Dennis Chapman

Preservation of Membranes in Anhydrobiotic Organisms: The Role of Trehalose

Trehalose is a nonreducing disaccharide of glucose commonly found at high concentrations in anhydrobiotic organisms. In the presence of trehalose, dry dipalmitoyl phosphatidylcholine (DPPC) had a transition temperature similar to that of the fully hydrated lipid, whereas DPPC dried without trehalose had a transition temperature about 30 degrees Kelvin higher. Results obtained with infrared spectroscopy indicate that trehalose and DPPC interact by hydrogen bonding between the OH groups in the carbohydrate and the polar head groups of DPPC. These and previous results show that this hydrogen bonding alters the spacing of the polar head groups and may thereby decrease van der Waals interactions in the hydrocarbon chains of the DPPC. This interaction between trehalose and DPPC is specific to trehalose. Hence this specificity may be an important factor in the ability of this molecule to stabilize dry membranes in anhydrobiotic organisms.

Biomembrane surfaces as models for polymer design: the potential for haemocompatibility☆

A major restriction in the application of polymeric biomaterials is the propensity of their surfaces to support thrombosis. Theoretical approaches to the design of thromboresistant polymers have been inadequate because of the complexity of surface thrombosis. We have developed a new, practical approach to this problem--the design of polymers which mimic the thromboresistant surfaces of blood cell membranes. Haemostatic processes are mediated by reactions which occur at membrane-plasma interfaces. The extra-cellular surfaces of the plasma membranes of red blood cells and quiescent platelets are thromboresistant; in contrast, their cytoplasmic surfaces are thrombogenic. The simplest common feature among the blood-compatible cellular and model membranes is the high content of the electrically neutral phospholipids which contain the phosphorylcholine head group. We have developed model systems of biological membranes which utilize polymerizable phosphatidylcholines and which mimic nonreactive cell surfaces. Polymeri phospholipids represent a new class of hybrid biomaterials with characteristics both of biomembranes (polar surfaces, nonthrombogenic, low antigenic potential and low permeability) and of synthetic polymers (chemical and physical stability).

Intrinsic protein—lipid interactions: Physical and biochemical evidence

The present consensus view of biomembrane structure is that a lipid bilayer is the basic matrix into which and around which the various proteins are situated. Not only can the proteins be attached to the outside of the lipid bilayer (the extrinsic proteins) but in many cases the proteins (the intrinsic proteins) are embedded within and can span the bilayer. Associated with this consensus view of biomembrane structure is the idea that in many, but not in all, cases the lipid matrix is in a fluid condition where the lipids are essentially above their Tc transition tenlperature and able to diffuse along the bilayer length. Not all biomembranes are highly fluid of course, and some contain crystalline lipid at the growth temperature of the cell. Many cell membrane systems (those without cholesterol) can undergo crystallisation of their biomembrane lipids when they are cooled slowly to lower tem~ratures [ 1,2f.

Phospholipid phase transitions. Effects of n-alcohols, n-monocarboxylic acids, phenylalkyl alcohols and quatenary ammonium compounds

The interactions of a series of alcohols, acids and quaternary ammonium salts with a phosphatidylcholine-water model biomembrane (dipalmitoyl phosphatidycholine) system have been studied using differential scanning calorimetry. In particular the effects of these molecules upon the lipid endothermic phase transitions were investigated over a range of concentrations. A variety of effects was observed. (a) Those molecules which shift or broaden the main lipid transition can also remove the pretransition endotherm. (b) n-Alcohols and n-monocarboxylic acids containing the same number of carbon atoms have very similar effects at molar concentrations up to 40%. Those molecules containing 12 or more carbon atoms raise the main lipid phase transition whilst those molecules containing 10 or less carbon atoms lower this transition temperature. (c) The phase diagram of stearoyl alcohol in the phosphatidylcholine-water system shows the formation of lipid-alcohol complexes. (d) Alkyl trimethyl ammonium bromides showed behaviour which differs considerably from n-alcohols and n-carboxylic acids of the same chain length. (e) Other alkyltrialkyl and tetraalkylammonium bromides show that a variety of effects on the lipid phase transition can be obtained. (f) With the homologous series of phenyl-alkyl alcohols from benzyl alcohol to 4-phenyl butanol increasing the number of methylenes between the terminal OH and the benzene ring leads to greater interaction between solute and bilayer. The range of different effects obtained with the compounds studied offers a means for introducing various degrees and types of perturbation into membrane systems.

