Interactions In Biological Membranes Research Articles

Fine-tuned protein-lipid interactions in biological membranes: exploration and implications of the ORMDL-ceramide negative feedback loop in the endoplasmic reticulum.

Biological membranes consist of a lipid bilayer in which integral membrane proteins are embedded. Based on the compositional complexity of the lipid species found in membranes, and on their specific and selective interactions with membrane proteins, we recently suggested that membrane bilayers can be best described as "finely-tuned molecular machines." We now discuss one such set of lipid-protein interactions by describing a negative feedback mechanism operating in the de novo sphingolipid biosynthetic pathway, which occurs in the membrane of the endoplasmic reticulum, and describe the atomic interactions between the first enzyme in the pathway, namely serine palmitoyl transferase, and the product of the fourth enzyme in the pathway, ceramide. We explore how hydrogen-bonding and hydrophobic interactions formed between Asn13 and Phe63 in the serine palmitoyl transferase complex and ceramide can influence the ceramide content of the endoplasmic reticulum. This example of finely-tuned biochemical interactions raises intriguing mechanistic questions about how sphingolipids and their biosynthetic enzymes could have evolved, particularly in light of their metabolic co-dependence.

Translocation of Antimicrobial Peptides across Model Membranes: The Role of Peptide Chain Length.

Cushioned lipid bilayers are structures consisting of a lipid bilayer supported on a solid substrate with an intervening layer of soft material. They offer possibilities for studying the behavior and interactions of biological membranes more accurately under physiological conditions. In this work, we continue our studies of cushion formation induced by histatin 5 (24Hst5), focusing on the effect of the length of the peptide chain. 24Hst5 is a short, positively charged, intrinsically disordered saliva peptide, and here, both a shorter (14Hst5) and a longer (48Hst5) peptide variant were evaluated. Experimental surface active techniques were combined with coarse-grained Monte Carlo simulations to obtain information about these peptides. Results show that at 10 mM NaCl, both the shorter and the longer peptide variants behave like 24Hst5 and a cushion below the bilayer is formed. At 150 mM NaCl, however, no interaction is observed for 24Hst5. On the contrary, a cushion is formed both in the case of 14Hst5 and 48Hst5, and in the latter, an additional thick, diffuse, and highly hydrated layer of peptide and lipid molecules is formed, on top of the bilayer. Similar trends were observed from the simulations, which allowed us to hypothesize that positively charged patches of the amino acids lysine and arginine in all three peptides are essential for them to interact with and translocate over the bilayer. We therefore hypothesize that electrostatic interactions are important for the interaction between the solid-supported lipid bilayers and the peptide depending on the linear charge density through the primary sequence and the positively charged patches in the sequence. The understanding of how, why, and when the cushion is formed opens up the possibility for this system to be used in the research and development of new drugs and pharmaceuticals.

Free-energy landscapes of transmembrane homodimers by bias-exchange adaptively biased molecular dynamics

Membrane proteins play essential roles in various biological functions within the cell. One of the most common functional regulations involves the dimerization of two single-pass transmembrane (TM) helices. Glycophorin A (GpA) and amyloid precursor protein (APP) form TM homodimers in the membrane, which have been investigated both experimentally and computationally. The homodimer structures are well characterized using only four collective variables (CVs) when each TM helix is stable. The CVs are the interhelical distance, the crossing angle, and the Crick angles for two TM helices. However, conformational sampling with multi-dimensional replica-exchange umbrella sampling (REUS) requires too many replicas to sample all the CVs for exploring the conformational landscapes. Here, we show that the bias-exchange adaptively biased molecular dynamics (BE-ABMD) with the four CVs effectively explores the free-energy landscapes of the TM helix dimers of GpA, wild-type APP and its mutants in the IMM1 implicit membrane. Compared to the original ABMD, the bias-exchange algorithm in BE-ABMD can provide a more rapidly converged conformational landscape. The BE-ABMD simulations could also reveal TM packing interfaces of the membrane proteins and the dependence of the free-energy landscapes on the membrane thickness. This approach is valuable for numerous other applications, including those involving explicit solvent and a lipid bilayer in all-atom force fields or Martini coarse-grained models, and enhances our understanding of protein-protein interactions in biological membranes.

Bacterial Membranes Are More Perturbed by the Asymmetric Versus Symmetric Loading of Amphiphilic Molecules.

