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Membrane Proteins Diffuse as Dynamic Complexes with Lipids
Abstract
We describe how membrane proteins diffuse laterally in the membrane plane together with the lipids surrounding them. We find a number of intriguing phenomena. The lateral displacements of the protein and the lipids are strongly correlated, as the protein and the neighboring lipids form a dynamical protein-lipid complex, consisting of approximately 50-100 lipids. The diffusion of the lipids in the complex is much slower compared to the rest of the lipids. We also find a strong directional correlation between the movements of the protein and the lipids in its vicinity. The results imply that in crowded membrane environments there are no "free" lipids, as they are all influenced by the protein structure and dynamics. Our results indicate that, in studies of cell membranes, protein and lipid dynamics have to be considered together.
... Many biological relevant processes take place on the nanometer length scale (Ramadurai et al., 2009;Busch et al., 2010) and at short times of a few nanoseconds. Consequently, it is not surprising that the complementary methods, quasielastic neutron scattering (QENS) experiments and molecular dynamics (MD) simulations, which cover these short time (from picoseconds up to several nanoseconds) and length (from Ångstrøm up to nanometers) scales, are perfectly suited to observe dynamical processes in membranes (Heller et al., 1993;Niemelä et al., 2010;Jeon et al., 2012;Pluhackova and Böckmann, 2015;Pluhackova et al., 2016b;Lautner et al., 2017;Srinivasan et al., 2019). ...
... On the other hand very little is known about the short-time transmembrane dynamics of proteins and peptides in membranes. Niemelä et al. (2010) used MD simulations to study the dynamics of a single Kv1.2 protein embedded in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayer in the liquid-crystalline phase. They observed a kind of transient complex formed by the protein and its neighboring lipids which diffuses laterally in the plane of the membrane. ...
... Finally, we introduce an estimation of the radius of the peptide influence on the lipid dynamics. Our experimental results of TFRC in DMPC membranes coincide with the findings of Kv1.2 protein embedded in POPC by MD simulations supporting the formation of transient complexes formed by the proteins and its neighboring lipids (Niemelä et al., 2010). ...
Lipids and proteins, as essential components of biological cell membranes, exhibit a significant degree of freedom for different kinds of motions including lateral long-range mobility. Due to their interactions, they not only preserve the cellular membrane but also contribute to many important cellular functions as e.g., signal transport or molecular exchange of the cell with its surrounding. Many of these processes take place on a short time (up to some nanoseconds) and length scale (up to some nanometers) which is perfectly accessible by quasielastic neutron scattering (QENS) experiments and molecular dynamics (MD) simulations. In order to probe the influence of a peptide, a transmembrane sequence of the transferrin receptor (TFRC) protein, on the dynamics of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) large unilamellar vesicles (LUVs) on a nanosecond time scale, high-resolution QENS experiments and complementary MD simulations have been utilized. By using different scattering contrasts in the experiment (chain-deuterated lipids and protonated lipids, respectively), a model could be developed which allows to examine the lipid and peptide dynamics separately. The experimental results revealed a restricted lipid lateral mobility in the presence of the TFRC transmembrane peptides. Also the apparent self-diffusion coefficient of the lateral movement of the peptide molecules could be determined quantitatively for the probed short-time regime. The findings could be confirmed very precisely by MD simulations. Furthermore, the article presents an estimation for the radius of influence of the peptides on the lipid long-range dynamics which could be determined by consistently combining results from experiment and simulation.
... We found that the presence of cholesterol at the interface results in new protein-protein interactions between adjacent AQP0 tetramers that may increase the stability of their association, but we wondered whether the cholesterol itself could contribute to the increased force needed to separate associated tetramers. Surface complementarity has previously been suggested to play a key role in modulating lipid-protein interactions (Aponte-Santamaría et al., 2012 ;Niemelä et al., 2010 ) with particular relevance for cholesterol (Kurth et al., 2020 ), prompting us to analyze the surface complementarity between AQP0 and cholesterol. We quantified surface complementarity as the contact area between the sandwiched lipids (either cholesterol or sphingomyelin) and the two tetramers, A Contact , and normalized it by the surface area of the lipid in question, A Lipid , i.e., a larger A Contact /A Lipid ratio would indicate a higher surface complementarity between the lipid and the proteins (see Methods for details of this calculation and Figure 8E for a schematic diagram). ...
... Nevertheless, cholesterol was rather depleted from the Chol1 position in AQP1. The shape of the protein surface has been shown to critically define the positions where lipids non-specifically associate with AQP0 (Aponte- Santamaría et al., 2012 ;Briones et al., 2017 ) and also other proteins (e.g., ion channels (Niemelä et al., 2010 ) and GPCRs (Kurth et al., 2020 ), among many others reviewed in (Corradi et al., 2019 )). This principle presumably applies here, too, i.e., the different surface of AQP0 at the Chol1 position, compared to that of AQP1, may result in a greater association of cholesterol over sphingolipids in this position (Figure 8-figure supplement 1). ...
