Structural determinants that target the hepatitis C virus core protein to lipid droplets

Hepatitis C virus core protein is targeted to lipid droplets, which serve as intracellular storage organelles, by its C-terminal domain, termed D2. From circular dichroism and nuclear magnetic resonance analyses, we demonstrate that the major structural elements within D2 consist of two amphipathic alpha-helices (Helix I and Helix II) separated by a hydrophobic loop. Both helices require a hydrophobic environment for folding, indicating that lipid interactions contribute to their structural integrity. Mutational studies revealed that a combination of Helix I, the hydrophobic loop, and Helix II is essential for efficient lipid droplet association and pointed to an in-plane membrane interaction of the two helices at the phospholipid layer interface. Aside from lipid droplet association, membrane interaction of D2 is necessary for folding and stability of core following maturation at the endoplasmic reticulum membrane by signal peptide peptidase. These studies identify critical determinants within a targeting domain that enable trafficking and attachment of a viral protein to lipid droplets. They also serve as a unique model for elucidating the specificity of protein-lipid interactions between two membrane-bound organelles.     

Fluorescence study of protein-lipid complexes with a new symmetric squarylium probe

The novel symmetric squarylium derivative SQ-1 has been synthesized and tested for its sensitivity to the formation of protein-lipid complexes. SQ-1 binding to the model membranes composed of zwitterionic lipid phosphatidylcholine (PC) and its mixtures with anionic lipid cardiolipin (CL) in different molar ratios was found to be controlled mainly by hydrophobic interactions. Lysozyme (Lz) and ribonuclease A (RNase) exerted an influence on the probe association with lipid vesicles resulting presumably from the competition between SQ-1 and the proteins for bilayer free volume and modification of its properties. The magnitude of this effect was much higher for lysozyme which may stem from the amphipathy of protein alpha-helix involved in the membrane binding. Varying membrane composition provides evidence for the dye sensitivity to both hydrophobic and electrostatic protein-lipid interactions. Fluorescence anisotropy studies uncovered the restriction of SQ-1 rotational mobility in lipid environment in the presence of Lz and RNase being indicative of the incorporation of the proteins into bilayer interior. The results of binding, fluorescence quenching and kinetic experiments suggested lysozyme-induced local lipid demixing upon protein association with negatively charged membranes with threshold concentration of CL for the lipid demixing being 10 mol%.     

Interfacial polar interactions affect gramicidin channel kinetics

Critical to biological processes such as secretion and transport, protein-lipid interactions within the membrane and at the membrane-water interface still raise many questions. Here we examine the role of lipid headgroups in these interactions by using gramicidin A (gA) channels in planar bilayers as a probe. We show that although headgroup demethylation from phosphatidylcholine (DOPC) to phosphatidylethanolamine decreases the lifetime of gA channels by an order of magnitude in accordance with the currently accepted hydrophobic mismatch mechanism, our findings with diether-DOPC suggest the importance of the headgroup-peptide interactions. According to our x-ray diffraction measurements, this lipid has the same hydrophobic thickness as DOPC but increases gA lifetime by a factor of 2. Thus we demonstrate that peptide-headgroup interactions may dominate over the effect of hydrophobic mismatch in regulating protein function.     

Hydration at the membrane protein-lipid interface.