Characterizing the biophysical properties of bacterial membranes is critical for understanding the protective nature of the microbial envelope, interaction of biological membranes with exogenous materials, and designing new antibacterial agents. Presented here are molecular dynamics simulations for two cationic quaternary ammonium compounds, and the anionic and nonionic form of a fatty acid molecule interacting with a Staphylococcus aureus bacterial inner membrane. The effect of the tested materials on the properties of the model membranes are evaluated with respect to various structural properties such as the lateral pressure profile, lipid tail order parameter, and the bilayer’s electrostatic potential. Conducting asymmetric loading of molecules in only one leaflet, it was observed that anionic and cationic amphiphiles have a large impact on the Staphylococcus aureus membrane’s electrostatic potential and lateral pressure profile as compared to a symmetric distribution. Nonintuitively, we find that the cationic and anionic molecules induce a similar change in the electrostatic potential, which points to the complexity of membrane interfaces, and how asymmetry can induce biophysical consequences. Finally, we link changes in membrane structure to the rate of electroporation for the membranes, and again find a crucial impact of introducing asymmetry to the system. Understanding these physical mechanisms provides critical insights and viable pathways for the rational design of membrane-active molecules, where controlling the localization is key.

Probing lipids in biological membranes using SERS

A selective and sensitive experimental approach to the examination of lipids as well as lipid–protein interactions in biological membranes using surface-enhanced Raman spectroscopy has been proposed. This approach has been used for probing the intact mitochondria, membranes of erythrocyte ghosts and liposomes.

Photosystem I oligomerization affects lipid composition in Synechocystis sp. PCC 6803

In cyanobacteria, increasing growth temperature decreases lipid unsaturation and the ratio of monomer/trimer photosystem I (PSI) complexes. In the present study we applied Fourier-transform infrared (FTIR) spectroscopy and lipidomic analysis to study the effects of PSI monomer/oligomer ratio on the physical properties and lipid composition of thylakoids. To enhance the presence of monomeric PSI, a Synechocystis sp. PCC6803/ΔpsaL mutant strain (PsaL) was used which, unlike both trimeric and monomeric PSI-containing wild type (WT) cells, contain only the monomeric form. The protein-to-lipid ratio remained unchanged in the mutant but, due to an increase in the lipid disorder in its thylakoids, the gel to liquid-crystalline phase transition temperature (Tm) is lower than in the WT. In thylakoid membranes of the mutant, digalactosyldiacylglycerol (DGDG), the most abundant bilayer-forming lipid is accumulated, whereas those in the WT contain more monogalactosyldiacylglycerol (MGDG), the only non-bilayer-forming lipid in cyanobacteria. In PsaL cells, the unsaturation level of sulphoquinovosyldiacylglycerol (SQDG), a regulatory anionic lipid, has increased. It seems that merely a change in the oligomerization level of a membrane protein complex (PSI), and thus the altered protein-lipid interface, can affect the lipid composition and, in addition, the whole dynamics of the membrane. Singular value decomposition (SVD) analysis has shown that in PsaL thylakoidal protein-lipid interactions are less stable than in the WT, and proteins start losing their native secondary structure at much milder lipid packing perturbations. Conclusions drawn from this system should be generally applicable for protein-lipid interactions in biological membranes.

Biomolecule displacement by Brownian step

Interactions in biological membranes cause the molecular mobility to behave specifically. The random walk of a molecule at short times is not described by the Einstein relation since the character of motion evolves from ballistic to diffusive. This phenomenon is detected because of the long length of the Brownian step which can be comparable to the size of the membrane confined domain and hence has a great influence on the diffusion coefficient in restricted areas and subsequent subdiffusive behavior in longer times. The experimental values of Brownian step length are analyzed. A model is formulated connecting the step length with the real-to-thermal speed ratio of the diffusing tagged molecule. A method is derived to estimate the diffusing biomolecule Brownian step length on the base of the measured diffusion coefficient divided by the thermal speed of tagged molecule. The method seems to be reasonable in a wide interval of diffusion coefficient.

Probing protein–lipid interactions by FRET between membrane fluorophores

Förster resonance energy transfer (FRET) is a powerful fluorescence technique that has found numerous applications in medicine and biology. One area where FRET proved to be especially informative involves the intermolecular interactions in biological membranes. The present study was focused on developing and verifying a Monte-Carlo approach to analyzing the results of FRET between the membrane-bound fluorophores. This approach was employed to quantify FRET from benzanthrone dye ABM to squaraine dye SQ-1 in the model protein–lipid system containing a polycationic globular protein lysozyme and negatively charged lipid vesicles composed of phosphatidylcholine and phosphatidylglycerol. It was found that acceptor redistribution between the lipid bilayer and protein binding sites resulted in the decrease of FRET efficiency. Quantification of this effect in terms of the proposed methodology yielded both structural and binding parameters of lysozyme-lipid complexes.

