycholate (NaDC) and sodium ursodeoxycholate (NaUDC) was studied for the critical micelle concentration (CMC), the micelle aggregation number, and the degree of counterion binding to micelle, where sodium cholate (NaC) was used as a reference. The fluorescence probe technique of pyrene was employed to determine accurately the CMC values for the bile salts, which indicated that a certain concentration range of CMC and a stepwise aggregation for micellization were reasonable. The temperature dependences of micellization for NaDC and NaUDC were studied at 288.2, 298.2, 308.2, and 318.2 K by aqueous solubility change with solution pH. Aggregations of the bile salt anions were analyzed using the stepwise association model and found to grow in size with increasing concentration, which confirmed that the mass action model worked quite well. The average aggregation number was found to be 2.5 (NaUDC) and 10.5 (NaDC) at the concentration of 20 mM and at 308.2 K. The aggregation number determined by static light scattering also agreed well with those by the solubility method in the order of size: NaUDC相似文献   

Adsorption of bile salts by the cestodes, Hymenolepis diminuta and H. microstoma.

The accumulation of purified sodium taurocholate (NaTC) and sodium glycocholate (NaGC) by Hymenolepis diminuta and Hymenolepis microstoma (Cestoda: Cyclophyllidea) was determined using radioactive bile salts. H. diminuta reached equilibrium levels of approximately 120 nmoles NaTC/g dry wt and 300 nmoles NaGC/g dry wt. Presentation of the bile salts in mixed micelles with 0.35 mM oleic acid did not alter these values. With H. microstoma, the maxima were 195 nmoles NaTC/g dry wt and 614 nmoles NaCG/g dry wt. These values were similarly unaffected by the addition of 0.35 mM oleic acid to the micelles. Equilibrium values of this magnitude, in media containing as much as 25 or 30 mM bile salt, and the maintenance of this level during incubations of 15 to 60 min eliminated the possibility that the accumulation was by diffusion or by any form of mediated transport into the worm. The accumulation on NaTC by H. diminuta was [Na+] independent, and insensitive to ouabain, DNP, and high [K+]. These observations, the maintenance of different levels of NaTC and NaGC, and the failure of the 2 bile salts to compete indicated that there was no active excretion mechanism operating in a fashion similar to the active transport of bile salts in the vertebrate small intestine. It was concluded that the accumulation of NaTC by H. diminuta was actually adsorption to the tegument. Comparable, although more limited, experiments extended this conclusion to the accumulation of NaGC by H. diminuta and of NaTC and NaGC by H. microstoma. It is suggested that bile salt monomers, rather than intact micelles, adsorb to specific loci on the tegument.     

Orientation of gramicidin D incorporated into phospholipid multibilayers: a Fourier transform infrared-attenuated total reflection spectroscopic study

Polarized Fourier transform infrared (FTIR)-attenuated total reflection (ATR) spectroscopy was applied to study the orientation of the linear pentadecapeptide antibiotic gramicidin D incorporated into phospholipid multibilayers, which were cast on a germanium ATR plate from chloroform solution. In DMPC and DPPC multibilayers, the CH2 stretching bands of lipid hydrocarbon chains were slightly shifted to the higher frequency side and bandwidth was increased in the presence of gramicidin. However, in DPPE multibilayers, frequencies and bandwidths of these bands were unaltered. In each case, gramicidin produced little effect on the orientation of lipid hydrocarbon chains, suggesting that gramicidin penetrates into lipid layers without noticeable perturbations. Upon incubation of cast films in contact with water above the gel-liquid-crystalline transition temperature (Tc) of lipids, the reorientation of gramicidin in lipid multibilayers occurred, the degree thereof depending upon the fluidity of the lipid hydrocarbon chains and the amount of surrounding water. In DMPC multibilayers, the helix axis of gramicidin was oriented almost parallel to the lipid hydrocarbon chains after incubation. In DPPC multibilayers, on the other hand, the helix axis of gramicidin was tilted on average about 15 degrees from the lipid hydrocarbon chains after incubation. However, in DPPE multibilayers, which are known to have the most rigid bilayer structures, the reorientation of gramicidin could not be seen.     