Aquaporin-0 (AQP0) tetramers form square arrays in lens membranes through a yet unknown mechanism, but lens membranes are enriched in sphingomyelin and cholesterol. Here, we determined electron crystallographic structures of AQP0 in sphingomyelin/ cholesterol membranes and performed molecular dynamics (MD) simulations to establish that the observed cholesterol positions represent those seen around an isolated AQP0 tetramer and that the AQP0 tetramer largely defines the location and orientation of most of its associated cholesterol molecules. At a high concentration, cholesterol increases the hydrophobic thickness of the annular lipid shell around AQP0 tetramers, which may thus cluster to mitigate the resulting hydrophobic mismatch. Moreover, neighboring AQP0 tetramers sandwich a cholesterol deep in the center of the membrane. MD simulations show that the association of two AQP0 tetramers is necessary to maintain the deep cholesterol in its position and that the deep cholesterol increases the force required to laterally detach two AQP0 tetramers, not only due to protein–protein contacts but also due to increased lipid–protein complementarity. Since each tetramer interacts with four such ‘glue’ cholesterols, avidity effects may stabilize larger arrays. The principles proposed to drive AQP0 array formation could also underlie protein clustering in lipid rafts.
... Molecular dynamics simulations (MDS) allow an increasingly faithful representation of the lipid bilayer environment sheathing IMPs at the molecular scale . These studies have corroborated theoretical predictions and provided support for experimental evidence, together suggesting considerable perturbation of the lipid bilayer thickness, lateral packing and mobility in the immediate proximity of IMPs and at significant distance from them (Ebersberger et al., 2020;Marrink et al., 2019;Mouritsen and Bloom, 1984;Niemela et al., 2010;Phillips et al., 2009). ...
... distance, implying co-diffusion with the TMD, corroborated byMDS (Ebersberger et al., 2020), in full agreement with earlier MDS studies(Niemela et al., 2010). Recent progress in MS-based lipidomics of IMPs extracted in SMA-based native nanodisks (Teo et al., 2019) may be a promising avenue for experimentally define lipid fingerprints. ...
Theoretical work suggests that collective spatiotemporal behaviour of integral membrane proteins (IMPs) can be modulated by annular lipids sheathing their hydrophobic moiety. Here, we present evidence for this prediction in a natural membrane by investigating the mechanism that maintains steady amount of active isoform of Lck kinase (LckA) by Lck trans-autophosphorylation offset by the phosphatase CD45. We gauged experimental suitability by quantitation of CD45 and LckA subcellular localisation, LckA generation as a function of Lck and pharmacological perturbation. Steady LckA was challenged by swapping Lck membrane anchor with structurally divergent ones expected to substantially modify Lck annular lipids, such as that of Src or the transmembrane domains of LAT, CD4, palmitoylation-defective CD4 and CD45, respectively. The data showed only small alteration of LckA, except for CD45 hydrophobic anchor that thwarted LckA, due to excessive lateral proximity to CD45. The data are best explained by annular lipids facilitating or penalising IMPs' lateral proximity, hence modulating IMPs protein-protein functional interactions. Our findings can contribute to improve the understanding of biomembranes' organisation.
... The choice of the radius value does remain questionable. Numerical simulations suggest that one should rather consider the diffusion of a complex made of a protein with neighboring lipids [65] i.e. an effective protein radius, a eff . It is reasonable to postulate that the number of lipids diffusing with a protein should depend on its hydrophobic domain i.e. the "smoother" the hydrophobic part the smaller the effective radius. ...
... Finally if one assumes that the effective radius of each protein should lead to an average viscosity value determined above from lipid diffusion, <μ m (h/2) > ≈ 110 mPa.s, then it will reciprocally lead to an effective radius for TolC of a eff = 18 nm and for OprM of a eff = 65 nm. Simulations of the even more irregular hydrophobic part of voltage-gated channel Kv1.2, suggest that lipids diffuse with the protein up to 4 nm from the lipid-protein interface with an irregular distribution following the protein shape [65]. If applied to OprM this would result in an a eff no larger than 6 nm. ...
The viscosity of lipid bilayers is a property relevant to biological function, as it affects the diffusion of membrane macromolecules. To determine its value, and hence portray the membrane, various literature-reported techniques lead to significantly different results. Herein we compare the results issuing from two widely used techniques to determine the viscosity of membranes: the Fluorescence Lifetime Imaging Microscopy (FLIM), and Fluorescence Recovery After Photobleaching (FRAP). FLIM relates the time of rotation of a molecular rotor inserted into the membrane to the macroscopic viscosity of a fluid. Whereas FRAP measures molecular diffusion coefficients. This approach is based on a hydrodynamic model connecting the mobility of a membrane inclusion to the viscosity of the membrane.
