Carsten Selle

FTIR-spectroscopic characterization of phosphocholine-headgroup model compounds

Abstract The polar headgroups are an important molecular subregion of phospholipids since they mediate a substantial part of the interactions emerging with other constituents of biological membranes, be it inherent macromolecules (proteins) or water as forming the natural environment. FTIR spectroscopy is well proven for characterizing aspects of weak interactions, above all hydrogen bonding. We have used this method to study solid deposits of a number of selected lipid models, such as choline, acetylcholine and methylphosphocholine (MePC), and compared them with the common phospholipid dimyristoylglycerophosphocholine (DMPC), at different degrees of hydration, which were varied via relative humidity (RH). At low RHs, only MePC and DMPC take up considerable amounts of water, thus elucidating the essential role of phosphate groups in the first stages of lipid hydration. The progress of PC-headgroup hydration can be sensitively monitored by the wavenumber decrease of the band owing to antisymmetric PO 2 − stretching vibration. Concomitant variations of the spectral parameters of ν CH bands of MePC reveal that conformational changes may simultaneously occur in the headgroup. Surprisingly to us, the model compounds display qualitative differences in the appearance of their ν CH bands, which are most probably a result of substituent effects.

Hydration of DMPC and DPPC at 4°C produces a novel subgel phase with convex–concave bilayer curvatures

Abstract Hydration of dimyristoyl- and dipalmitoylphosphatidylcholines at 4°C results in the formation of a characteristic subgel phase designated Pcc. Examination of the phase by freeze-fracture electron microscopy shows convex–concave deformations of the planar bilayer which are of two types. A smaller type with a radius of curvature of about 20 nm predominates in DMPC, and a larger type with about 70 nm radii of curvatures dominates in DPPC. The Pcc phase can also be formed in samples hydrated at temperatures above the main phase transition if the dispersion is frozen slowly and subsequently incubated at 4°C for several days. The subgel Pcc phase was distinguished from the subgel Lc phase by the temperature of transition, packing of the acyl chains on the basis of wide-angle X-ray diffraction, and 2H-NMR spectra characteristic of a ‘solid-ordered’ phase. Vibrational spectra of the carbonyl and phosphate regions are consistent with a partially reduced hydration state. The origin of the convex–concave bilayer deformation is believed to result from constraints imposed by limiting hydration of the headgroup and a frustration arising from the spontaneous curvature of both monolayers.

COMPARATIVE FTIR SPECTROSCOPIC STUDY UPON THE HYDRATION OF LECITHINS AND CEPHALINS

Abstract Fourier-transform infrared (FTIR) spectroscopy has been used to study the gradual hydration of films prepared from some ubiquitous phospholipids. The diacyl lecithins (PCs, DPPC and DOPC) and cephalins (PEs, DPPE and DOPE) are representative for compounds with saturated (palmitoyl) and unsaturated (oleoyl) hydrocarbon chains, respectively. The adsorption isotherms obtained spectroscopically reveal that lecithins take up more water than cephalins, independently of the nature of their acyl chains. Furthermore, whereas the two lecithins exhibit more or less substantial and continuous wavenumber shifts for the well-assigned infrared absorption bands arising from the vibrations of their polar parts, the cephalins show a significantly diverse pattern with characteristic differences determined by the chemical nature of their tails. DPPE is, as far as reflected by its IR-band parameters, completely invariant against hydration, i.e. no influence of the water imbibed by the film is visible. Such a finding can be interpreted in terms of a tight hydrogen-bonding network formed between the phosphate and ammonium groups of DPPE. This explanation is confirmed by the results of relevant quantum-chemical AM1 calculations another part of which is also suitable to rationalize the wavenumber downwards shifts of the PO−2 and CO stretching-vibration bands of lecithins observed upon hydration. DOPE differs from DPPE by undergoing rather dramatic hydration-induced wavenumber shifts of the IR bands due to its polar parts. Contrarily to the lecithins, however, these displacements are restricted to a very narrow range of water activities. This behaviour suggests the existence of a lyotropic phase transition ascribed to a conversion from H∥ to Pα occurring when the water content of DOPE is decreased.