Infrared and time-resolved fluorescence spectroscopic studies of the polymorphic phase behavior of phosphatidylethanolamine/diacylglycerol lipid mixtures

Fourier transform infrared (FTIR) and time-resolved fluorescence spectroscopy have been employed to examine the structural dynamics of lipid fatty acyl chains and lipid/water interfacial region of a binary lipid mixture containing unsaturated phosphatidylethanolamine (PE) and diacylglycerol (DG). Infrared vibrational frequencies of the CH2 symmetric stretching and the C = O stretching bands of the lipids were measured at different lipid compositions and temperatures. For 0% DG, the lamellar gel to lamellar liquid crystalline (L beta-L alpha) and the L alpha to inverted hexagonal (L alpha-HII) phase transitions were observed at approximately 15 degrees and 55 degrees C, respectively. As the DG content increased gradually from 0% to 15%, the L alpha-HII phase transition temperature decreased drastically while the L beta-L alpha phase transition temperature decreased only slightly. At 10% DG, a merge of these two phase transitions was noticed at approximately 10 degrees C. For the composition study at 23 degrees C, the L alpha-HII transition occurred at approximately 6-10% DG as indicated by abrupt increases in both the CH2 and C = O stretching frequencies at those DG contents. Using time-resolved fluorescence spectroscopy, abrupt decreases in both the normalized long time residual and the initial slope of the anisotropy decay function of lipid probes, 1-palmitoyl-2-[[2-[4-(6-phenyl-trans-1,3,5- hexatrienyl)phenyl]ethyl]carbonyl]-3-sn-phosphatidylcholine, in these PE/DG mixtures were observed at the L alpha-HII phase transition. These changes in the anisotropy decay parameters suggested that the rotational dynamics and orientational packing of the lipids were altered at the composition-induced L alpha-HII transition, and agreed with a previous temperature-induced L alpha-HII transition study on pure unsaturated PE (Cheng (1989) Biophys. J. 55, 1025-1031). The fluorescence lifetime of water soluble probes, 8,1-anilinonapthalenes sulfonate acid, in PE/DG mixtures increased abruptly at the L alpha-HII phase transition, suggesting that the conformation and hydration of the lipid/water interfacial region also undergo significant changes at the L alpha-HII transition.     

Effect of bile salts on the absorption of glucose and oleic acid by the cestodes, Hymenolepis diminuta and H. microstoma.

The uptake of 2 mM 14C-glucose by H. diminuta during 1-min incubations was inhibited by addition of 10 mM sodium taurocholate (NaTC) to the incubation media. Preincubation in 10 mM NaTC for 30 min did not increase the inhibition, suggesting that the inhibition was competitive. This was confirmed with a standard Lineweaver-Burk experiment. Addition of 0.35 mM oleic acid to the NaTC micelles did not alter the level of inhibition. Sodium glycocholate (NaGC) did not inhibit the uptake of glucose by H. diminuta. The uptake of glucose by H. microstoma was also inhibited by NaTC, and was not affected by NaGC. H. diminuta absorbed 3.62 mumoles of oleic acid/g dry wt during 15-min incubations in mixed micelles of 10 mM NaTC and 0.35 mM oleic acid. The total uptake was determined as the sum of the ethanol extractable and nonextractable 3H-oleic acid. In 15 mM NaTC, the uptake of oleic acid was reduced by 50%; at 30 mM NaTC the uptake of oleic acid decreased by half again. Substituting NaGC for NaTC, the greatest uptake of oleic acid, 2.63 mumoles/g dry wt, was from mixed micelles of 15 mM NaGC and 0.35 mM oleic acid. Lesser amounts of oleic acid were absorbed from mixed micelles at 5 or 30 mM NaGC. H. microstoma exhibited a similar pattern of oleic acid uptake from mixed micelles with NaTC and NaGC. At all bile salt concentrations tested, H. microstoma absorbed more oleic acid than H. diminuta and incorporated more oleic acid into the nonextractable pool. The possible roles of bile salts in the absorption of oleic acid as indicated by the results herein are discussed.     