We show that:
-The first method is very sensitive to local changes in viscosity; however, most often it would only provide the viscosity of the hydrophobic part of the membrane.
-The membrane viscosity is adequately estimated when the hydrodynamic model approach is applied to the mobility of micrometric size membrane inclusion but not for nanometric size inclusions such as lipids or proteins. In this case, the calculated value extracted from the same hydrodynamic model characterizes the interaction of the given nano-inclusion with the bilayer instead of the bilayer viscosity.
This article emphasizes the pitfalls to be avoided and the rules to be observed in order to obtain a value of the bilayer viscosity that characterizes the bilayer instead of interactions between the bilayer and the embedded probe.
... Despite all of the differences between the PM and the NM (e.g., lipid packing, single bilayer versus double bilayer, and others), making analogies between the two structures can help us to think about the NM as a conduit for mechanical signals. Of central importance is the notion that integral membrane proteins immobilized by an underlying matrix act as obstacles to lipid flow or diffusion (Nakada et al. 2003, Niemelä et al. 2010). The PM, and perhaps regions of the NM, may form so-called picket fences (Kusumi et al. 2012) that limit flow between (relaxed) regions of high lateral lipid pressure to (tense) regions of low lateral lipid pressure (Shi et al. 2018). ...
... By contrast, the mechanical consequences of solid-liquid coupling between integral membrane proteins and their surrounding lipid bilayers are more complex. Integral membrane proteins modulate the mobility of nearby lipids by forming shells of ∼50-100 lipids with reduced diffusivity around them (Niemelä et al. 2010). Anchored to underlying cytoskeletal structures, membrane-embedded proteins act as diffusion barriers (Nakada et al. 2003) and limit tension propagation within the membrane. ...
The cell nucleus is best known as the container of the genome. Its envelope provides a barrier for passive macromolecule diffusion, which enhances the control of gene expression. As its largest and stiffest organelle, the nucleus also defines the minimal space requirements of a cell. Internal or external pressures that deform a cell to its physical limits cause a corresponding nuclear deformation. Evidence is consolidating that the nucleus, in addition to its genetic functions, serves as a physical sensing device for critical cell body deformation. Nuclear mechanotransduction allows cells to adapt their acute behaviors, mechanical stability, paracrine signaling, and fate to their physical surroundings. This review summarizes the basic chemical and mechanical properties of nuclear components, and how these properties are thought to be utilized for mechanosensing.
Expected final online publication date for the Annual Review of Cell and Developmental Biology, Volume 37 is October 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... When SEDM was used to analyze biological membrane mimetics it was found that nanoscopic raft-like assemblies self-assembled through the attractive interactions of proteins and lipids. The experimental groups observed nanoscopic multicomponent biomolecular structures that consisted of small (3-6 nm) membrane proteins with encompassing pools of lipids (~5 nm) [89]. ...
... Shiga toxin) interact with ganglioside lipid headgroups they can induce the formation of curved lipid rafts, or caveolae[537,542,[1015][1016][1017][1018]. The ganglioside molecules move to areas of high positive curvature such as the rim of caveolae where their presence reduces the line tension energy and promotes large-scale membrane reshaping processes[671][672][673].Through the application of sophisticated microscopy methods, it was shown that lipid rafts can have a range of different sizes ranging from approximately 10 nm[89] through to 200 nm, i.e. an order of magnitude difference[348,1029]. Coarse-grained molecular dynamics simulations with the Martini forcefield have corroborated the inference that ganglioside molecules can form large lipid rafts when they cluster together[363,[497][498]. The coarsegrained Martini molecular dynamics simulations showed that coarse-grained ganglioside lipids can spontaneously self-assemble into clusters that have high intrinsic spontaneous curvature and further, that these glycosphingolipid clusters promote membrane curvature generation ...
Gram-negative bacteria have an unusual cell envelope that contains an inner cytoplasmic lipid membrane and an outer bacterial lipid membrane. The outer bacterial lipid membrane produces outer membrane vesicles that regulate bacterial pathogenesis processes. The outer membrane vesicles transport virulence factors from bacteria to host cell surfaces and the vesicles then move into the host cell cytosol. Computer simulations were conducted here in this thesis to understand how outer membrane vesicles pass through host cell surfaces independently of any membrane protein effects. The simulations suggest that outer membrane vesicles enter cells via lipidmediated endocytosis processes and interestingly, that the host membrane wrapping interactions depend on the length of the lipopolysaccharide macromolecules. Additional simulations were conducted to understand how polymyxin B1 peptides affect the inner and outer membranes of Gramnegative bacteria and how cohesive intermolecular interactions between lipopolysaccharide lipids can affect the durability of Gram-negative bacterial membranes. The simulation studies are by no means disparate; the simulations provide general insights into disease transmission. The simulations clarify how lipopolysaccharide macromolecules promote the spread of disease and conversely how antibiotics can curb it.