Evidence has been found for the existence water at the protein-lipid hydrophobic interface of the membrane proteins, gramicidin and apocytochrome C, using two related fluorescence spectroscopic approaches. The first approach exploited the fact that the presence of water in the excited state solvent cage of a fluorophore increases the rate of decay. For 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-palmitoyl-2-[[2-[4-(6-phenyl-trans-1,3,5- hexatrienyl)phenyl]ethyl]carbonyl]-3-sn-PC (DPH-PC), where the fluorophores are located in the hydrophobic core of the lipid bilayer, the introduction of gramicidin reduced the fluorescence lifetime, indicative of an increased presence of water in the bilayer. Since a high protein:lipid ratio was used, the fluorophores were forced to be adjacent to the protein hydrophobic surface, hence the presence of water in this region could be inferred. Cholesterol is known to reduce the water content of lipid bilayers and this effect was maintained at the protein-lipid interface with both gramicidin and apocytochrome C, again suggesting hydration in this region. The second approach was to use the fluorescence enhancement induced by exchanging deuterium oxide (D2O) for H2O. Both the fluorescence intensities of trimethylammonium-DPH, located in the lipid head group region, and of the gramicidin intrinsic tryptophans were greater in a D2O buffer compared with H2O, showing that the fluorophores were exposed to water in the bilayer at the protein-lipid interface. In the presence of cholesterol the fluorescence intensity ratio of D2O to H2O decreased, indicating a removal of water by the cholesterol, in keeping with the lifetime data.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Interactions of cytochromes b5 and c with phospholipid monolayers

Monolayers of charged and neutral phospholipids at the air/water interface containing the cytochromes b5 and c are studied by film balance techniques and by fluorescence microscopy. A new technique is introduced to obtain a defined and homogeneous protein distribution within the membrane. It is shown that both proteins preferentially partition into the fluid membrane phases coexisting with solid lipid domains, thus allowing formation of periodic protein distributions. Protein reconstitution in protein/lipid ratios up to 1:50 does not change the pressure, pi c, corresponding to the main lipid transition but changes the slope in the pressure/area isotherms. It also affects the pressure-induced lipid crystallization, in that the monolayer can be viewed as segregated into a protein-free and a protein-enriched phase. Whereas penetration of cytochrome c into the monolayer is highly dependent on lipid head group charge, this does not hold for cytochrome b. In both cases, monolayer penetration is monotonously reduced with increasing surface pressure, pointing to the dependence of hydrophobic protein-lipid interactions on hydrocarbon chain density.     

Mechanisms of lipid regulation and lipid gating in TRPC channels

TRPC proteins form cation channels that integrate and relay cellular signals by mechanisms involving lipid recognition and lipid-dependent gating. The lipohilic/amphiphilic molecules that function as cellular activators or modulators of TRPC proteins span a wide range of chemical structures. In this context, cellular redox balance is likely linked to the lipid recognition/gating features of TRPC channels. Both classical ligand-protein interactions as well as indirect and promiscuous sensory mechanisms have been proposed. Some of the recognition processes are suggested to involve ancillary lipid-binding scaffolds or regulators as well as dynamic protein–protein interactions determined by bilayer architecture. A complex interplay of protein–protein and protein-lipid interactions is likely to govern the gating and/or plasma membrane recruitment of TRPC channels, thereby providing a distinguished platform for signal integration and coincident signal detection. Both the primary molecular event(s) of lipid recognition by TRPC channels as well as the transformation of these events into distinct gating movements is poorly understood at the molecular level, and it remains elusive whether lipid sensing in TRPCs is conferred to a distinct sensor domain. Recent structural information on the molecular action of lipophilic activators in distantly related members of the TRP superfamily encourages speculations on TRPC gating mechanisms involved in lipid recognition/gating. This review aims to provide an update on the current understanding of the lipid–dependent control of TRPC channels with focus on the TRPC lipid sensing, signal-integration hub and a short discussion of potential links to redox signaling.     

Three-dimensional structure of rat surfactant protein A trimers in association with phospholipid monolayers