Lipid-induced conformational transitions of beta-lactoglobulin

Bovine beta-lactoglobulin (betaLG) provides an excellent model protein system for beta-to-alpha conformational change, but its behavior varies when the change is induced by alcohols, surfactants, or lipid vesicles. Here the interaction and orientation of betaLG in association with various artificial lipid vesicles at neutral and acidic pH have been studied by use of several complementary spectroscopic techniques. Circular dichroism (CD) and Fourier transform infrared (FTIR) spectra demonstrated that betaLG acquires a non-native alpha-helical structure upon binding with anionic lipids, while zwitterionic lipids do not have a significant effect on its conformation. The degree of induced alpha-helix depends on the lipid concentration and is strongly affected by the charge of the protein and lipids as well as the ionic strength of the solution. Near-UV CD and Trp emission spectra revealed that the tertiary structure of lipid-bound betaLG is highly expanded but not completely disrupted. Fluorescence quenching together with a Trp emission blue shift showed that the Trp residues remain largely shielded from the solvent when interacting with DMPG, which would be consistent with at least some portions of betaLG having been inserted into the lipid membrane. The orientations of the alpha-helix and beta-sheet axes in membrane-bound betaLG were found to be parallel and perpendicular, respectively, to the membrane film normal, as determined by use of polarized attenuated total reflection (ATR) FTIR spectra. Our findings reveal that the lipid-induced beta-to-alpha transition in betaLG, accompanied by a substantial disruption in tertiary structure, is mainly driven by strong electrostatic interactions. Once the tightly packed betaLG is disrupted, hydrophobic residues become exposed and available for insertion into the lipid bilayer, where hydrophobic interaction with the lipids may play a role in stabilizing the helical components.     

Infrared spectroscopic characterization of the structural changes connected with the E1----E2 transition in the Ca2+-ATPase of sarcoplasmic reticulum

The Ca2+-transporting ATPase (EC 3.6.1.38) of sarcoplasmic reticulum alternates between several conformational states during ATP-dependent Ca2+ transport. The E1 conformation is stabilized by 0.1 mM Ca2+ and the E2 conformation by vanadate in a Ca2+-free medium. Fourier transform infrared spectroscopy reveals significant differences between the two states that indicate differences in the protein secondary structure. The two states and the corresponding spectra can be interconverted reversibly by changing the Ca2+ concentration of the medium. The infrared spectral changes indicate the appearance of a new alpha-helical substructure connected with the E1----E2 conversion accompanied by small changes in beta-turns, while the beta-sheet content remains essentially unchanged. There are also differences between the E1 and E2 states in the C = O stretching vibrations of the ester carbonyl groups of phospholipids in intact sarcoplasmic reticulum that are not observed under identical conditions in isolated sarcoplasmic reticulum lipid dispersions. These observations imply an effect of proteins on the structure of the interfacial regions of the phospholipids that is dependent on the conformational state of the Ca2+-ATPase. The CH2- and CH3-stretching frequencies of the membrane lipids are not affected significantly by the E1----E2 transition. The Fourier transform infrared spectra of sarcoplasmic reticulum vesicles in the presence of 20 mM Ca2+ suggest the stabilization of a protein conformation similar to the E2 state except for differences in the behavior of COO- and phospholipid ester C = O groups that may reflect charge effects of the bound Ca2+.     

Incorporation of cholesterol in sphingomyelin- phosphatidylcholine vesicles has profound effects on detergent-induced phase transitions

Vesicle <--> micelle transitions are important phenomena during bile formation and intestinal lipid processing. The hepatocyte canalicular membrane outer leaflet contains appreciable amounts of phosphatidylcholine (PC) and sphingomyelin (SM), and both phospholipids are found in the human diet. Dietary SM enrichment inhibits intestinal cholesterol absorption. We therefore studied detergent-induced vesicle --> micelle transitions in SM-PC vesicles. Phase transitions were evaluated by spectrophotometry and cryotransmission electron microscopy (cryo-TEM) after addition of taurocholate (3-7 mM) to SM-PC vesicles (4 mM phospholipid, SM/PC 40%/60%, without or with 1.6 mM cholesterol). After addition of excess (5-7 mM) taurocholate, SM-PC vesicles were more sensitive to micellization than PC vesicles. As shown by sequential cryo-TEM, addition of equimolar (4 mM) taurocholate to SM-PC vesicles induced formation of open vesicles, then (at the absorbance peak) fusion of bilayer fragments into large open structures (around 200 nm diameter) coexisting with some multilamellar or fused vesicles and thread-like micelles and, finally, transformation into an uniform picture with long thread-like micelles. Incorporation of cholesterol in the SM/PC bilayer changed initial vesicular shape from spherical into ellipsoid and profoundly increased detergent resistance. Disk-like micelles and multilamellar vesicles, and then extremely large vesicular structures, were observed by sequential cryo-TEM under these circumstances, with persistently increased absorbance values by spectrophotometry. These findings may be relevant for bile formation and intestinal lipid processing. Inhibition of intestinal cholesterol absorption by dietary SM enrichment may relate to high resistance against bile salt-induced micellization of intestinal lipids in presence of the sphingolipid.     

