Lipid-protein interactions in membranes

The interactions of lipids with integral and peripheral proteins can be studied in reconstituted and natural membranes using spin label electron spin resonance (ESR) spectroscopy. The ESR spectra reveal a reduction in mobility of the spin-labelled lipid species, and in certain cases evidence is obtained for a partial penetration of the peripheral proteins into the membrane. The latter may be relevant to the import mechanism of apocytochrome c into mitochondria. Integral proteins induce a more direct motional restriction of the spin-labelled lipid chains, allowing the stoichiometry and specificity of the interaction, and the lipid exchange rate at the protein interface to be determined from the ESR spectra. In this way, a population of very slowly exchanging cardiolipin associated with the mitochondrial ADP-ATP carrier has been identified. The residues involved in the specificity for charged lipids of the myelin proteolipid protein have been localized to the deletion in the DM-20 mutant, and the difference in lipid-protein interactions with the beta-sheet and alpha-helical conformations of the M-13 coat protein, has been characterized.     

Lipid-protein interactions in biological membranes: a structural perspective

Lipid molecules bound to membrane proteins are resolved in some high-resolution structures of membrane proteins. An analysis of these structures provides a framework within which to analyse the nature of lipid-protein interactions within membranes. Membrane proteins are surrounded by a shell or annulus of lipid molecules, equivalent to the solvent layer surrounding a water-soluble protein. The lipid bilayer extends right up to the membrane protein, with a uniform thickness around the protein. The surface of a membrane protein contains many shallow grooves and protrusions to which the fatty acyl chains of the surrounding lipids conform to provide tight packing into the membrane. An individual lipid molecule will remain in the annular shell around a protein for only a short period of time. Binding to the annular shell shows relatively little structural specificity. As well as the annular lipid, there is evidence for other lipid molecules bound between the transmembrane α-helices of the protein; these lipids are referred to as non-annular lipids. The average thickness of the hydrophobic domain of a membrane protein is about 29 Å, with a few proteins having significantly smaller or greater thicknesses than the average. Hydrophobic mismatch between a membrane protein and the surrounding lipid bilayer generally leads to only small changes in membrane thickness. Possible adaptations in the protein to minimise mismatch include tilting of the helices and rotation of side chains at the ends of the helices. Packing of transmembrane α-helices is dependent on the chain length of the surrounding phospholipids. The function of membrane proteins is dependent on the thickness of the surrounding lipid bilayer, sometimes on the presence of specific, usually anionic, phospholipids, and sometimes on the phase of the phospholipid.     

Lipid-protein interactions in biological membranes: a structural perspective

Lipid molecules bound to membrane proteins are resolved in some high-resolution structures of membrane proteins. An analysis of these structures provides a framework within which to analyse the nature of lipid-protein interactions within membranes. Membrane proteins are surrounded by a shell or annulus of lipid molecules, equivalent to the solvent layer surrounding a water-soluble protein. The lipid bilayer extends right up to the membrane protein, with a uniform thickness around the protein. The surface of a membrane protein contains many shallow grooves and protrusions to which the fatty acyl chains of the surrounding lipids conform to provide tight packing into the membrane. An individual lipid molecule will remain in the annular shell around a protein for only a short period of time. Binding to the annular shell shows relatively little structural specificity. As well as the annular lipid, there is evidence for other lipid molecules bound between the transmembrane alpha-helices of the protein; these lipids are referred to as non-annular lipids. The average thickness of the hydrophobic domain of a membrane protein is about 29 A, with a few proteins having significantly smaller or greater thicknesses than the average. Hydrophobic mismatch between a membrane protein and the surrounding lipid bilayer generally leads to only small changes in membrane thickness. Possible adaptations in the protein to minimise mismatch include tilting of the helices and rotation of side chains at the ends of the helices. Packing of transmembrane alpha-helices is dependent on the chain length of the surrounding phospholipids. The function of membrane proteins is dependent on the thickness of the surrounding lipid bilayer, sometimes on the presence of specific, usually anionic, phospholipids, and sometimes on the phase of the phospholipid.     

Lipid-protein interactions in reconstituted membranes containing acetylcholine receptor

Functional membranes containing purified Torpedo californica acetylcholine receptor and dioleoylphosphatidylcholine (DOPC) were prepared by a cholate dialysis procedure with lipid to protein ratios of 100-400 to 1 (mol/mol). Spin-labeled lipids were incorporated into the reconstituted membranes and into native membranes prepared from Torpedo electroplax, and electron paramagnetic resonance (EPR) spectra were recorded between 0 and 20 degrees C. The spin-labels included nitroxide derivatives of stearic acid (16-doxylstearic acid), androstane, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidic acid (PA). The phospholipid spin-labels had 16-doxylstearic acid in the sn-2 position. All the spectra showed two components corresponding to a relatively mobile bilayer component and a motionally restricted "protein-perturbed" component. The relative amounts of mobile and perturbed components were quantitated by spectral subtraction and integration techniques. The mobile/perturbed ratio was somewhat temperature dependent, and the results are discussed in terms of exchange between mobile and perturbed environments. Plots of the mobile/perturbed ratios vs. lipid/protein ratios at 1 degree C gave straight lines from which the relative binding affinity of each spin-label and the number of perturbed lipids per receptor protein could be calculated. All the spin-labels gave similar values for the number of perturbed lipids (40 +/- 7), a number close to the number of lipids that will fit around the intramembranous perimeter of the receptor. The affinities of the spin-labeled lipids for the receptor relative to DOPC were androstane (K = 4.3) congruent to 16-doxylstearic acid (4.1) greater than PA (2.7) greater than PE (1.1) approximately PC (1.0) approximately PS (0.7). The lipids having the highest affinity for the acetylcholine receptor were also those that have the largest effects on the ion flux functional properties of the receptor, and the results are discussed in terms of lipid effects on receptor function.     