... Of note, transmembrane proteins are known to associate with an annular ring of lipids on a nanoscale [14,15]. Up to a hundred lipids may co-diffuse with the transmembrane protein forming a ring of reduced mobility extending several nanometers from the protein surface [16]. ...
... The side length of the simulated square was set to 750 nm, which ensured that all observed results were insensitive to changes in the size of the simulated area. To emulate the size of large transmembrane proteins including a ring of annular lipids [16,23,24] or hypothesized lipid rafts [25], nanofeatures were simulated with a radius R = 2 nm to 15 nm. The tracer diffusion constant in absence of nanofeatures was set to D out = 0.4 µm 2 s −1 , a typical value for lipid diffusion in the plasma membrane of a living cell [26]. ...
Nanoscopic features of reduced diffusivity have long been suggested to contribute to plasma membrane heterogeneity. Two prominent examples of this are highly dynamic lipid-mediated assemblies ('membrane rafts') and shells of annular lipids surrounding transmembrane proteins. Here, we simulated a micropatterning experiment, where such nanoscopic features are immobilized in specific areas within the live cell plasma membrane. We evaluated the effect of patterned nanofeatures of different sizes and diffusivities on the spatial distribution and two-dimensional mobility of tracer molecules. From this, we derive empirical models that describe the long-range tracer mobility as a function of the nanofeature density. In turn, our results facilitate the determination of nanofeature dimensions from micropatterning experiments.
... D shows a linear dependence on the depth of embedding into the membrane (h) and a logarithmic dependence on the particle radius (R) ( Fig. 2A). The particle radius for the transmembrane protein is determined not only by its own structure, but also by the tightly associated annular lipid shell [65,66]. Validity of Saffman-Delbr€ uck model was experimentally confirmed, for example, by measuring diffusion coefficients of membrane proteins of different sizes of diffusion in homogeneous lipid bilayers at low protein:lipid ratios [58,64]. ...
... However, Saffman-Delbr€ uck model does not account for the heterogeneous composition of native cellular membranes and specific protein:lipid contacts, which may occur even in model membranes. For instance, individual membrane proteins change their mobility when forming clusters together with the proximal solvating lipids [66,67]. Changes in the membrane thickness and the associated hydrophobic mismatch between lipids and incorporated proteins can modulate the diffusion behaviour in a complicated manner, up to the transition from a weak dependence D~ln(1/R) to the more pronounced size-dependent Stokes-like diffusion, where D~1/R [45,[67][68][69]. ...
Proteins are essential and abundant components of cellular membranes. Being densely packed within the limited surface area, proteins fulfil essential tasks for life, which include transport, signalling and maintenance of cellular homeostasis. The high protein density promotes nonspecific interactions, which affect the dynamics of the membrane‐associated processes, but also contribute to higher levels of membrane organization. Here, we provide a comprehensive summary of the most recent findings of diverse effects resulting from high protein densities in both living membranes and reconstituted systems and display why the crowding phenomenon should be considered and assessed when studying cellular pathways. Biochemical, biophysical and computational studies reveal effects of crowding on the translational mobility of proteins and lipids, oligomerization and clustering of integral membrane proteins, and also folding and aggregation of proteins at the lipid membrane interface. The effects of crowding pervade to larger length scales, where interfacial and transmembrane crowding shapes the lipid membrane. Finally, we discuss the design and development of fluorescence‐based sensors for macromolecular crowding and the perspectives to use those in application to cellular membranes and suggest some emerging topics in studying crowding at biological interfaces.
... It is well known that membrane proteins diffuse on the lipid membrane [47,48]. The diffusion constant is estimated to vary over a large range [49, Table 1]. ...
Elastic properties of nanoscale extracellular vesicles (EVs) are believed to influence their cellular interactions, thus having a profound implication in intercellular communication. Yet, an accurate quantification of the elasticity of such small lipid vesicles is difficult even with AFM-based nanoindentation experiments as it crucially depends on the reliability of the theoretical interpretation of such measurements. We developed here a unified model and experimental procedure to reliably estimate the elastic modulus of EVs. Further, we experimentally demonstrate that the quantification of EV-elastic modulus from AFM-based force spectroscopy measurement is marred by the interplay of their compositionally inhomogeneous fluid membrane with the adhesion forces from the substrate and thermal effects, two exquisite phenomena which could thus far only be theoretically predicted. The effects result in a large spreading of elastic modulus even for a single EV. Our unified model is then applied to genetically engineered classes of EVs to understand how the alterations in tetraspanin expression may influence their elastic modulus.