Surfactant protein A (SP-A) is a C-type lectin found primarily in the lung and plays a role in innate immunity and the maintenance of surfactant integrity. To determine the three-dimensional (3D) structure of SP-A in association with a lipid ligand, we have used single particle electron crystallography and computational 3D reconstruction in combination with molecular modeling. Recombinant rat SP-A, containing a deletion of the collagen-like domain, was incubated with dipalmitoylphosphatidylcholine:egg phosphatidylcholine (1:1, wt/wt) lipid monolayers in the presence of calcium, negatively stained, and examined by transmission electron microscopy. Images of SP-A-lipid complexes with different angular orientations were used to reconstruct the 3D structure of the protein. These results showed that SP-A subunits readily formed trimers and interacted with lipid monolayers exclusively via the globular domains. A homology-based molecular model of SP-A was generated and fitted into the electron density map of the protein. The plane of the putative lipid-protein interface was relatively flat and perpendicular to the hydrophobic neck region, and the cleft region in the middle of the trimer had no apparent charge clusters. Amino acid residues that are known to affect lipid interactions, Glu(195) and Arg(197), were located at the protein-lipid interface. The molecular model indicated that the hydrophobic neck region of the SP-A did not interact with lipid monolayers but was instead involved in intratrimeric subunit interactions. The glycosylation site of SP-A was located at the side of each subunit, suggesting that the covalently linked carbohydrate moiety probably occupies the spaces between the adjacent globular domains, a location that would not sterically interfere with ligand binding.     

Regulation of channel function due to physical energetic coupling with a lipid bilayer

Regulation of membrane protein functions due to hydrophobic coupling with a lipid bilayer has been investigated. An energy formula describing interactions between lipid bilayer and integral ion channels with different structures, which is based on the screened Coulomb interaction approximation, has been developed. Here the interaction energy is represented as being due to charge-based interactions between channel and lipid bilayer. The hydrophobic bilayer thickness channel length mismatch is found to induce channel destabilization exponentially while negative lipid curvature linearly. Experimental parameters related to channel dynamics are consistent with theoretical predictions. To measure comparable energy parameters directly in the system and to elucidate the mechanism at an atomistic level we performed molecular dynamics (MD) simulations of the ion channel forming peptide–lipid complexes. MD simulations indicate that peptides and lipids experience electrostatic and van der Waals interactions for short period of time when found within each other’s proximity. The energies from these two interactions are found to be similar to the energies derived theoretically using the screened Coulomb and the van der Waals interactions between peptides (in ion channel) and lipids (in lipid bilayer) due to mainly their charge properties. The results of in silico MD studies taken together with experimental observable parameters and theoretical energetic predictions suggest that the peptides induce ion channels inside lipid membranes due to peptide–lipid physical interactions. This study provides a new insight helping better understand of the underlying mechanisms of membrane protein functions in cell membrane leading to important biological implications.     

Intermolecular interactions involved in the association of the variant surface glycoprotein of Trypanosoma brucei

Trypanosomes in their mammalian host are covered by the densely packed variant surface glycoprotein (VSG). Depending on the presence or absence of a glycosyl-phosphatidyl inositol anchor. VSG is accessible as soluble globular protein (sVSG), or as insoluble membrane form (mfVSG). In order to get insight into the two-dimensional association of VSG within the surface layer, protein-protein interactions were investigated in a wide range of protein concentrations. No self-assembly of sVSG could be detected even at protein concentrations close to the local packing in the surface layer. The absence of preferential interactions with soybean phospholipid or lysolecithin monolayers (spread on a Langmuir trough) suggests that the soluble form of the protein is not integrated into a model lipid-water interface. Thus, the two-dimensional arrangement of the protein in situ seems to be determined by hydrophobic interactions of the lipid components rather than protein-lipid interactions. In contrast to sVSG, the membrane form (mfVSG) undergoes aggregation and shows a strong tendency to absorb to surfaces and chromatographic matrices, thus interfering with standard techniques of protein purification.     