Fourier-transform infrared spectroscopic evidence for a novel lyotropic phase transition occurring in dioleoylphosphatidylethanolamine

Abstract The hydration of 1,2-dioleoyl-sn-glycerophosphoethanolamine (DOPE) has been studied by Fourier-ttransform infrared spectroscopy applied to macroscopically oriented films in comparison to related phospholipids (DPPE and DPPC). DOPE differs from the other lipids mainly in one respect, since it displays a number of relatively drastic, correlated spectroscopic changes within a distinct very narrow range of water activities at an ambient relative humidity of ∼ 50%. These striking alterations can be observed especially for a set of infrared bands due to the headgroup (phosphoethanolamine) moieties whose parameters are simultaneously changed at a certain degree of hydration estimated to be less than one in the water-per-DOPE molecular ratio. This unique spectral behaviour being much more dramatic than, for instance, that observed for the main transition of lipids seems to reflect the occurrence of considerable structural reorientations within the polar part of DOPE molecules and may be explained as indicating the existence of a lyotropic phase transition. Discussion of the peculiar nature of this transformation results in a tentative assignment of the participating phases as belonging to the non-lamellar aggregations enclosing most probably the inverse hexagonal phase (at higher hydration) and the inverse fluid ribbon phase (at lower hydration).

Fourier transform infrared spectroscopy as a probe for the study of the hydration of lipid self-assemblies. II. Water binding versus phase transitions.

The gradual hydration of phospholipid films can be effectively probed by Fourier transform infrared (FTIR) spectroscopy (cf. part I of this series). The hydration-induced changes observed for lipid IR-absorption bands are probably composed of contributions arising from the effects of both the direct binding of water molecules and the thereby caused conformational changes and phase transitions in the lipid molecules and assemblies, respectively. In this article, an attempt is made to attribute some of the more indicative spectroscopic results to these molecular and supermolecular processes with a view to separating their individual contributions to the relevant spectroscopic data. This is done by considering a series of suitable PLs consisting of the palmitoyl and oleoyl lecithins, DPPC, DOPC, POPC, and OPPC, and one cephalin, DOPE. This choice of PCs and DOPE means that at room temperature and different degrees of hydration, several phase states including lamellar gel and liquid crystalline as well as certain nonlamellar phases are covered. The separation of the water-binding and phase-transition contributions to the FTIR-spectroscopic data, we believe, is clearly demonstrated by interpreting the hydration-dependent wavenumber shifts of the nu C=O band of the PCs. Carbonyl groups are affected to a more significant degree for lipids arrayed in the L alpha phase than in the gel phase. A number of spectral features reveal the lyotropically triggered chain-melting transition as well as other structural rearrangements of PCs. This is discussed in detail and demonstrates the excellent sensitivity of the FTIR methodology for the study of such systems.

Chain-length dependence of the hydration properties of saturated phosphatidylcholines as revealed by FTIR spectroscopy

Abstract Phospholipids — as biomembrane constituents — are an interesting class of molecules, and among them, phosphatidylcholines (PCs) or lecithins are most abundant in eucaryotic organisms. We have used FTIR spectroscopy to explore hydration-induced phenomena in symmetric, even-numbered, saturated diacyl PCs with varying chain length, n , of 10–22. These compounds were studied at room temperature as multilamellar films dependent on water activity which was regulated via the ambient relative humidity (RH). The PCs were compared in terms of water-uptake capacity by analyzing adsorption isotherms obtained from IR spectra. As demonstrated in the data, this parameter is governed by the phase type adopted in the lipid assemblies rather than by the sheer chain length. Short-chain lipids with n ≤14 which are shown to undergo lyotropic main transitions at room temperature and to adopt the lamellar liquid–crystalline phase can imbibe considerably more water than the other PCs maintaining a rigid phase at any RH. Chain melting is not systematically correlated with spectral features due to bands assigned to polar IR-active groups (phosphate, carbonyl).

FTIR spectroscopic characterization of a cationic lipid–DNA complex and its components

FTIR spectroscopy is used to study structural aspects of ternary complexes formed by the cationic lipid dimyristoyltrimethylammoniumpropane (DMTAP), the zwitterionic lipid dimyristoylphosphatidylcholine (DMPC), and deoxyribonucleic acid (DNA). Spectra of the single components are compared with those obtained for both equimolar DMPC–DMTAP mixture and lipid–DNA complex. The IR spectra of mixed lipid–DNA phases are strongly dominated by the lipidic absorption bands. This allows one to easily monitor, in particular, the thermotropic phase behaviour of lipid within the complex. The IR spectra of DNA intercalated between cationic lipid bilayers are determined by subtracting corresponding pure lipid spectra from lipid–DNA complex spectra. These difference spectra indicate deviations of lipid–associated DNA from B-form DNA. Furthermore, two additional water bands arise at positions different from those known for lipid- and DNA-bound water which are indicative of two distinct states of hydration in lipid–DNA complexes. The pure lipid DMTAP exhibits unusual spectroscopic features at the temperature of chain melting, Tm, near 53°C, which are attributed to the existence of a crystalline, headgroup-interdigitated phase existing at temperatures below Tm, in accordance with X-ray diffraction and differential scanning calorimetry (DSC) data.