Physicochemical and physiological properties of 5alpha-cyprinol sulfate,the toxic bile salt of cyprinid fish

5alpha-Cyprinol sulfate was isolated from bile of the Asiatic carp, Cyprinus carpio. 5alpha-Cyprinol sulfate was surface active and formed micelles; its critical micellization concentration (CMC) in 0.15 M Na+ using the maximum bubble pressure device was 1.5 mM; by dye solubilization, its CMC was approximately 4 mM. At concentrations >1 mM, 5alpha-cyprinol sulfate solubilized monooleylglycerol efficiently (2.1 molecules per mol micellar bile salt). When infused intravenously into the anesthetized rat, 5alpha-cyprinol sulfate was hemolytic, cholestatic, and toxic. In the isolated rat liver, it underwent little biotransformation and was poorly transported (Tmax congruent with 0.5 micromol/min/kg) as compared with taurocholate. 5alpha-Cyprinol, its bile alcohol moiety, was oxidized to its corresponding C27 bile acid and to allocholic acid (the latter was then conjugated with taurine); these metabolites were efficiently transported. 5alpha-Cyprinol sulfate inhibited taurocholate uptake in COS-7 cells transfected with rat asbt, the apical bile salt transporter of the ileal enterocyte. 5alpha-Cyprinol had limited aqueous solubility (0.3 mM) and was poorly absorbed from the perfused rat jejunum or ileum. Sampling of carp intestinal content indicated that 5alpha-cyprinol sulfate was present at micellar concentrations, and that it did not undergo hydrolysis during intestinal transit. These studies indicate that 5alpha-cyprinol sulfate is an excellent digestive detergent and suggest that a micellar phase is present during digestion in cyprinid fish.     

Micellization and interactions with phospholipid vesicles of the lipopeptide iturin a,as monitored by time-resolved fluorescence of a D-tyrosyl residue

The micellization and the interactions with lipid vesicles of the antifungal cyclic lipopeptide iturin A have been investigated by nanosecond pulse fluorometry of a D-tyrosyl residue. We show that this lipopeptide has three conformers in solution whose proportions are modified during the micellization process. Below the critical micellar concentration (CMC) iturin A does not self-associate inside the bilayer. Above the CMC all the molecules of iturin A interact with the vesicles and self-associate inside the membrane.     

Vibrational spectroscopic studies of newly developed synthetic biopolymers

Vibrational spectroscopic techniques such as near‐infrared (NIR), Fourier transform infrared (FTIR), and Raman spectroscopy are valuable diagnostic tools that can be used to elucidate comprehensive structural information of numerous biological samples. In this review article, we have highlighted the advantages of nanotechnology and biophotonics in conjunction with vibrational spectroscopic techniques in order to understand the various aspects of new kind of synthetic biopolymers termed as polyethylene glycol (PEG)ylated lipids. In contrast to conventional phospholipids, these novel lipids spontaneously form liposomes or nanovesicles upon hydration, without the supply of external activation energy. The amphiphiles considered in this study differ in their hydrophobic acyl chain length and contain different units of PEG hydrophilic headgroups. We have further explored the thermotropic phase behaviors and associated changes in the conformational order/disorder of such lipids by using variable‐temperature FTIR and Raman spectroscopy. Phase transition temperature profiles and correlation between various spectral indicators have been identified by either monitoring the shifts in the vibrational peak positions or plotting vibrational peak intensity ratios in the C? H stretching region as a function of temperature. To supplement our observations of phase transformations, a thermodynamic approach known as differential scanning calorimetry (DSC) has been applied and revealed a good agreement with the infrared and Raman spectroscopic data. Finally, the investigation of thermal properties of lipids is extremely crucial for numerous purposes, thus the results obtained in this work may find application in a wide variety of studies including the development of PEGylated lipid based drug and substances delivery vehicles. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 403–417, 2010. 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     

Ethanol effects on the stratum corneum lipid phase behavior.