A review of traditional and emerging methods to characterize lipid–protein interactions in biological membranes

Lipid–protein interactions are essential for modulating membrane protein structures and biological functions in the cell plasma membrane. In this review we describe the salient features of classical and emerging methodologies for studying protein–lipid interactions and their limitations.

Membrane dipole potential is sensitive to cholesterol stereospecificity: Implications for receptor function

Dipole potential is the potential difference within the membrane bilayer, which originates due to the nonrandom arrangement of lipid dipoles and water molecules at the membrane interface. Cholesterol, an essential lipid in higher eukaryotic membranes, has previously been shown to increase membrane dipole potential. In this work, we explored the effect of stereoisomers of cholesterol, ent-cholesterol and epi-cholesterol, on membrane dipole potential, monitored by the dual wavelength ratiometric approach utilizing the probe di-8-ANEPPS. Our results show that cholesterol and ent-cholesterol share comparable ability in increasing membrane dipole potential. In contrast, epi-cholesterol displays a slight reduction in membrane dipole potential. Our results constitute the first report on the effect of stereoisomers of cholesterol on membrane dipole potential, and imply that an extremely subtle change in sterol structure can significantly alter the dipolar field at the membrane interface. These results assume relevance in the context of differential abilities of these stereoisomers of cholesterol in supporting the activity of the serotonin1A receptor, a representative G protein-coupled receptor. The close correlation between membrane dipole potential and receptor activity provides new insight in receptor-cholesterol interaction in terms of stereospecificity. We envision that membrane dipole potential could prove to be a sensitive indicator of lipid-protein interactions in biological membranes.

An Analysis for Causal Properties of Membrane Interactions

We present here an analysis, based on Abstract Interpretation [P. Cousot and R. Cousot. Abstract Interpretation: A Unified Lattice Model for Static Analysis of Programs by Construction or Approximation of Fixpoints. In Proc. Fourth ACM Symp. Principles of Programming Languages (POPLʼ77), pp. 238–252, 1977.], obtained by defining a finite computable approximation of the causal semantics proposed in [N. Busi. Towards a Causal Semantics for Brane Calculi. In What is it About Government that Americans Dislike, pp. 1945–1965, University Press, 2007.] for the Mate/Bud/Drip (MBD) Brane Calculus [L. Cardelli. Brane calculi - interactions of biological membranes. In Proc. of Computational Methods in Systems Biology (CMSBʼ04), LNCS 3082, pp. 257–280, 2005.]. The causal semantics in [N. Busi. Towards a Causal Semantics for Brane Calculi. In What is it About Government that Americans Dislike, pp. 1945–1965, University Press, 2007.] is able to address different kinds of causal dependencies: structural, synchronisation and the so-called environment causality. Our analysis, being a safe (over)-approximation of such causal semantics, besides modelling all possible evolutions of the system, can be used to formally prove causal properties among membrane interactions.

Polar/Ionizable residues in transmembrane segments: effects on helix-helix packing.

The vast majority of membrane proteins are anchored to biological membranes through hydrophobic α-helices. Sequence analysis of high-resolution membrane protein structures show that ionizable amino acid residues are present in transmembrane (TM) helices, often with a functional and/or structural role. Here, using as scaffold the hydrophobic TM domain of the model membrane protein glycophorin A (GpA), we address the consequences of replacing specific residues by ionizable amino acids on TM helix insertion and packing, both in detergent micelles and in biological membranes. Our findings demonstrate that ionizable residues are stably inserted in hydrophobic environments, and tolerated in the dimerization process when oriented toward the lipid face, emphasizing the complexity of protein-lipid interactions in biological membranes.

