Derek Marsh

Different membrane anchoring positions of tryptophan and lysine in synthetic transmembrane α-helical peptides.

Specific interactions of membrane proteins with the membrane interfacial region potentially define protein position with respect to the lipid environment. We investigated the proposed roles of tryptophan and lysine side chains as “anchoring” residues of transmembrane proteins. Model systems were employed, consisting of phosphatidylcholine lipids and hydrophobic α-helical peptides, flanked either by tryptophans or lysines. Peptides were incorporated in bilayers of different thickness, and effects on lipid structure were analyzed. Induction of nonbilayer phases and also increases in bilayer thickness were observed that could be explained by a tendency of Trp as well as Lys residues to maintain interactions with the interfacial region. However, effects of the two peptides were remarkably different, indicating affinities of Trp and Lys for different sites at the interface. Our data support a model in which the Trp side chain has a specific affinity for a well defined site near the lipid carbonyl region, while the lysine side chain prefers to be located closer to the aqueous phase, near the lipid phosphate group. The information obtained in this study may further our understanding of the architecture of transmembrane proteins and may prove useful for refining prediction methods for transmembrane segments.

Electron spin resonance: spin labels.

In general biological membranes possess no intrinsic paramagnetism and hence in the unlabeled state do not give rise to an electron spin resonance (ESR) spectrum. The introduction of a stable free radical (“spin label”) thus enables one to use ESR spectroscopy to study specific environments within the membrane. The spin label which is invariably used is the nitroxide radical, which has a three-line nitrogen hyperfine structure whose splitting varies with the orientation of the magnetic field relative to the nitroxide axes. It is this spectral anisotropy which has made spin label ESR such a powerful tool in the study of the molecular motions which are the characteristic feature of the highly dynamic structure of biological membranes. The nitroxide hyperfine splittings are partially averaged by the anisotropic motion, which gives a measure of the motional amplitude, and the linewidths are differentially broadened by an extent which depends on the rate of molecular motion. Other important features of the spin label spectra are the broadening by intermolecular label-label interactions and the ability to detect compartmentation of the label by quantitating spectral lineheights after selectively removing accessible spin-label signals by chemical reducing agents. Label-label interactions arise from two sources: the Heisenberg exchange interaction which is essentially a contact interaction and is therefore capable of measuring translational diffusion; and the dipole-dipole interaction which depends on the distance apart of the labels and is therefore capable of measuring intermolecular separations. Quantitation after treatment with reducing agents is chiefly concerned with the measurement of transport properties: of spin-label substrates or reducing agents, or the translocation of labelled lipid molecules.

Protein modulation of lipids, and vice-versa, in membranes.

This review describes: (i) perturbations of the membrane lipids that are induced by integral membrane proteins, and reciprocally, (ii) the effects that the lipids may have on the function of membrane-associated proteins. Topics of the first category that are covered include: stoichiometry and selectivity of the first shell of lipids associated at the intramembranous perimeter of transmembrane proteins; the chain configuration and exchange rates of the first-shell lipids; the effects of transmembrane peptides on transbilayer movement of lipids (flip-flop); the effects of membrane proteins on lipid polymorphism and formation of non-lamellar phases; and the effects of hydrophobic mismatch on lipid chain configuration, phase stability and selectivity of lipid-protein association. Topics of the second category are: the influence of lipid selectivity on conformational changes in the protein; the effects of elastic fluctuations of the lipid bilayer on protein insertion and orientation in membranes; the effects of hydrophobic matching on intramembrane protein-protein association; and the effects of intrinsic lipid curvature on membrane integration, oligomer formation and activity of membrane proteins.

Structure, dynamics and composition of the lipid-protein interface. Perspectives from spin-labelling.

Implications of the data on lipid-protein interactions involving integral proteins that are obtained from EPR spectroscopy with spin-labelled lipids in membranes are reviewed. The lipid stoichiometry, selectivity and exchange dynamics at the lipid-protein interface can be determined, in addition to information on the configuration and rotational dynamics of the protein-associated lipid chains. These parameters, particularly the stoichiometry and selectivity, are directly related to the intramembranous structure and degree of oligomerisation of the integral protein, and conversely may be used to study the state of assembly of such proteins in the membrane. Insertion of proteins into membranes can be studied by analogous methods. Comparison with the results obtained from integral proteins helps to define the extent of membrane penetration and degree of transmembrane crossing that are relevant to protein translocation mechanisms.

Cholesterol-induced fluid membrane domains: A compendium of lipid-raft ternary phase diagrams.