... This is most likely what inhibits free rearrangement of the lipids in the supported membranes and leads to the ruptures observed by the AFM. Reduced lipid diffusivity was also observed in an MD study [40] for the WALP23 dimer, where a small pool of slowly diffusing lipids was identified. All this preceding and current evidence demonstrates that non-specific interactions of a TM domain with lipids have important implications for the structure, dynamics, and macroscopic mechanical properties of lipid membranes. ...
... In addition, strong attractions of TG with protein residues can further reduce the rate of diffusion (Figure 2d). We note that the slower diffusion of the lipids that are near the protein have been discussed in previous papers (Javanainen et al., 2017;Niemelä et al., 2010). The mean squared displacement of the lumenal PLs trapped inside the seipin ring, referred to as proteinized PLs, leveled off at later simulation times due to confinement (Figure 2-figure supplement 3). ...
Lipid droplets (LDs) are organelles formed in the endoplasmic reticulum (ER) to store triacylglycerol (TG) and sterol esters. The ER protein seipin is key for LD biogenesis. Seipin forms a cage-like structure, with each seipin monomer containing a conserved hydrophobic helix (HH) and two transmembrane (TM) segments. How the different parts of seipin function in TG nucleation and LD budding is poorly understood. Here, we utilized molecular dynamics simulations of human seipin, along with cell-based experiments, to study seipin's functions in protein-lipid interactions, lipid diffusion, and LD maturation. An all-atom (AA) simulation indicates that seipin TM segment residues and hydrophobic helices residues located in the phospholipid (PL) tail region of the bilayer attract TG. Simulating larger, growing LDs with coarse-grained (CG) models, we find that the seipin TM segments form a constricted neck structure to facilitate conversion of a flat oil lens into a budding LD. Using cell experiments and simulations, we also show that conserved, positively charged residues at the end of seipin's TM segments affect LD maturation. We propose a model in which seipin TM segments critically function in TG nucleation and LD growth.
... It is estimated that a mean residence time in the annulus is 10 times higher than for the surrounding lipid layer [100]. A more detailed insight into this issue was performed by Niemelä et al. [101] who applied all-atom (for voltage dependent potassium channel) and coarse grain (lactose permease in a POPC bilayer and WALP23 dimer in a DPPC bilayer) MD simulations to describe the behavior of lipids in close proximity to proteins. As deduced from mean lateral displacement tracking, for all investigated systems, lipid molecules can be divided into two groups depending on their mobility. ...
Lipids, together with molecules such as DNA and proteins, are one of the most relevant systems responsible for the existence of life. Selected lipids are able to assembly into various organized structures, such as lipid membranes. The unique properties of lipid membranes determine their complex functions, not only to separate biological environments, but also to participate in regulatory functions, absorption of nutrients, cell-cell communication, endocytosis, cell signaling, and many others. Despite numerous scientific efforts, still little is known about the reason underlying the variability within lipid membranes, and its biochemical significance. In this review, we discuss the structural complexity of lipid membranes, as well as the importance to simplify studied systems in order to understand phenomena occurring in natural, complex membranes. Such systems require a model interface to be analyzed. Therefore, here we focused on analytical studies of artificial systems at various interfaces. The molecular structure of lipid membranes, specifically the nanometric thickens of molecular bilayer, limits in a major extent the choice of highly sensitive methods suitable to study such structures. Therefore, we focused on methods that combine high sensitivity, and/or chemical selectivity, and/or nanometric spatial resolution, such as atomic force microscopy, nanospectroscopy (tip-enhanced Raman spectroscopy, infrared nanospectroscopy), phase modulation infrared reflection-absorption spectroscopy, sum-frequency generation spectroscopy. We summarized experimental and theoretical approaches providing information about molecular structure and composition, lipid spatial distribution (phase separation), organization (domain shape, molecular orientation) of lipid membranes, and real-time visualization of the influence of various molecules (proteins, drugs) on their integrity. An integral part of this review discusses the latest achievements in the field of lipid layer-based biosensors.
... It has been shown that diffusion of lipids in the proximity of proteins is noticeably decreased compared to freely diffusing ones. [98][99][100] Ap rotein determines the formation of an anometre-scale lipid patch of reduced mobility,r evoking the compartmentalisation of membranes described above.Onthe other hand, the mobility of the formed receptor/ligand complex was shown to depend on the strength of proteinlipid association, [101] further illustrating the reciprocal action of proteins and surrounding lipids. ...