Phospholipid polymers--synthesis and spectral characteristics.

A new approach has been developed for the study of model and natural biomembranes. This involves the cross-linking of diacetylene groups after ultraviolet irradiation. For the study of model biomembranes, pure phospholipids (phosphatidylcholines) have been synthesized containing diacetylene groups in each acyl chain. The physical properties of these lipids have been examined and the conditions under which they polymerise have been determined. Polymerisation occurs when the lipid is in a crystalline phase, either compressed in KBr, dispersed in water (liposomes) or deposited on a suitable support (multilayers). The resultant polymer contains a conjugated backbone and is coloured. The visible spectrum of the phospholipid polymer is sensitive to its environment. Preliminary experiments show that similar polymerisation can be induced in Acholeplasma laidlawii cells grown on diacetylenic fatty acid.

Interactions of helical polypeptide segments which span the hydrocarbon region of lipid bilayers: Studies of the gramicidin alipid-water system

Abstract The polypeptide gramicidin A in a dimeric form is considered to form a helical structure which spans the hydrocarbon region of lipid bilayers. In the present investigation it is used as a model for the interactions of the polypeptide segments of transmembrane proteins within the hydrocarbon region of the lipid bilayers of biomembrane structures. A variety of physical techniques (X-ray diffraction, differential scanning calorimetry, optical and electron microscopy, Raman and electron spin resonance spectroscopy) are applied to a study of the interactions of this polypeptide within the phospholipid bilayers of dimyristoyl and dipalmitoyl lecithins in water, at temperatures both above and below the main endothermic phase transition of the pure lipids. Above the transition temperature of the lipid, the Raman studies show that the polypeptide perturbs the fluid lipid environment and causes a marked decrease in the number of gauche isomers of the lipid hydrocarbon chains, even at quite low relative molar concentrations of the polypeptide to lipid (1:150). At concentrations of phospholipid to polypeptide of less than 5:1, the electron spin resonance studies show the existence of two lipid regions within the bilayer. One region corresponds to the relatively fluid lipid region normally observed at these temperatures and the other to a relatively rigid lipid region. The latter is considered to arise from clusters of the polypeptide in which some of the lipid is entrapped. Below the lipid phase transition temperature, the pretransition endotherm observed with pure lipid-water systems is removed by small molar concentrations of the polypeptide (1:50) and the rippled appearance observed in freeze-fracture electron micrographs with pure dimyristoyl lecithin-water dispersions is replaced by a smooth appearance. The main lipid phase transition becomes broadened by the presence of increasing amounts of the polypeptide within the lipid bilayer as indicated by calorimetry, and electron spin resonance spectroscopy. The enthalpy of the lipid transition decreases linearly with increasing amounts of the polypeptide until, with dipalmitoyl lecithin, a concentration of approximately 20 lipids per polypeptide is reached. This is considered to correspond to the onset of an aggregation process which produces localised polypeptide-lipid clusters within the plane of the membrane. At concentrations of polypeptide less than five lipids per polypeptide, freezefracture electron microscopy shows the presence of liposomes with smooth fracture faces. At higher polypeptide concentrations, sheet-like structures are observed with smooth fracture faces. When a mixed lipid-water system (dilauroyl and dipalmitoyl lecithin) containing low concentrations of the polypeptide is slowly cooled, the calorimetric evidence shows that the polypeptide moves preferentially into the lower melting region of the bilayer, whereas at higher polypcptide eoncentrations a mixing of the two lipids takes place. The various results are discussed to provide insight pertinent to the organisation, interactions, aggregation properties, boundary layer and packing arrangements of helical polypeptides and proteins in reconstituted systems and natural biomembranes.

Intrinsic protein-lipid interactions: Infrared spectroscopic studies of gramicidin A, bacteriorhodopsin and Ca2+-ATPase in biomembranes and reconstituted systems