The biophysical underpinning of the lipid-raft concept in cellular membranes is the liquid-ordered phase that is induced by moderately high concentrations of cholesterol. Although the crucial feature is the coexistence of phase-separated fluid domains, direct evidence for this in mixtures of cholesterol with a single lipid is extremely sparse. More extensive evidence comes from ternary mixtures of a high chain-melting lipid and a low chain-melting lipid with cholesterol, including those containing sphingomyelin that are taken to be a raft paradigm. There is, however, not complete agreement between the various phase diagrams and their interpretation. In this review, the different ternary phase diagrams of cholesterol-containing systems are presented in a uniform way, using simple x,y-coordinates to increase accessibility for the non-specialist. It is then possible to appreciate the common features and examine critically the discrepancies and hence what direct biophysical evidence there is that supports the raft concept.

Control of the structure and fluidity of phosphatidylglycerol bilayers by pH titration

Abstract Titration of the single dissociable proton in phosphatidylglycerol bilayers not only shifts the ordered-fluid phase transition but also changes the bilayer fluidity in the region above the phase transition, and gives rise to a quite different bilayer structure in the region below the phase transition: 1. 1. The ordered-fluid phase transition temperatures of dimyristoyl and dipalmitoyl phosphatidylglycerol bilayers have been measured as a function of bulk pH in 0.1 M salt using water-lipid partitioning spin labels. From the dissociation curve obtained, it is found that the one titrable proton has an apparent p K a of 2.9 for bilayers of both lipids. 2. 2. In the fully ionized state, these phosphatidylglycerols not only give very similar transition temperatures (23°C for dimyristoyl and 40°C for dipalmitoyl chains) and pre-transition temperatures to the similar chain length phosphatidylcholines, but also show identical bilayer structures at corresponding temperatures when examined by freeze-fracture electron microscopy: i.e. defects, ripples and jumbled patterns, in the ordered, pre-transitional and fluid phases, respectively. 3. 3. When the phosphatidylglycerols are fully protonated, the bilayer pre-transition, as monitored by the spin labels, is absent and the main transition is somewhat broader and increased in temperature by approx. 17°C. Exclusively smooth bilayers are observed by electron microscopy at all temperatures. This suggests that, contrary to the situation in the charged bilayers, the phosphatidylglycerol molecules are not tilted relative to the bilayer normal in the ordered phase. 4. 4. In fluid bilayers, above the main transition temperature, the fluidity is found to be greater when the phosphatidylglycerol molecules are charged than when uncharged, due to the increased intermolecular separation caused by electrostatic repulsion. The results demonstrate that the structure and fluidity of charged lipid bilayer membranes can be changed isothermally without the mediation of the ordered-fluid phase transition.

Polarity and permeation profiles in lipid membranes

The isotropic 14N-hyperfine coupling constant, a\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{o}^{N}}}\end{equation*}\end{document}, of nitroxide spin labels is dependent on the local environmental polarity. The dependence of a\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{o}^{N}}}\end{equation*}\end{document} in fluid phospholipid bilayer membranes on the C-atom position, n, of the nitroxide in the sn-2 chain of a spin-labeled diacyl glycerophospholipid therefore determines the transmembrane polarity profile. The polarity variation in phospholipid membranes, with and without equimolar cholesterol, is characterized by a sigmoidal, trough-like profile of the form {1 + exp [(n − no)/λ]}−1, where n = no is the point of maximum gradient, or polarity midpoint, beyond which the free energy of permeation decreases linearly with n, on a characteristic length-scale, λ. Integration over this profile yields a corresponding expression for the permeability barrier to polar solutes. For fluid membranes without cholesterol, no ≈ 8 and λ ≈ 0.5–1 CH2 units, and the permeability barrier introduces an additional diffusive resistance that is equivalent to increasing the effective membrane thickness by 35–80%, depending on the lipid. For membranes containing equimolar cholesterol, no ≈ 9–10, and the total change in polarity is greater than for membranes without cholesterol, increasing the permeability barrier by a factor of 2, whereas the decay length remains similar. The permeation of oxygen into fluid lipid membranes (determined by spin-label relaxation enhancements) displays a profile similar to that of the transmembrane polarity but of opposite sense. For fluid membranes without cholesterol no ≈ 8 and λ ≈ 1 CH2 units, also for oxygen. The permeation profile for polar paramagnetic ion complexes is closer to a single exponential decay, i.e., no lies outside the acyl-chain region of the membrane. These results are relevant not only to the permeation of water and polar solutes into membranes and their permeabilities, but also to depth determinations of site-specifically spin-labeled protein residues by using paramagnetic relaxation agents.