Numerous key biological processes rely on the concept of multivalency, where ligands achieve stable binding only upon engaging multiple receptors. These processes, like viral entry or immune synapse formation, occur on the diffusive cellular membrane. One crucial, yet underexplored aspect of multivalent binding is the mobility of coupled receptors. Here, we discuss the consequences of mobility in multivalent processes from four perspectives: (I) The facilitation of receptor recruitment by the multivalent ligand due to their diffusivity prior to binding. (II) The effects of receptor preassembly, which allows their local accumulation. (III) The consequences of changes in mobility upon the formation of receptor/ligand complex. (IV) The changes in the diffusivity of lipid environment surrounding engaged receptors. We demonstrate how understanding mobility is essential for fully unravelling the principles of multivalent membrane processes, leading to further development in studies on receptor interactions, and guide the design of new generations of multivalent ligands.
... Biological membranes are composed of numerous proteins at concentrations reaching as high as 25% area occupancy, resulting in crowded environments [55]. Over the last years, it has become increasingly apparent that lipids within biological membranes have intricate relationships with membrane proteins, and their interaction with the membranes is fundamental to comprehend the structure of biomembranes and many physiological phenomena [61,62]. ...
The amounts of antibiotics of anthropogenic origin released and accumulated in the environment are known to have a negative impact on local communities of microorganisms, which leads to disturbances in the course of the biodegradation process and to growing antimicrobial resistance. This mini-review covers up-to-date information regarding problems related to the omnipresence of antibiotics and their consequences for the world of bacteria. In order to understand the interaction of antibiotics with bacterial membranes, it is necessary to explain their interaction mechanism at the molecular level. Such molecular-level interactions can be probed with Langmuir monolayers representing the cell membrane. This mini-review describes monolayer experiments undertaken to investigate the impact of selected antibiotics on components of biomembranes, with particular emphasis on the role and content of individual phospholipids and lipopolysaccharides (LPS). It is shown that the Langmuir technique may provide information about the interactions between antibiotics and lipids at the mixed film surface (π–A isotherm) and about the penetration of the active substances into the phospholipid monolayer model membranes (relaxation of the monolayer). Effects induced by antibiotics on the bacterial membrane may be correlated with their bactericidal activity, which may be vital for the selection of appropriate bacterial consortia that would ensure a high degradation efficiency of pharmaceuticals in the environment.
... The anomalous nature of lipid diffusion is typically reported to be sub-diffusive with α < 1 when the lipids are crowded by large external molecules such as proteins or peptides . Anomalous diffusion has been widely observed in lipid dynamics of multicomponent membranes induced by compositional heterogeneity as well as the presence of transmembrane proteins (Niemelä et al., 2010;Jeon et al., 2016;Javanainen et al., 2017). Thus the manner in which the free particle motion is modulated in these different environments have been referred to as either trapped or hop diffusion to primarily differentiate between the underlying restrictions to particle motion. ...
Pore forming proteins are a broad class of pathogenic proteins secreted by organisms as virulence factors due to their ability to form pores on the target cell membrane. Bacterial pore forming toxins (PFTs) belong to a subclass of pore forming proteins widely implicated in bacterial infections. Although the action of PFTs on target cells have been widely investigated, the underlying membrane response of lipids during membrane binding and pore formation has received less attention. With the advent of superresolution microscopy as well as the ability to carry out molecular dynamics (MD) simulations of the large protein membrane assemblies, novel microscopic insights on the pore forming mechanism have emerged over the last decade. In this review, we focus primarily on results collated in our laboratory which probe dynamic lipid reorganization induced in the plasma membrane during various stages of pore formation by two archetypal bacterial PFTs, cytolysin A (ClyA), an α -toxin and listeriolysin O (LLO), a β -toxin. The extent of lipid perturbation is dependent on both the secondary structure of the membrane inserted motifs of pore complex as well as the topological variations of the pore complex. Using confocal and superresolution stimulated emission depletion (STED) fluorescence correlation spectroscopy (FCS) and MD simulations, lipid diffusion, cholesterol reorganization and deviations from Brownian diffusion are correlated with the oligomeric state of the membrane bound protein as well as the underlying membrane composition. Deviations from free diffusion are typically observed at length scales below ∼130 nm to reveal the presence of local dynamical heterogeneities that emerge at the nanoscale—driven in part by preferential protein binding to cholesterol and domains present in the lipid membrane. Interrogating the lipid dynamics at the nanoscale allows us further differentiate between binding and pore formation of β - and α -PFTs to specific domains in the membrane. The molecular insights gained from the intricate coupling that occurs between proteins and membrane lipids and receptors during pore formation are expected to improve our understanding of the virulent action of PFTs.