Role of buried polar residues in helix bundle stability and lipid binding of apolipophorin III: destabilization by threonine 31

Apolipophorin III (apoLp-III) from Locusta migratoria is a model exchangeable apolipoprotein that plays a key role in neutral lipid transport. The protein is comprised of a bundle of five amphipathic alpha-helices, with most hydrophobic residues buried in the protein interior. The low stability of apoLp-III is thought to be crucial for lipid-induced helix bundle opening, to allow protein-lipid interactions. The presence of polar residues in the hydrophobic protein interior may facilitate this role. To test this, two buried polar residues, Thr-31 and Thr-144, were changed into alanine by site-directed mutagenesis. Secondary structure analysis and GdnHCl- and temperature-induced denaturation studies indicated an increase in alpha-helical content and protein stability for T31A apoLp-III compared to wild-type apoLp-III. In contrast, T144A had a decreased alpha-helical content and protein stability, while tryptophan fluorescence indicated increased exposure of the hydrophobic interior to buffer. Two mutant proteins that had lysine residues introduced in the hydrophobic core displayed a more pronounced decrease in secondary structure and protein stability. Lipid binding studies using phospholipid vesicles showed that T31A apoLp-III was able to transform phospholipid vesicles into discoidal particles but at a 3-fold reduced rate compared to wild-type apoLp-III. In contrast, the less stable apoLp-III mutants displayed an increased ability to transform phospholipid vesicles. These results demonstrate the inverse correlation between protein stability and the ability to transform phospholipid vesicles into discoidal protein-lipid complexes and that Thr-31 is a key determinant of the relatively low protein stability, thereby promoting apoLp-III to interact with lipid surfaces.     

How proteins adapt to a membrane-water interface

Membrane proteins present a hydrophobic surface to the surrounding lipid, whereas portions protruding into the aqueous milieu expose a polar surface. But how have proteins evolved to deal with the complex environment at the membrane-water interface? Some insights have been provided by high-resolution structures of membrane proteins, and recent studies of the role of individual amino acids in mediating protein-lipid contacts have shed further light on this issue. It now appears clear that the polar-aromatic residues Trp and Tyr have a specific affinity for a region near the lipid carbonyls, whereas positively charged residues extend into the lipid phosphate region.     

Estimation of lipid regions in a cytochrome oxidase-lipid complex using spin labeling electron spin resonance: Distribution effects on the spin label

The distribution of lipid in the cytochrome oxidase-lipid complex from beef heart mitochondria has been studied by the spin labeling electron spin resonance technique. The spectra of a phospholipid spin label incorporated in the complex reveals an immobilized (on the ESR time scale) component in addition to the fluid component which is found in aqueous dispersions of the extracted lipids. The first component corresponds to the domain of lipid influenced by the protein, and the second component to the remaining lipid. A theory taking into account not only the sizes of the lipid regions in which the spin label molecule distributes itself, but also the different affinities of the label for the two domains, has been developed. Taking advantage of the variation in spectra obtained with increasing amounts of spin label, computer calculations have been performed to estimate the distribution of lipid in the different regions of the cytochrome oxidase-lipid complex. An extrapolation of the amount of immobilized spin-labeled phospholipid to zero concentration of label allows a calculation of the number of fatty acid residues interacting with the protein to be made. It has been found that the number of aliphatic chains influenced by the protein is higher than that calculated for a single boundary layer around the protein. The approach used in this paper can be useful for studies of protein-lipid interactions in other systems.     

Modulation of pig kidney Na+/K+-ATPase activity by cholesterol: role of hydration

Cholesterol is known to affect the activity of membrane-bound enzymes, including Na(+)/K(+)-ATPase. To gain insight into the mechanism of cholesterol's effect, we have used various hydrophobic fluorescent probes which insert into different regions of the membrane bilayer and report on the degree of hydration of their environment. Specifially, we have measured the generalized polarization of Laurdan and the lifetime of DPH and derivatives of DPH inserted into membranes from pig kidneys enriched in Na(+)/K(+)-ATPase. Spectral measurements were also carried out on these membranes after modification of their cholesterol content. The generalized polarization of Laurdan increased with increasing cholesterol, showing an abrupt modification at the native cholesterol content. The fluorescence lifetimes of DPH and the DPH derivatives were analyzed using a distribution model. The center value of these lifetime distributions and their widths also changed with increasing cholesterol. One DPH derivative, DPH-PC, showed a minimum value for the lifetime center at the native cholesterol concentration, whereas the other derivatives showed a maximum value for the lifetime center at that cholesterol concentration. DPH-PC is known to sense the protein-lipid interface, whereas the other derivatives sense the bulk lipid phase. These data suggest that hydration at the protein-lipid interface is maximal at the native cholesterol concentration as is the enzymatic activity. Hydration at the protein-lipid interface is therefore proposed to be required for activity. These results are in agreement with current models of membrane dynamics and thermodynamics of protein function.     

