The role of membrane thickness in charged protein-lipid interactions

Charged amino acids are known to be important in controlling the actions of integral and peripheral membrane proteins and cell disrupting peptides. Atomistic molecular dynamics studies have shed much light on the mechanisms of membrane binding and translocation of charged protein groups, yet the impact of the full diversity of membrane physico-chemical properties and topologies has yet to be explored. Here we have performed a systematic study of an arginine (Arg) side chain analog moving across saturated phosphatidylcholine (PC) bilayers of variable hydrocarbon tail length from 10 to 18 carbons. For all bilayers we observe similar ion-induced defects, where Arg draws water molecules and lipid head groups into the bilayers to avoid large dehydration energy costs. The free energy profiles all exhibit sharp climbs with increasing penetration into the hydrocarbon core, with predictable shifts between bilayers of different thickness, leading to barrier reduction from 26 kcal/mol for 18 carbons to 6 kcal/mol for 10 carbons. For lipids of 10 and 12 carbons we observe narrow transmembrane pores and corresponding plateaus in the free energy profiles. Allowing for movements of the protein and side chain snorkeling, we argue that the energetic cost for burying Arg inside a thin bilayer will be small, consistent with recent experiments, also leading to a dramatic reduction in pK(a) shifts for Arg. We provide evidence that Arg translocation occurs via an ion-induced defect mechanism, except in thick bilayers (of at least 18 carbons) where solubility-diffusion becomes energetically favored. Our findings shed light on the mechanisms of ion movement through membranes of varying composition, with implications for a range of charged protein-lipid interactions and the actions of cell-perturbing peptides. This article is part of a Special Issue entitled: Membrane protein structure and function.     

The role of retinal in the long-range protein-lipid interactions in bacteriorhodopsin-phosphatidylcholine vesicles

The effects of bacteriorhodopsin analogues and the analogues of a bacteriorhodopsin mutant (D96N) on the lateral organization of lipids have been investigated with lipid species with a variety of acyl chain lengths. The analogues, obtained by regeneration of bacterioopsin or mutant opsin with 14-, 12-, 10-, or 8-fluororetinal, were reconstituted with 1,2-didodecanoyl-sn-glycero-3-phosphocholine, 1,2-ditetradecanoyl-sn-glycero-3-phosphocholine, 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine, and 1,2-dioctadecanoyl-sn-glycero-3-phosphocholine. The phase behavior of the protein-lipid systems was investigated at different temperatures and different protein/lipid molar ratios by analyzing the fluorescence and phase properties of the 1-acyl-2-[8-(2-anthroyl)octanol]-sn-glycero-3-phosphocholine probe. The (8,10,12)-bacteriorhodopsins had a similar effect on the lipid phase transition to that induced by native bacteriorhodopsin: a rigidifying effect on the three shorter lipid species and a fluidifying effect on the longest-chain lipids used. The substitution of retinal with 14-fluororetinal resulted in much stronger effects of the protein on the lipids: a more pronounced up-shift of the lipid phase transition temperature, a rigidifying effect on all the lipids used, and an elongation of the distance over which the hydrophobic thickness of the lipid bilayer was perturbed by the protein. Evidence was provided that retinal contributed to the long-range protein-lipid interactions in bacteriorhodopsin-phosphatidylcholine vesicles. The extent of this contribution was dependent on the retinal structure in close vicinity to the Shiff base and on the compactness of the protein structure.     

Effects of protein-protein and protein-lipid interactions on heme site conformation in the mitochondrial b cytochromes

Removal of lipid from detergent-solubilized succinate cytochrome c reductase by a mild method leads to a series of changes in the optical and EPR spectra of the b cytochromes. This culminates in a state that resembles purified b cytochromes from the same source and bisimidazole ferriheme model complexes. Reconstitution of the lipid-depleted complex with phospholipid restores the native spectra in a significant fraction of the complexes in the early stages of lipid depletion. Once the final state has been reached, however, reconstitution has so far been incapable of restoring described in this communication can be related to a model for integral membrane cytochromes.     

Lipid dynamics and protein-lipid interactions in rat colonic epithelial cell basolateral membranes

Lipid dynamics and lipid-protein interactions were examined in basolateral membranes prepared from rat proximal and distal colonic epithelial cells. The results demonstrate that: (1) these membranes have a high lipid fluidity, as assessed by steady-state fluorescence polarization studies using seven fluorescent probes; (2) lipid compositional differences exist between these membranes but their fluidity is similar; (3) fluorescence polarizations studies, using diphenylhexatriene (DPH), detect a thermotropic transition at 22–23°C in each membrane; (4) several membrane protein activities, including adenylate cyclase and sodium-potassium dependent adenosine triphosphatase ($\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$) appear to be functionally dependent on the physical state of the proximal basolateral membrane's lipid.     