... A more accurate view is by considering a mutual impact of lipid-protein interactions on the lateral organization of biological membranes (Poveda et al., 2008;Lingwood and Simons, 2010;Groger et al., 2012). Furthermore, membrane proteins appear to diffuse in a concerted manner with numerous lipids around them, predicting only a few if any free lipids due to molecular crowing in membranes (Niemela et al., 2010). In this context, oligomerization of membrane proteins such as respiratory complexes can represent an important factor in lipid domain formation (Figure 3). ...
... Transmembrane proteins are known to reduce lipid diffusion of vicinal lipids [6,7,104]. Here, we have investigated the effect of a transmembrane protein on the trans fraction of the lipid tail dihedral angles at AA resolution and the difference in the order parameters of CG lipids, thus investigating the ordering effect of AQP1 on neighboring lipids. Interestingly, the ordering effect in AA resolution is not linear with temperature. ...
Background
Lipid-protein interactions stabilize protein oligomers, shape their structure, and modulate their function. Whereas in vitro experiments already account for the functional importance of lipids by using natural lipid extracts, in silico methods lack behind by embedding proteins in single component lipid bilayers. However, to accurately complement in vitro experiments with molecular details at very high spatio-temporal resolution, molecular dynamics simulations have to be performed in natural(-like) lipid environments.
Results
To enable more accurate MD simulations, we have prepared four membrane models of E. coli polar lipid extract, a typical model organism, each at all-atom (CHARMM36) and coarse-grained (Martini3) representations. These models contain all main lipid headgroup types of the E. coli inner membrane, i.e., phosphatidylethanolamines, phosphatidylglycerols, and cardiolipins, symmetrically distributed between the membrane leaflets. The lipid tail (un)saturation and propanylation stereochemistry represent the bacterial lipid tail composition of E. coli grown at 37 ∘ C until 3/4 of the log growth phase. The comparison of the Simple three lipid component models to the complex 14-lipid component model Avanti over a broad range of physiologically relevant temperatures revealed that the balance of lipid tail unsaturation and propanylation in different positions and inclusion of lipid tails of various length maintain realistic values for lipid mobility, membrane area compressibility, lipid ordering, lipid volume and area, and the bilayer thickness. The only Simple model that was able to satisfactory reproduce most of the structural properties of the complex Avanti model showed worse agreement of the activation energy of basal water permeation with the here performed measurements. The Martini3 models reflect extremely well both experimental and atomistic behavior of the E. coli polar lipid extract membranes. Aquaporin-1 embedded in our native(-like) membranes causes partial lipid ordering and membrane thinning in its vicinity. Moreover, aquaporin-1 attracts and temporarily binds negatively charged lipids, mainly cardiolipins, with a distinct cardiolipin binding site in the crevice at the contact site between two monomers, most probably stabilizing the tetrameric protein assembly.
Conclusions
The here prepared and validated membrane models of E. coli polar lipids extract revealed that lipid tail complexity, in terms of double bond and cyclopropane location and varying lipid tail length, is key to stabilize membrane properties over a broad temperature range. In addition, they build a solid basis for manifold future simulation studies on more realistic lipid membranes bridging the gap between simulations and experiments.
... Article times; since different lipid species, as well as the protein(s), must be given sufficient time to mix (diffuse) in the matrix. 91 Simple POPC lipid matrices were used on the basis of experimental results on oligomerization of UCPs 3,21 and scarcity of computational studies of UCP2 in these lipids. In this protein−lipid system, average root-mean-squaredeviations (RMSDs), radius of gyration (R g ), principal component analysis (PCA), solvent accessible surface area (SASA) per chain, and pore radius and free radius were determined. ...
Stoichiometry of uncoupling proteins (UCPs) and their coexistence as functional monomeric and associated forms in lipid membranes remain intriguing open questions. In this study, tertiary and quaternary structures of UCP2 were analyzed experimentally and through molecular dynamics (MD) simulations. UCP2 was overexpressed in the inner membrane of Escherichia coli, then purified and reconstituted in lipid vesicles. Structure and proton transport function of UCP2 were characterized by circular dichroism (CD) spectroscopy and fluorescence methods. Findings suggest a tetrameric functional form for UCP2. MD simulations conclude that tetrameric UCP2 is a dimer of dimers, is more stable than its monomeric and dimeric forms, is asymmetrical and induces asymmetry in the membrane's lipid structure, and a biphasic on-off switch between the dimeric units is its possible mode of transport. MD simulations also show that the water density inside the UCP2 monomer is asymmetric, with the cytoplasmic side having a higher water density and a wider radius. In contrast, the structurally comparable adenosine 5'-diphosphate (ADP)/adenosine 5'-triphosphate (ATP) carrier (AAC1) did not form tetramers, implying that tetramerization cannot be generalized to all mitochondrial carriers.