Different membrane anchoring positions of tryptophan and lysine in synthetic transmembrane alpha-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 alpha-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.     

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.     

Lateral diffusion of gramicidin S,M-13 coat protein and glycophorin in bilayers of saturated phospholipids: Mean field and Monte Carlo studies

We have developed a general model that relates the lateral diffusion coefficient of one isolated large intrinsic molecule (mol. wt. ?1000) in a phosphatidylcholine bilayer to the static lipid hydrocarbon chain order. We have studied how protein lateral diffusion can depend upon protein-lipid interactions but have not investigated possible non-specific contributions from gel-state lattice defects. The model has been used in Monte Carlo simulations or in mean-field approximations to study the lateral diffusion coefficients of Gramicidin S, the M-13 coat protein and glycophorin in dimyristoyl- and dipalmitoylphosphatidylcholine (DMPC and DPPC) bilayers as functions of temperature. Our calculated lateral diffusion coefficients for Gramicidin S and the M-13 coat protein are in good agreement with what has been observed and suggest that Gramicidin S is in a dimeric form in DMPC bilayers. In the case of glycophorin we find that the ‘ice breaker’ effect can be understood as a consequence of perturbation of the lipid polar region around the protein. In order to understand this effect is is necessary that the protein hydrophilic section perturb the polar regions of at least approx. 24 lipid molecules, in good agreement with the numbers of 29–30 measured using ³¹P-NMR. Because of lipid-lipid interactions this effect extends itself out to four or five lipid layers away from the protein so that the hydrocarbon chains of between approx. 74 and approx. 108 lipid molecules are more disordered in the gel phase, so contributing less to the transition enthalpy, in agreement with the numbers of 80–100 deduced from differential scanning calorimetry (DSC). An understanding of the abrupt change in the diffusion coefficient at a temperature below the main bilayer transition temperature requires an additional mechanism. We propose that this change may be a consequence of a ‘coupling-uncoupling’ transition involving the protein hydrophilic section and the lipid polar regions, which may be triggered by the lipid bilayer pretransition. Our calculation of the average number of gauche bonds per lipid chain as a function of temperature and distance away from an isolated polypeptide or integral protein shows the extent of statically disordered lipid around such molecules. The range of this disorder depends upon temperature, particularly near the main transition.     

Elastic response of bilayers with intrinsic proteins

Intrinsic membrane proteins affect the ordering of neighbouring lipid chains. We have used a model of protein-lipid interactions in bilayers proposed by Owicki et al. (Owicki, J.C., Springgate, M.W. and McConnell, H.M. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 1616–1619) to show that near the lipid phase transition this effect may significantly increase the magnitude of a membrane's lateral compressibility (or correspondingly, decrease the magnitude of the membrane's elastic moduli).     

A theoretical study of lipid-protein interactions in bilayers.

We present a theoretical study of the effect of different types of lipid-protein interactions on the thermodynamic properties of protein-containing lipid bilayers. The basis of this work is a theoretical model for pure lipid bilayer phase transitions developed earlier by Scott. Simple assumptions on the nature of the lipid conformations near a protein strongly affect the predicted properties of the model. Here we consider (a) random protein-lipid contacts, (b) enhanced contact between protein and lipid with a number of gauche bonds, and (c) enhanced contact between protein and all-trans but tilted lipid chains. Comparison of predicted results with experimental data seems to favor point c above but, by itself point c does not work well at larger protein concentrations. The results are discussed in the light of spectroscopic data, lipid-protein (plus annular lipid) miscibility, and interprotein forces.