Identifying Key Juxtamembrane Interactions in Cell Membranes Using AraC-based Transcriptional Reporter Assay (AraTM)

Dimerization is a key regulatory mechanism in activation of transmembrane (TM) receptors during signal transduction. This process involves a coordinated interplay between extracellular (EX), TM, and cytoplasmic (CYTO) regions to form a specific interface required for both ligand binding and intracellular signaling to occur. While several transcriptional activator-based methods exist for investigating TM interactions in bacterial membranes, expression of TM chimera in these methods occurs in a reverse orientation, and are limited to only TM domains for proper membrane trafficking and integration. We therefore developed a new, AraC-based transcriptional reporter assay (AraTM) that expresses EX-TM-CYTO chimera in their native orientation, thereby enabling membrane trafficking to occur independent of the TM chimera used as well as permitting analysis of EX-TM-CYTO interactions in biological membranes. Using integrin α(IIb) TM-CYTO as a model, we observe a large increase in homodimerization for the constitutively active TM mutant L980A relative to wild-type in the TM-CYTO construct (A963-E1008). We also characterized the receptor for advanced glycation endproducts (RAGE), whose homooligomeric state is critical in ligand recognition, and find the specific juxtamembrane region within the CYTO (A375-P394) mediates homodimerization, and is dominant over effects observed when the extracellular C2 domain is included. Furthermore, we find good agreement between our AraTM measurements in bacterial membranes and BRET measurements made on corresponding RAGE constructs expressed in transfected HEK293 cells. Overall, the AraTM assay provides a new approach to identify specific interactions between receptor EX-TM-CYTO domains in biological membranes that are important in regulation of signal transduction.

MALDI-TOF Mass Spectrometry Analysis of Amphipol-Trapped Membrane Proteins

Amphipols (APols) are amphipathic polymers with the ability to substitute detergents to keep membrane proteins (MPs) soluble and functional in aqueous solutions. APols also protect MPs against denaturation. Here, we have examined the ability of APol-trapped MPs to be analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). For that purpose, we have used ionic and nonionic APols and as model proteins (i) the transmembrane domain of Escherichia coli outer membrane protein A, a β-barrel, eubacterial MP, (ii) Halobacterium salinarum bacteriorhodopsin, an α-helical archaebacterial MP with a single cofactor, and (iii, iv) two eukaryotic MP complexes comprising multiple subunits and many cofactors, cytochrome b(6)f from the chloroplast of the green alga Chlamydomonas reinhardtii and cytochrome bc(1) from beef heart mitochondria. We show that these MP/APol complexes can be readily analyzed by MALDI-TOF-MS; most of the subunits and some lipids and cofactors were identified. APols alone, even ionic ones, had no deleterious effects on MS signals and were not detected in mass spectra. Thus, the combination of MP stabilization by APols and MS analyses provides an interesting new approach to investigating supramolecular interactions in biological membranes.

Multi-Tox: Application of the ToxR-transcriptional reporter assay to the study of multi-pass protein transmembrane domain oligomerization

ToxR-based transcriptional reporter assays allow the strength of transmembrane helix interactions in biological membranes to be measured. Previously, these assays have only been used to study single-pass transmembrane systems. To facilitate investigation of polytopic transmembrane domain (TMD) oligomerization, we applied the ToxR methodology to the study of multi-pass TMD oligomerization to give ‘Multi-Tox’. Association propensities of the viral oncoprotein, latent membrane protein-1 (LMP-1), and the E. coli membrane-integral diacylglycerol kinase (DAGK) were studied by Multi-Tox, highlighting residues of particular mechanistic importance. Both homo- and hetero-oligomerizations were studied.

Proteome-wide detection of phospholipid–protein interactions in mitochondria by photocrosslinking and click chemistry

Photoactivatable lipid analogues are uniquely suited for the detection of lipid-protein interactions in biological membranes. Based on photocrosslinking, new methodology has been developed for the proteome-wide detection of lipid-protein interactions. Bifunctional lipid analogues containing a tag for click chemistry in addition to the photoactivatable moiety enable the enrichment of the crosslinked proteins that is required for subsequent identification by mass spectrometry. In principle the phospholipid interaction-based membrane protein proteomics approach is applicable to any biomembrane and any lipid. Here, we review the background and the development of the new methodology. Results obtained with photocrosslinking in purified mitochondrial membranes from the yeast Saccharomyces cerevisiae are summarized and future perspectives discussed.