Abstract Infrared spectroscopy has been applied to the study of a number of aqueous systems of model and natural biomembranes. The absorption bands arising from water and buffer solutions were eliminated by means of an infrared spectrometer data station. Spectra were examined using H 2 O and 2 H 2 O aqueous buffer systems. Pure lecithin-water systems, and various model biomembranes containing cholesterol, gramicidin A, bacteriorhodopsin or Ca 2+ -ATPase were examined. The infrared spectra of the reconstituted biomembranes were compared with those of the corresponding natural biomembranes, i.e. the purple membrane of Halobacterium halobium and also sarcoplasmic reticulum membranes, respectively. Changes in lipid chain conformation caused by the various intrinsic molecules incorporated within the model lipid bilayer structures were monitored by studying the shifts in frequency (cm −1 ) of the CH 2 symmetric and asymmetric absorption bands arising from the lipid chains. The effect of gramicidin A and also the intrinsic proteins, as indicated by the shift of band frequencies, are quite different from that of cholesterol at temperatures above the main lipid transition temperature t c . Cholesterol causes a reduction in gauche isomers which increases with concentration of cholesterol within the lipid bilayer. Whilst gramicidin A and the intrinsic proteins at low concentration cause a reduction of gauche isomers, at higher concentrations of these molecules, however, there is little difference in gauche isomer content when the intrinsic molecule is present compared with that of the fluid lipid alone. These results are considered and compared with previously published studies using deuterium nuclear magnetic resonance spectroscopy on similar model biomembrane systems. Below the lipid t c value, all the intrinsic molecules produce an increase in gauche isomers presumably by disturbing the lipid chain packing in the crystalline lipid arrangement. Information about the polypeptide structure within gramicidin A. the reconstituted proteins and also the proteins in the natural biomembranes was obtained by examining the region of the infrared spectrum between 1600 and 1700 cm −1 associated with the amide I and amide II bands. An examination of the infrared band frequencies of the different systems in this region leads to the conclusions: (1) that gramicidin A within a phospholipid bilayer structure probably has a single helix rather than a double helix structure; (2) that there are differences in band widths of the reconstituted Ca 2+ -ATPase and bacteriorhodopsin compared with the spectra of the corresponding sarcoplasmic reticulum and purple membrane; (3) different membrane proteins adopt different conformations as evinced by a comparison of the spectra of the sarcoplasmic reticulum and purple membrane; (4) the polypeptide arrangement in the purple membrane is mainly helical but the abnormal frequency of the amide I band suggests that some distortion of the helix occurs: and (5) the sarcoplasmic reticulum membrane contains unordered as well as α-helix polypeptide arrangements.

Does Fourier-transform infrared spectroscopy provide useful information on protein structures ?

Abstract Interest in growing in the application of Fourier-transform infrared (FTIR) spectroscopy to the study of biomolecules in an aqueous environment. The increasing popularity of the technique is due to its ease of application to determine the secondary structure of peptides, proteins and also membrane proteins within their native lipid bilayer matrix. The ability to probe structural changes at a molecular level as a function of light, heat, pH or other external factors using difference spectroscopy is making a detailed understanding of the mechanism of action of different proteins possible. The use of isotopically labelled molecules helps in both the assignment of spectra and the attainment of greater specificity, and provides a new approach for the study of protein-protein interactions.

Infrared spectroscopic studies on interactions of water and carbohydrates with a biological membrane

Infrared spectroscopy was used to investigate the changes in bands assigned to phospholipids and proteins in dehydrated and rehydrated sarcoplasmic reticulum. The changes in CH2 and CH3 stretching bands, amide bands, and phosphate stretching bands are similar to shifts in frequency seen for those bands in phospholipid and protein preparations during thermotropic phase transitions and hydration. IR studies on dry trehalose-sarcoplasmic reticulum mixtures show similar results; with increasing trehalose concentration in the dry mixtures, amide and phosphate bands shift to frequencies characteristic of hydrated samples. Changes in bands assigned to OH deformations in the trehalose suggest that the interaction between the carbohydrate and membrane is by means of hydrogen bonding between these -OH groups and membrane components.

Conformational transitions in poly (L-lysine): studies using Fourier transform infrared spectroscopy

Abstract FT-IR spectroscopy has been applied to the study of the pH-induced conformational transitions which occur with poly( l -lysines) of varying molecular weight. The studies were performed in aqueous media under conditions believed from previous CD studies to induce the polypeptide to adopt a particular type of secondary structure. Each different type of secondary structure gives amide I bands with different frequencies. The α-helix secondary structure shows a strong band at 1638 cm−1, random structures a strong band at 1644 cm−1 and the β-sheet arrangement a strong absorption at 1610 cm−1 with a weaker band at 1680 cm−1. The low band frequency of the polypeptide when in an α-helical conformation compared to the band characteristic of α-helical structures in proteins is discussed. Additional components are observed when second derivative analysis is applied to the spectra corresponding to the random and helical conformations. The number of these additional components increases with the α-helical conformation as the molecular weight of the polymer increases. Estimations of the area of the amide I band in the various conformations are made.