Effects of nitric oxide on protein-lipid interactions in the membranes of the myelinated nerve fiber

The influence of nitric oxide (NO) on the myelinated nerve fiber and the impact of modification of SHgroups of axon and myelin membrane proteins on the amplitude and propagation velocity of action potential (AP), amount of the membrane-bound calcium (Ca _mb ²⁺ , viscosity of the axon membrane, and saturation factor of phospholipid fatty acids (Sf) of myelin have been investigated. We established that the decrease in the number of extracellular SH-groups in membrane proteins induced by p-chloromercuribenzoate (pCMB, 10^?4 M), led to a decrease in the AP amplitude and a reversible desorption of Ca _mb ²⁺ but did not affect the axolemma viscosity and Sf. Nitric oxide (NO) caused a decrease in the AP amplitude and propagation velocity, an increase in the axolemma viscosity and a decrease in Sf of myelin; it also induced a reversible desorption of Ca _mb ²⁺ . Pretreatment of the nerve fiber with pCMB weakened the NO-induced desorption of Pretreatment of the nerve fiber with K⁺-channel blocker tetraethylammonium (10^?2 M) completely abolished the NO-induced change in the amount of Ca _mb ²⁺ . We suppose that NO-mediated changes in axolemma viscosity, Sf of myelin and desorption of Ca _mb ²⁺ affect protein-lipid interactions in axolemma and myelin, which in their turn influence the propagation of AP.     

A light-harvesting antenna protein retains its folded conformation in the absence of protein-lipid and protein-pigment interactions

The first study by nmr of the integral membrane protein, the bacterial light-harvesting (LH) antenna protein LH1 beta, is reported. The photosynthetic apparatus of purple bacteria contains two different kinds of antenna complexes (LH1 and LH2), which consist of two small integral membrane proteins alpha and beta, each of approximately 6 kDa, and bacteriochlorophyll and carotenoid pigments. We have purified the antenna polypeptide LH1 beta from Rhodobacter sphaeroides, and have recorded CD spectra and a series of two-dimensional nmr spectra. A comparison of CD spectra of LH1 beta observed in organic solvents and detergent micelles shows that the helical character of the peptide does not change appreciably between the two milieus. A significantly high-field shifted methyl signal was observed both in organic solvents and in detergent micelles, implying that a similar three-dimensional structure is present in each case. However, the 1H-nmr signals observed in organic solvents had a narrower line width and better resolution, and it is shown that in this case organic solvents provide a better medium for nmr studies than detergent micelles. A sequential assignment has been carried out on the C-terminal transmembrane region, which is the region in which the pigment is bound. The region is shown to have a helical structure by the chemical shift values of the alpha-CH protons and the presence of nuclear Overhauser effects characteristic of helices. An analysis of the amide proton chemical shifts of the residues surrounding the histidine chlorophyll ligand suggests that the local structure is well ordered even in the absence of protein-lipid and protein-pigment interactions. Its structure was determined from 348 nmr-derived constraints by using distance geometry calculations. The polypeptide contains an alpha-helix extending from Leu19 (position of cytoplasmic surface) to Trp44 (position of periplasmic surface). The helix is bent, as expected from the amide proton chemical shifts, and it is similar to the polypeptide fold of the previously determined crystal structure of Rhodopseudomonas acidophila Ac10050 LH2 beta (S. M. Prince et al., Journal of Molecular Biology, 1997, Vol. 268, pp. 412-423). It is concluded that the polypeptide conformation of this region may facilitate assembly of the LH complex.     

Compartmentation of gramicidin 5 in membranes of sensitive bacteria and protein-lipid interactions]

Gramicidin S is sorbed on the isolated membranes of granicidin-sensitive Micrococcus lysodeikticus strain. The antibiotic inhibits the membrane malate dehydrogenase within the temperature range of 9--42 degrees C, i.e. under conditions of gel and liquid-crystalline lipid state; however its effect at 10 degrees C is 10 times as low as is observed at 42 degrees C. The inhibitory effect of gramicidin S on malate dehydrogenase can be eliminated and the antibiotic can be removed from the membrane by an excess of different phospholipids. No transfer of the membrane components on exogenous phospholipids is observed. A prolonged (about 2 hrs, 30 degrees C) incubation of the membranes with gramicidin S results in irreversible inactivation of malate dehydrogenase, although the antibiotic can be still eliminated by an addition of phospholipid emulsions. It is suggested that gramicidin S forms complexes with phospholipids, in which the antibiotic is oriented to water. These complexes disturb the lipid-protein interactions, resulting in relaxation of the binding between the boundary phospholipids and proteins, in the loosening of near-protein lipid zones and simultaneous condensation of acid phospholipids in the whole membrane. Destruction of the lipid zone is accompanied by changes in the enzyme activity, by separation of lipid and protein regions and by transphase enzyme transitions (expulsion or immersion). A slow formation of secondary protein-protein associates may be irreversible.     