The stratum corneum is considered to be the diffusional barrier of mammalian skin for water and most solutes. The intercellular lipid multilayer domains of the stratum corneum are believed to be the diffusional pathway for most lipophilic solutes. Fluidization of the lipid multilayers in the presence of ethanol is frequently conceived to result in enhanced permeation. Current investigations address the effect of ethanol on the phase behavior in terms of stratum corneum lipid alkyl chain packing, mobility and conformational order as measured by Fourier transform infrared (FTIR) spectroscopy. Phospholipid multilamellar vesicles were also studied as model systems. There appeared to be no effect of ethanol on either the solid-solid phase transition or the gel phase interchain coupling of the stratum corneum lipids. However, there was a reduction in the mobility of the alkyl chains in the presence of ethanol. Possible mechanistic relationships between the current FTIR spectroscopic results with available literature data of ethanol induced lipophilic solute penetration enhancement through the skin are discussed.     

Differential scanning calorimetry and (2)H nuclear magnetic resonance and Fourier transform infrared spectroscopy studies of the effects of transmembrane alpha-helical peptides on the organization of phosphatidylcholine bilayers

We have studied the effects of the incorporation of the alpha-helical transmembrane peptides Ac-K(2)-L(24)-K(2)-amide (L(24)) and Ac-K(2)-(L-A)(12)-K(2)-amide ((LA)(12)) on the thermotropic phase behavior of 1,2-dipalmitoyl-d(62)-sn-glycero-3-phosphocholine (DPPC-d(62)) and 1-palmitoyl-d(31)-2-oleoyl-sn-glycero-3-phosphocholine (POPC-d(31)) lipid bilayer model membranes by differential scanning calorimetry (DSC) and the conformational and orientational order of the phospholipid chains by Fourier transform infrared (FTIR) spectroscopy and (2)H nuclear magnetic resonance ((2)H-NMR) spectroscopy, respectively. Our DSC and FTIR spectroscopic studies indicate that the peptides L(24) and (LA)(12) both decrease the temperature and enthalpy of the gel/liquid-crystalline phase transition of DPPC-d(62) bilayers, with (LA)(12) having the greater effect in this regard. An examination of the frequencies of the CH(2) and CD(2) symmetric stretching bands of the infrared spectra of liquid-crystalline states of the peptide-free and peptide-containing DPPC-d(62) and POPC-d(31) samples, and a comparison with the orientational order as measured by (2)H-NMR spectroscopy as well as with the chain order as measured by electron spin resonance spectroscopy, lead us to conclude that the CH(2) (or CD(2)) stretching frequencies of lipid hydrocarbon chains are not a reliable measure of chain conformational order in lipid bilayers containing significant amounts of peptides or other lipophilic inclusions. In contrast, the results of our (2)H-NMR spectroscopic studies present a consistent picture in which both L(24) and (LA)(12) increased in a similar way the time-averaged orientational order of the lipid chains of their liquid-crystalline lipid bilayer hosts. The comparison of the effects L(24) and (LA)(12) on phosphatidylcholine bilayers indicates that the gel-to-liquid-crystalline phase transition appears to be more sensitive to small changes in transmembrane peptide surface topology than hydrocarbon carbon chain orientational order in the liquid-crystalline state.     

Micelle formation of sodium chenodeoxycholate and solubilization into the micelles: comparison with other unconjugated bile salts

Micellization of sodium chenodeoxycholate (NaCDC) was studied for the critical micelle concentration (CMC), the micelle aggregation number, and the degree of counterion binding to micelle at 288.2, 298.2, 308.2, and 318.2 K. They were compared with those of three other unconjugated bile salts; sodium cholate (NaC), sodium deoxycholate (NaDC), and sodium ursodeoxycholate (NaUDC). The I(1)/I(3) ratio of pyrene fluorescence and the solubility dependence of solution pH were employed to determine the CMC values. As the results, a certain concentration range for the CMC and a stepwise molecular aggregation for micellization were found reasonable. Using a stepwise association model of the bile salt anions, the mean aggregation number (n) of NaCDC micelles was found to increase with the total anion concentration, while the n values decreased with increasing temperature; 9.1, 8.1, 7.4, and 6.3 at 288.2, 298.2, 308.2, and 318.2 K, respectively, at 50 mmol dm(-3). The results from four unconjugated bile salts indicate that the number, location, and orientation of hydroxyl groups in the steroid nucleus are quite important for growth of the micelles. Activity of the counterion (Na(+)) was determined by a sodium ion selective electrode in order to confirm the low counterion binding to micelles. The solubilized amount of cholesterol into the aqueous bile salt solutions increased in the order of NaUDC相似文献   

A study on the C-terminal membrane anchoring of Escherichia coli penicillin-binding protein 5.