Lipid-protein interactions of integral membrane proteins: a comparative simulation study

The interactions between membrane proteins and their lipid bilayer environment play important roles in the stability and function of such proteins. Extended (15-20 ns) molecular dynamics simulations have been used to explore the interactions of two membrane proteins with phosphatidylcholine bilayers. One protein (KcsA) is an alpha-helix bundle and embedded in a palmitoyl oleoyl phosphatidylcholine bilayer; the other (OmpA) is a beta-barrel outer-membrane protein and is in a dimyristoyl phosphatidylcholine bilayer. The simulations enable analysis in detail of a number of aspects of lipid-protein interactions. In particular, the interactions of aromatic amphipathic side chains (i.e., Trp, Tyr) with lipid headgroups, and "snorkeling" interactions of basic side chains (i.e., Lys, Arg) with phosphate groups are explored. Analysis of the number of contacts and of H-bonds reveal fluctuations on an approximately 1- to 5-ns timescale. There are two clear bands of interacting residues on the surface of KcsA, whereas there are three such bands on OmpA. A large number of Arg-phosphate interactions are seen for KcsA; for OmpA, the number of basic-phosphate interactions is smaller and shows more marked fluctuations with respect to time. Both classes of interaction occur in clearly defined interfacial regions of width approximately 1 nm. Analysis of lateral diffusion of lipid molecules reveals that "boundary" lipid molecules diffuse at about half the rate of bulk lipid. Overall, these simulations present a dynamic picture of lipid-protein interactions: there are a number of more specific interactions but even these fluctuate on an approximately 1- to 5-ns timescale.     

Lipid-protein interactions in lipovitellin

The refined molecular structure of lipovitellin is described using synchrotron cryocrystallographic data to 1.9 A resolution. Lipovitellin is the predominant lipoprotein found in the yolk of egg-laying animals and is involved in lipid and metal storage. It is thought to be related in amino acid sequence to segments of apolipoprotein B and the microsomal transfer protein responsible for the assembly of low-density lipoproteins. Lipovitellin contains a heterogeneous mixture of about 16% (w/w) noncovalently bound lipid, mostly phospholipid. Previous X-ray structural studies at ambient temperature described several different protein domains including a large cavity in each subunit of the dimeric protein. The cavity was free of any visible electron density for lipid molecules at room temperature, suggesting that only dynamic interactions exist with the protein. An important result from this crystallographic study at 100 K is the appearance of some bound ordered lipid along the walls of the binding cavity. The precise identification of the lipid type is difficult because of discontinuities in the electron density. Nonetheless, the conformations of 7 phospholipids and 43 segments of hydrocarbon chains greater than 5 atoms in length have been discovered. The conformations of the bound lipid and the interactions between protein and lipid provide insights into the factors governing lipoprotein formation.     

Understanding nucleosomal histone and DNA interactions: a biophysical study

Histones are associated with DNA to form nucleosome essential for chromatin structure and major nuclear processes like gene regulation and expression. Histones consist of H1, H2A, H2B and H3, H4 type proteins. In the present study, combined histones from calf thymus were complexed with ct DNA and their binding affinities were measured fluorimetrically. All the five histones were resolved on SDS page and their binding with DNA was visualized. The values of biding affinities varied with pH and salt concentration. Highest affinity (4.0?×?10⁵ M^?1) was recorded at pH 6.5 in 50 mM phosphate buffer and 1.5?×?10⁴ M^?1 in 2 M NaCl at pH 7.0. The CD spectra support the highest binding affinity with maximum conformational changes at pH 7.0. The time-resolved fluorescence data recorded two life times for histone tyrosine residues at 300 nm emission in phosphate buffer pH 6.5. These life times did not show much change upon binding with DNA in buffer as well as in 2 M NaCl. The isothermal calorimetric studies yielded thermodynamic parameters ΔG, ΔH and ΔS as ?1.6?×?10⁵ cal/mol, ?1.13?×?10³ cal/mol and ?3.80 cal/mol/deg, respectively, evidencing a spontaneous exothermic reaction. The dominant binding forces in building the nucleosome are electrostatic interactions.     