Spin label saturation transfer ESR studies of protein-lipid interactions in Photosystem II-enriched membranes

Saturation transfer ESR has been used to study the dynamic behaviour of lipids in the appressed regions of thylakoid membranes from pea seedlings. Four different phospho- and galacto-lipid spin labels (phosphatidylcholine labelled at the 12 or 14 C-atom positions of the sn-2 chain, phosphatidylglycerol labelled at the 14-position of the sn-2 chain, and monogalactosyldiacylglycerol labelled at the 12-position of the sn-2 chain) were used to probe the lipid environment in photosystem II-enriched membranes prepared by detergent extraction. The ESR spectra show that the majority of the lipid in these preparations is strongly motionally restricted. Values for the effective rotational correlation times of the labelled chains were deduced from the lineheight ratios and integrals of thhe saturation transfer ESR spectra. The effective rotational correlation times were found to be in the 10⁵ range, indicating a very low lipid chain mobility which correlates with the low lipid content of these preparations. Comparison of the effective rotational correlation times deduced from the different diagnostic regions of the spectrum revealed little anisotropy in the chain mobility, indicating that the dominant motional mode was trans-gauche isomerization. The effective rotational correlation times deduced from the spectral integrals were similar to those deduced from the lineheight ratios, consistent with the absence of any appreciable fluid lipid component in these preparations. The results also indicate some selectivity of interaction between the lipid species, with phosphatidylcholine exhibiting appreciably slower motion than either phosphatidylglycerol or monogalactosyldiacylglycerol.     

New fluorescent octadecapentaenoic acids as probes of lipid membranes and protein-lipid interactions.

The chemical and spectroscopic properties of the new fluorescent acids all(E)-8, 10, 12, 14, 16-octadecapentaenoic acid (t-COPA) and its (8Z)-isomer (c-COPA) have been characterized in solvents of different polarity, synthetic lipid bilayers, and lipid/protein systems. These compounds are reasonably photostable in solution, present an intense UV absorption band (epsilon(350 nm) approximately 10(5) M(-1) cm(-1)) strongly overlapped by tryptophan fluorescence and their emission, centered at 470 nm, is strongly polarized (r(O) = 0.385 +/- 0.005) and decays with a major component (85%) of lifetime 23 ns and a faster minor one of lifetime 2 ns (D,L-alpha-dimyristoylphosphatidylcholine (DMPC), 15 degrees C). Both COPA isomers incorporate readily into vesicles and membranes (K(p) approximately 10(6)) and align parallel to the lipids. t-COPA distributes homogeneously between gel and fluid lipid domains and the changes in polarization accurately reflect the lipid T(m) values. From the decay of the fluorescence anisotropy in spherical bilayers of DMPC and POPC it is shown that t-COPA also correctly reflects the lipid order parameters, determined by 2H NMR techniques. Resonance energy transfer from tryptophan to the bound pentaenoic acid in serum albumin in solution, and from the tryptophan residues of gramicidin in lipid bilayers also containing the pentaenoic acid, show that this probe is a useful acceptor of protein tryptophan excitation, with R(O) values of 30-34 A.     

Determinants of specificity at the protein-lipid interface in membranes

The complexity of pro- and eukaryotic lipidomes is increasingly appreciated mainly owing to the advance of mass spectrometric methods. Biophysical approaches have revealed that the large number of lipid classes and molecular species detected have implications for the self-organizing potential of biological membranes, resulting in the formation of lateral heterogeneous phases. How membrane proteins are able to adapt specifically to their surrounding heterogeneous matrix, and whether this environment affects protein targeting and function, is therefore a matter of particular interest. Here, we review specific protein-lipid interactions, focusing on the molecular mechanisms that determine specificity at the protein-lipid interface, and on membrane proteins that require lipids as cofactors for their architecture and function.     