Cooperativity of the phase transition in single- and multibilayer lipid vesicles

The effect of membrane morphology on the cooperativity of the ordered-fluid, lipid phase transition has been investigated by comparing the transition widths in extended, multibilayer dispersons of dimyristoyl phosphatidyl-choline, and also of dipalmitoyl phosphatidylcholine, with those in the small, single-bilayer vesicles obtained by sonication. The electron spin resonance spectra of three different spin-labelled probes, 2,2,6,6-tetramethylpiperdine-N-oxyl, phosphatidylcholine and stearic acid, and also 90 degrees light scattering and optical turbidity measurements were used as indicators of the phase transition. In all cases the transition was broader in the single-bilayer vesicles than in the multibilayer dispersions, corresponding to a decreased cooperativity on going to the small vesicles. Comparison of the light scattering properties of centrifuged and uncentrifuged, sonicated vesicles suggests that these are particularly sensitive to the presence of intermediate-size particles, and thus the spin label measurements are likely to give a more reliable measure of the degree of cooperativity of the small, single-bilayer vesicles. Application of the Zimm and Bragg theory ((1959) J. Chem. Phys. 31, 526-535) of cooperative transitions to the two-dimensional bilayer system shows that the size of the cooperative unit, 1/square root sigma, is a measure of the mean number of molecules per perimeter molecule, in a given region of ordered or fluid lipid at the centre of the transition. From this result it is found that it is the vesicle size which limits the cooperativity of the transition in the small, single-bilayer vesicles. The implications for the effect of membrane structure and morphology on the cooperativity of phase transitions in biological membranes, and for the possibility of achieving lateral communication in the plane of the membrane, are discussed.

Lipid membranes with grafted polymers: physicochemical aspects

Membranes grafted with water-soluble polymers resist protein adsorption and adhesion to cellular surfaces. Liposomes with surface-grafted polymers therefore find applications in drug delivery. The physicochemical properties of polymer-grafted lipid membranes are reviewed with mean-field and scaling theories from polymer physics. Topics covered are: mushroom-brush transitions, membrane expansion and elasticity, bilayer-micelle transitions, membrane-membrane interactions and protein-membrane interactions.

Rhodopsin-lipid associations in bovine rod outer segment membranes. Identification of immobilized lipid by spin-labels

Rhodopsin-lipid interactions have been studied in bovine rod outer segment (ROS) membranes by using spin-labels. Spin-labeled fatty acid, sterol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, and phosphatidic acid molecules all display a two-component spectrum when probing ROS membranes. One of the spectral components represents 33-43% of the total spectral intensity and is characteristic of a strongly immobilized nitroxide spin-label. This immobilized component is resolved from -4 to 37 O C . The remaining 67-57% of the integrated spectral intensity has a very similar form to the spectra of the same spin-labels in bilayers of extracted ROS membrane lipid. A small selectivity for the immobilized regions of ROS M a n y important biological functions are performed by proteins organized in lipid bilayer membranes (Sandemann, 1978), and an understanding of the interactions between these proteins and their lipids is therefore of particular importance. Rod outer segment (ROS’) membranes are well suited to the study of the interactions between integral membrane proteins and the lipid matrix for a variety of reasons. Firstly, rhodopsin constitutes 85-90% of the total membrane protein of bovine ROS membranes (Montal & Korenbrot, 1976; O’Brien, 1978; Daemen, 1973; Papermaster et al., 1976), and therefore any protein-lipid interactions will, in all probability, be directly due to rhodopsin-lipid associations. Such a level of single protein enrichment is normally achieved by reconstitution, recombination, or specific enrichment procedures which suffer from the hazards of either protein aggregation or denaturation or both and of detergent removal, which is often a long and incomplete process. ROS membranes are isolated without the use of detergents or of enrichment processes involving delipidation or protein loss. Secondly, a limited amount of structural information for rhodopsin is available. This has come from X-ray diffraction studies (Charbre, 1975), X-ray scattering (Sardet et al., 1976) and neutron-scattering (Osborne et al., 1978) data, and ultracentrifugation experiments (Lewis et al., 1974), and some dimensional information has been determined (Sardet et al., 1976; Osborne et al., 1978). Such parameters are useful when interpreting observations of the interactions between lipids and integral membrane proteins in structural terms. Thirdly, ROS membranes are particularly interesting since rhodopsin, within its membrane environment, is responsible for the primary step in visual perception, leading from light absorption to nerve excitation. This process involves both the full photolytic cycle of rhodopsin, including regeneration, and the consequent modulation of the cytoplasmic activity of an internal transmitter through conformational changes taking From the Max-Planck-Institut fur biophysikalische Chemie, Abteilung Spektroskopie, D-3400 Gottingen-Nikolausberg, Federal Republic of Germany. Received May 29, 1979. I.D.V. was the recipient of a DFG exchange stipend to the Abteilung Biochemische Kinetik.