Lipid-protein interactions: NMR study of melittin and its binding to lysophosphatidylcholine

Proton NMR of melittin differs according to the association state of the peptide in the monomer or tetramer. Melittin interacts with lysophosphatidylcholine micelles, whatever the association state of melittin; well resolved superimposed spectra from both components for all the lipid to peptide molar ratios are observed. Within the complexes, local mobility and fast exchange occurs. On binding concomitant shifts on Trp₁₉ indole lines and on the aliphatic CH₂ protons of the lipids are detected. The lipid perturbation is maximum for methylene groups in α and β of the ester bond, this could allow positionning of Trp₁₉ in the hydrophobic core of the lipids.     

Deuterium oxide stabilizes conformation of tubulin:a biophysical and biochemical study

The present study was aimed to elucidate the mechanism of stabilization of tubulin by deuterium oxide (D(2)O). Rate of decrease of tryptophan fluorescence during aging of tubulin at 4 degrees C and 37 degrees C was significantly lower in D(2)O than in H(2)O. Circular dichroism spectra of tubulin after incubation at 4 degrees C, suggested that complete stabilization of the secondary structure in D(2)O during the first 24 hours of incubation. The number of available cysteine measured by DTNB reaction was decreased to a lesser extent in D(2)O than in H(2)O. During the increase in temperature of tubulin, the rate of decrease of fluorescence at 335 nm and change of CD value at 222 nm was lesser in D(2)O. Differential Scanning calorimetric experiments showed that the T(m) values for tubulin unfolding in D(2)O were 58.6 degrees C and 62.17 degrees C, and in H(2)O those values were 55.4 degrees C and 59.35 degrees C.     

Lipid-protein interactions in GPCR-associated signaling

Signal transduction via G-protein-coupled receptors (GPCRs) is a fundamental pathway through which the functions of an individual cell can be integrated within the demands of a multicellular organism. Since this family of receptors first discovered, the proteins that constitute this signaling cascade and their interactions with one another have been studied intensely. In parallel, the pivotal role of lipids in the correct and efficient propagation of extracellular signals has attracted ever increasing attention. This is not surprising given that most of the signal transduction machinery is membrane-associated and therefore lipid-related. Hence, lipid-protein interactions exert a considerable influence on the activity of these proteins. This review focuses on the post-translational lipid modifications of GPCRs and G proteins (palmitoylation, myristoylation, and isoprenylation) and their significance for membrane binding, trafficking and signaling. Moreover, we address how the particular biophysical properties of different membrane structures may regulate the localization of these proteins and the potential functional consequences of this phenomenon in signal transduction. Finally, the interactions that occur between membrane lipids and GPCR effector enzymes such as PLC and PKC are also considered.     

Lipid-protein interactions in GPCR-associated signaling

Signal transduction via G-protein-coupled receptors (GPCRs) is a fundamental pathway through which the functions of an individual cell can be integrated within the demands of a multicellular organism. Since this family of receptors first discovered, the proteins that constitute this signaling cascade and their interactions with one another have been studied intensely. In parallel, the pivotal role of lipids in the correct and efficient propagation of extracellular signals has attracted ever increasing attention. This is not surprising given that most of the signal transduction machinery is membrane-associated and therefore lipid-related. Hence, lipid-protein interactions exert a considerable influence on the activity of these proteins. This review focuses on the post-translational lipid modifications of GPCRs and G proteins (palmitoylation, myristoylation, and isoprenylation) and their significance for membrane binding, trafficking and signaling. Moreover, we address how the particular biophysical properties of different membrane structures may regulate the localization of these proteins and the potential functional consequences of this phenomenon in signal transduction. Finally, the interactions that occur between membrane lipids and GPCR effector enzymes such as PLC and PKC are also considered.     

Lipid-protein interactions in blood serum]

The lipoprotein-fatty acid complexes show an electrophoretic mobility greater than that lipoproteins alone, but that phenomenon does not appear when those complexes have previously incubates for two days at 37 degrees C: an hypothesis on the evolution in time of the patterns of interaction binding lipoproteins and fatty acids has been confirmed by studies in isoelectric focusing.     

Lipid-protein interactions in model membranes from bovine brain white matter. An ESR spin label and electron microscopy study

Lipid-protein model membranes, prepared from bovine brain white matter and containing all the lipids and Folch-Lees proteolipids, have been studied in macroscopically oriented multibilayers. To examine the lipid environment the membranes were spin labeled with the cholestane spin label ($\text{3-spiro(2′-(N-}\text{oxyl-4′,4′-dimethyl-oxazolidine))5α-cholestane}$) and a fatty acid spin label ($\text{4′-,4′-dimethyloxazolidine-N-}\text{oxyl}$ derivative of 5-ketostearic acid). The ESR spectra exhibit two components arising from fairly well oriented and completely unoriented lipids. Up to a temperature of 55°C the amount of oriented lipids is almost constant, being about 35%. At higher temperatures this percentage drops rapidly to zero. It is shown that the presence of unoriented lipids arises mainly from disrupted areas in the lipid bilayer structure. This is confirmed by electron microscopy and from an analysis of the temperature dependence of the order parameters of the spin labels. The presence of locally disrupted lipid parts in the bilayer is discussed in relation to the interaction of the brain white matter lipids with Folch-Lees protein.