Escherichia coli penicillin-binding protein 5 (PBP5) anchors to the inner membrane in a pH-dependent manner via a C-terminal amphiphilic alpha-helix. Low pH was found to enhance both levels of PBP5 membrane anchoring and levels of alpha-helicity in an aqueous PBP5 C-terminal homologue, which led to the suggestion that levels of PBP5 membrane anchoring are related to levels of PBP5 C-terminal alpha-helicity. Here we have used Fourier-transformed infrared spectroscopy (FTIR) and a peptide homologue of the PBP5 C-terminal sequence to investigate the effect of pH on the conformational behavior of this sequence at a lipid interface and on its ability to interact with lipid. Our results suggest that the membrane-anchoring mechanism of PBP5 is unlikely to involve conformational change in the protein's C-terminal region and may therefore involve conformational changes in the protein's ectomembranous domain.     

Discrimination of single‐porin Escherichia (E.) coli mutants by ATR and transmission mode FTIR spectroscopy

Vibrational spectroscopy has long been used in bacterial identification with different levels of taxonomic discrimination but its true potential for intra‐species differentiation remains poorly explored. Herein, both transmission Fourier‐transform infrared (FTIR) and attenuated total reflectance (ATR)‐FTIR spectroscopy are used to analyse E. coli strains that differ solely in their porin expression profile. In this previously unreported approach, the applicability of both FTIR‐spectroscopy techniques is compared with the same collection of unique strains. ATR‐FTIR spectroscopy proved to reliably distinguish between several E. coli porin mutants with an accuracy not replicated by FTIR in transmission mode (using previously optimized procedures). Further studies should allow the identification of the individual contribution of the single porin channel to the overall bacterial infrared spectrum and of molecular predictive patterns of porin alterations. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Lipid organization in human and porcine stratum corneum differs widely, while lipid mixtures with porcine ceramides model human stratum corneum lipid organization very closely

The conformational disordering and lateral packing of lipids in porcine and human isolated stratum corneum (SC) was compared using Fourier transform infrared spectroscopy (FTIR). It was shown that SC of both species differ markedly, porcine SC lipids being arranged predominantly in a hexagonal lattice while lipids in human SC are predominantly packed in the denser orthorhombic lattice. However, the lipid organization of equimolar ceramide:cholesterol:free fatty acid (CER:CHOL:FFA) mixtures prepared with isolated porcine CER or human CER is very similar, only the transition temperatures differed being slightly lower in mixtures with porcine CER. Therefore, the difference in lateral packing between human and porcine stratum corneum is not due to the difference in CER composition. Furthermore, it is possible to use more readily available porcine CER in model lipid mixtures to mimic lipid organization in human SC. As the equimolar porcine CER:CHOL:FFA mixtures closely mimic the lipid organization in human SC, both human SC and this mixture were selected to examine the effect of glycerol on the lipid phase behaviour. It was found that high concentrations of glycerol change the lamellar organization slightly, while domains with an orthorhombic lateral packing are still observed.     

Lipid organization in human and porcine stratum corneum differs widely, while lipid mixtures with porcine ceramides model human stratum corneum lipid organization very closely

The conformational disordering and lateral packing of lipids in porcine and human isolated stratum corneum (SC) was compared using Fourier transform infrared spectroscopy (FTIR). It was shown that SC of both species differ markedly, porcine SC lipids being arranged predominantly in a hexagonal lattice while lipids in human SC are predominantly packed in the denser orthorhombic lattice. However, the lipid organization of equimolar ceramide:cholesterol:free fatty acid (CER:CHOL:FFA) mixtures prepared with isolated porcine CER or human CER is very similar, only the transition temperatures differed being slightly lower in mixtures with porcine CER. Therefore, the difference in lateral packing between human and porcine stratum corneum is not due to the difference in CER composition. Furthermore, it is possible to use more readily available porcine CER in model lipid mixtures to mimic lipid organization in human SC. As the equimolar porcine CER:CHOL:FFA mixtures closely mimic the lipid organization in human SC, both human SC and this mixture were selected to examine the effect of glycerol on the lipid phase behaviour. It was found that high concentrations of glycerol change the lamellar organization slightly, while domains with an orthorhombic lateral packing are still observed.