Measurement of diffusion in Langmuir monolayers by single-particle tracking

There is a great amount of literature available indicating that membranes are inhomogeneous, complex fluids. For instance, observation of diffusion in cell membranes demonstrated confined motion of membrane constituents and even subdiffusion. In order to circumvent the small dimensions of cells leading to weak statistics when investigating the diffusion properties of single membrane components, a technique based on optical microscopy employing Langmuir monolayers as membrane model systems has been developed in our lab. In earlier work, the motion of labeled single lipids was visualized. These measurements with long observation times, thus far only possible with this method, were combined with respective Monte-Carlo simulations. We could conclude that noise can lead in general to the assumption of subdiffusion while interpreting the results of single-particle-tracking (SPT) experiments within membranes in general. Since the packing density of lipids within monolayers at the air/water interface can be changed easily, inhomogeneity with regard to the phase state can be achieved by isothermal compression to coexistence regions. Surface charged polystyrene latexes were used as model proteins diffusing in inhomogeneous monolayers as biomembrane mimics. Epifluorescence microscopy coupled to SPT revealed that domain associated, dimensionally reduced diffusion can occur in these kinds of model systems. This was caused by an attractive potential generated by condensed domains within monolayers. Monte-Carlo simulations supported this view point. Moreover, long-time simulations show that diffusion coefficients of respective particles were dependent on the strength of the attractive potential present: a behavior reflecting altered dimensionality of diffusion. The widths of those potentials were also found to be affected by the domain size of the more ordered lipid phase. In biological membrane systems, cells could utilize these physical mechanisms to adjust diffusion properties of membrane components.

Comparative FTIR-spectroscopic studies of the hydration of diphytanoylphosphatidylcholine and -ethanolamine

Abstract Lipids with phytanyl or phytanoyl chains are of basic interest because of their natural occurrence in the membranes of extremophilic archaebacteria and frequent use in biochemical studies and applications as a model for stable bilayers. Since data from physico-chemical studies of phytanoyl lipids in terms of structural and phase properties are rare and partly discrepant, we have studied films of diphytanoylphosphatidylcholine (DPhPC) and its ethanolamine analogue (DPhPE) in dependence on water activity using Fourier-transform infrared spectroscopy. DPhPC imbibes much more water than DPhPE as substantiated by Karl–Fischer-titration and gravimetric data. Marker bands due to the methylene stretching vibrations indicate an extremely high disorder of the acyl chains of both lipids over the whole hydration range. The spectral features of DPhPE are different from those of DPhPC in many respects but similar to those of DPhPE. This similarity encourages us to conclude a predominance of the inverted hexagonal H II phase in DPhPE. Hydration-induced structural changes of DPhPC are indicated by discontinuous wavenumber shifts at water activities of 0.6–0.8, and towards very low water activities for DPhPE, suggesting nonlamellar–lamellar and nonlamellar–nonlamellar phase transitions, respectively, to proceed. Thus, nonlamellar phases may play an important role also in DPhPC.

Physicochemical characterisation of human stratum corneum lipid liposomes.

Liposomes were prepared from an extract of all human stratum corneum lipids (hSCL) and characterised in terms of temperature and the presence of Ca2+ by different physicochemical methods. Vesicle aggregation and lateral phase separation were induced by divalent cations with Ca2+ being more efficient than Mg2+. At 24.1 degrees C, i.e. well below physiological temperatures the suspensions consisted of a lamellar phase and crystalline cholesterol. At and above 37 degrees C, this cholesterol surplus was dissolved in the hSCL membranes. However, melting of the hSCL was not completed up to 60 degrees C. The presence of Ca2+ (> or = 9 mM) induced lateral phase separation and fusion of vesicles into extended multilamellar lipid sheets (MLLS) at and above 32.5 degrees C. Upon a subsequent cooling cycle recrystallisation of cholesterol occurred within the MLLS. Finally, membrane mixing of hSCL liposomes with vesicles made of synthetic lipids was investigated. No mixing was observed between either of DPPE/oleic acid, DPPC/DPPE, DPPC/lyso-PC and hSCL liposomes. Mixtures of DPPC/cholesterol hemisuccinate showed a temperature-dependent membrane mixing behaviour, whilst hSCL liposomes and phosphatidylserine liposomes fused temperature-independently with hSCL liposomes.