Membrane Proteins-bilayer Interplay: Insights From Coarse-grained Self-assembly And Potential Of Mean Force Simulations Of Rhodopsin In Model Bilayers

Due to the transient nature of typical protein-protein interactions in biological membranes and other technical limitations, it is extremely difficult to devise robust experimental approaches to elucidate atomic details of complex membrane systems. By offering alternative and complementary information, theoretical and computational approaches have become extremely useful and valuable tools. Here we will present our recent development of new molecular models, which, while conserving the physicochemical properties of the different components in a system, reduce the complexity of the description of a system down to chemical entities (3 to 6 heavy atoms). These coarse-grained models allow simulating systems of size and for time scales relevant to their biological function. The results obtained from self-assembly simulations of visual pigment rhodopsin in model membranes [1] and potentials of mean force (PMF) of rhodopsins association will be presented. The data show that the membrane bilayer responds to the presence of a protein by inhomogeneous and localized deformations at the surface of the protein and that, whereas the amount of deformation (hydrophobic mismatch) strongly affects the propensity of the proteins to aggregate, the location of the deformation correlates with protein-protein interfaces. The PMFs further demonstrate that protein binding energies are strongly correlated to the interfaces involved and that the membrane bilayer has a significant contribution to the energy barriers encountered during association. This work brings new insights to our understanding of the forces driving protein self-organization in membrane bilayers. [1] X. Periole, T. Huber, S.-J. Marrink, and T.P. Sakmar. 2007. G Protein-Coupled Receptors Self-Assemble in Dynamics Simulations of Model Bilayers. J. Am. Chem. Soc. 129, 10126-10132.

Mechanisms and Energetics of Protein/Peptide Interactions in Biological Membranes

Understanding the delicate balance of forces governing helix/β-hairpin interactions in transmembrane proteins is central to understanding membrane structure and function. These membrane constituent interactions play an essential role in determining the structure and function of membrane proteins, and protein interactions in membranes, and thus form the basis for many vital processes, including transmembrane signaling, transport of ions and small molecules, energy transduction, and cell-cell recognition.

Probing Molecular Interactions in Biological Membranes by Solid-State NMR

The interaction between integral membrane proteins and the lipid bilayer is vital in the regulation of many biological processes. Here we report preliminary solid-state NMR studies on two systems where interactions at the lipid/protein interface play an important role in the regulation and trafficking of integral membrane proteins.The anaesthetic octanol has been shown to interact with several sites on the nicotinic acetylcholine receptor (nAChR) one of which is in the lipid/protein interface. In preliminary studies we are investigating how octanol interacts with the lipid bilayer. Analysis of the 1H-13C dipolar couplings present in 2D dipolar/chemical shift spectra we have been able to measure changes in bilayer fluidity at pharmacologically relevant concentrations. We have been able to correlate these results with 2D-1H-NOE-MAS spectra enabling us to locate the position of the octanol within the lipid bilayer. As these methods function with isotopes at natural abundance levels, we are currently applying them to native nAChR membranes to see if changes in the order parameter profile observed in model lipid bilayers are reproduced in native receptor membranes.The interaction of proteins with lateral domains (rafts) in lipid bilayers has been proposed to play an important role in the trafficking of proteins within cells. We are utilizing exchange spectroscopy to study the partitioning of Fukutin, a protein linked to the onset of muscular dystrophy, into lipid rafts. Exchange measurements have enabled us to observe the restricted lateral diffusion of phospholipids in the presence of lateral domains formed in vesicles formed from ternary lipid mixtures. The affects of these lateral domains on the structure and oligomeric state of the Fukutin is also being probed.

Expressiveness Issues in Brane Calculi: A Survey

Brane calculi are a family of biologically inspired process calculi proposed in [L. Cardelli. Brane Calculi - Interactions of biological membranes. In Proc. Computational Methods in System Biology 2004 (CMSB 2004), LNCS 3082, Springer, 2005] for modeling the interactions of dynamically nested membranes. In [L. Cardelli. Brane Calculi - Interactions of biological membranes. In Proc. Computational Methods in System Biology 2004 (CMSB 2004), LNCS 3082, Springer, 2005] a basic calculus for membranes interactions – called Phago/Exo/Pino (PEP) – is proposed, whose primitives are inspired by endocytosis and exocytosis. An alternative basic calculus – called Mate/Bud/Drip (MBD) and inspired by membrane fusion and fission – is also outlined and shown to be encodable in Phago/Exo/Pino in [L. Cardelli. Brane Calculi - Interactions of biological membranes. In Proc. Computational Methods in System Biology 2004 (CMSB 2004), LNCS 3082, Springer, 2005]. In this paper we survey some results on the comparison of the expressivity of the PEP and the MBD calculi, w.r.t. their ability to act as computational devices.