Incorporation of outer membrane protein OmpG in lipid membranes: protein-lipid interactions and beta-barrel orientation

OmpG is an intermediate size, monomeric, outer membrane protein from Escherichia coli, with n beta = 14 beta-strands. It has a large pore that is amenable to modification by protein engineering. The stoichiometry ( N b = 20) and selectivity ( K r = 0.7-1.2) of lipid-protein interaction with OmpG incorporated in dimyristoyl phosphatidylcholine bilayer membranes was determined with various 14-position spin-labeled lipids by using EPR spectroscopy. The limited selectivity for different lipid species is consistent with the disposition of charged residues in the protein. The conformation and orientation (beta-strand tilt and beta-barrel order parameters) of OmpG in disaturated phosphatidylcholines of odd and even chain lengths from C(12:0) to C(17:0) was determined from polarized infrared spectroscopy of the amide I and amide II bands. A discontinuity in the protein orientation (deduced from the beta-barrel order parameters) is observed at the point of hydrophobic matching of the protein with lipid chain length. Compared with smaller (OmpA; n beta = 8) and larger (FhuA; n beta = 22) monomeric E. coli outer membrane proteins, the stoichiometry of motionally restricted lipids increases linearly with the number of beta-strands, the tilt (beta approximately 44 degrees ) of the beta-strands is comparable for the three proteins, and the order parameter of the beta-barrel increases regularly with n beta. These systematic features of the integration of monomeric beta-barrel proteins in lipid membranes could be useful for characterizing outer membrane proteins of unknown structure.     

Physicochemical studies of the protein-lipid interactions in melittin-containing micelles

Complexes of melittin with detergents and phospholipids have been characterized by fluorescence, circular dichroism, ultracentrifugation, quasi-elastic light scattering and 1H nuclear magnetic resonance (NMR) experiments. By ultracentrifugation and quasi-elastic light-scattering measurements it is shown that melittin forms stoichiometrically well-defined complexes with dodecylphosphocholine micelles consisting of one melittin molecule and approximately forty detergent molecules. Evidence from fluorescence, circular dichroism and 1H nuclear magnetic resonance experiments indicates that the conformation of melittin bound to micelles of various detergents or of diheptanoyl phosphatidylcholine is largely independent of the type of lipid and furthermore appears to be quite closely related to the conformation of melittin bound to phosphatidylcholine bilayers. 1H NMR is used to investigate the conformation of micelle-bound melittin in more detail and to compare certain aspects of the melittin conformation in the micelles with the spatial structures of monomeric and self-aggregated tetrameric melittin in aqueous solution. The experience gained with this system demonstrates that high resolution NMR of complexes of membrane proteins with micelles provides a viable method for conformational studies of membrane proteins.     

The role of hydrophobic interactions in positioning of peripheral proteins in membranes

Background  

Three-dimensional (3D) structures of numerous peripheral membrane proteins have been determined. Biological activity, stability, and conformations of these proteins depend on their spatial positions with respect to the lipid bilayer. However, these positions are usually undetermined.     

The modulation of Ca-ATPase activity and protein-lipid interactions in the sarcoplasmic reticulum by ATP

The time-course of ATP hydrolysis by Ca-ATPase of purified sarcoplasmic reticulum is biphasic with an initial rate over 1 to 2 min exceeding the subsequent rate. Hydrolysis of GTP and p-nitrophenylphosphate (pNPP) occurs at a slower but constant rate. Arrhenius plots of GTP, p-nitrophenylphosphate and initial rates of ATP hydrolysis all exhibit a discontinuity at about 20-24 degrees C; no breaks are observed in plots of the slower phase of ATP hydrolysis. The effect of substrate hydrolysis on the disposition of the enzyme in the membrane was examined by monitoring the quenching of tryptophan fluorescence by pyrene present in the hydrophobic domain of the membrane. The presence of ATP, but not GTP, prevents a temperature-dependent decrease in fluorescence quenching suggesting that ATP binding causes a change in the protein domain in contact with the membrane lipids.     

A biophysical study of protein-lipid interactions in membranes of Escherichia coli. Fluoromyristic acid as a probe.

Fluorine-19 nuclear magentic resonance spectroscopy and transport assays have been used to investigate and compare the membrane properties of unsaturated fatty acid auxotrophs of two strains of Escherichia coli, K1060B5 and ML 308-225-UFA-8. A fluorinated analog of myristic acid, 8, 8-difluoromyristic acid, can be incorporated into the membrane phospholipids by substitution for oleate in the growth medium. Growth for one generation on 8, 8-difluoromyristate results in a 20% content of fluorinated fatty acid in the membranes, changes in the protein to lipid ratio, and altered transport of methyl beta-D-thiogalactopyranoside. The differences in membrane composition and transport behavior seen in oleate supplemented E. coli K1060B5 relative to ML 308-225-UFA-8 are enhanced by the incorporation of 8, 8-difluoromyristate. The phase transition behavior becomes distinctly different and some differences in lipid organization persist above the transition temperature. Concomitantly, the rate and extent of concentration of methyl beta-D-thiogalactopyranoside are reduced two-fold more in E. coli K1060B5 compared to ML 308-225-UFA-8. Such behavior suggests that these fluorinated fatty acid supplemented strains of E. coli are useful to study subtle differences in protein-lipid interactions and their effects on the function of membrane-bound enzymes.