... Despite this ordering of acyl chains, experiments with SUVs composed of E. coli B LPS and phospholipid extract doped with spin-labelled PE and PG lipids, and S. typhimurium OM lipid preparations doped with a spin-labelled stearic acid probe, found that phospholipids remained freely diffusive and segregated away from LPS (197,228,229). The slower diffusion of phospholipids in the inner leaflet of the OM observed in the previous study (which retained its complement of OMPs) (225) may therefore be due to the transient clustering and reduced diffusion of lipids around embedded OMPs (157, [230][231][232]. These in vitro studies, although not on fully asymmetric membranes (207), broadly validate the observations of the above in silico studies. ...
β-barrel outer membrane proteins (OMPs) represent the major proteinaceous component of the outer membrane (OM) of Gram-negative bacteria. These proteins perform key roles in cell structure and morphology, nutrient acquisition, colonisation and invasion, and protection against external toxic threats such as antibiotics. To become functional, OMPs must fold and insert into a crowded and asymmetric OM that lacks much freely accessible lipid. This feat is accomplished in the absence of an external energy source and is thought to be driven by the high thermodynamic stability of folded OMPs in the OM. With such a stable fold, the challenge that bacteria face in assembling OMPs into the OM is how to overcome the initial energy barrier of membrane insertion. In this review, we highlight the roles of the lipid environment and the OM in modulating the OMP folding landscape and discuss the factors that guide folding in vitro and in vivo. We particularly focus on the composition, architecture and physical properties of the OM and how an understanding of the folding properties of OMPs in vitro can help explain the challenges they encounter during folding in vivo. Current models of OMP biogenesis in the cellular environment are still in flux, but the stakes for improving the accuracy of these models are high. Since OMP folding is an essential process in all Gram-negative bacteria, and considering the looming crisis of widespread microbial drug resistance, to bring down the powerful, OMP-supported barrier against antibiotics, we must first understand how bacterial cells build it.
... 16 This indicates that proteins undergo repeated collisions with other proteins, 17 which can further affect lipid motion via confinement and excluded area effects, indicating that essentially no "bulk" lipids exist in cellular membranes. 18,19 This picture is drastically different from the one considered in the derivation of the SD model, hinting that the model should be put to test also under crowded conditions. ...
Membrane proteins travel along cellular membranes and reorient themselves to form functional oligomers and protein–lipid complexes. Following the Saffman–Delbrück model, protein radius sets the rate of this diffusive motion. However, it is unclear how this model — derived for ideal and dilute membranes — performs under crowded conditions of cellular membranes. Here, we study the rotational motion of membrane proteins using molecular dynamics simulations of coarse-grained membranes and 2-dimensional Lennard-Jones fluids with varying levels of crowding. We find that the Saffman–Delbrück model captures the size-dependency of rotational diffusion under dilute conditions, whereas a stronger scaling laws arise under crowding. Together with our recent work on lateral diffusion, our results reshape the description of protein dynamics in their native membrane environments: the translational and rotational motion of proteins with small transmembrane domains is rapid, whereas larger proteins or protein complexes display substantially slower dynamics.
... Given that the typical assembly takes 0.5-1.5 µs, the lipids have an opportunity to leave the cavity during the assembly simulation. Third, lipids are known to co-diffuse with membrane proteins (Niemelä et al., 2010), and thus might be dragged by the half-rings pulled by the biasing potential. In assembly simulations with biasing potential coefficients of 0.01 kJ/(mol·nm 2 ) most of the lipids that are eventually trapped inside the c-ring are initially positioned in between the c 7 halfrings ( Figure 6A). ...
Rotor ATPases are large multisubunit membrane protein complexes found in all kingdoms of life. The membrane parts of these ATPases include a ring-like assembly, so-called c-ring, consisting of several subunits c, plugged by a patch of phospholipids. In this report, we use a nature-inspired approach to model the assembly of the spinach (Spinacia oleracea) c14 ring protein-lipid complex, where partially assembled oligomers are pulled toward each other using a biasing potential. The resulting assemblies contain 23 to 26 encapsulated plug lipids, general position of which corresponds well to experimental maps. However, best fit to experimental data is achieved with 15 to 17 lipids inside the c-ring. In all of the simulations, the lipids from one leaflet (loop side of the c subunit) are ordered and static, whereas the lipids from the other leaflet are disordered and dynamic. Spontaneous permeation of water molecules toward Glu61 at the active site is also observed. The presented assembly approach is expected to be generalizable to other protein complexes with encapsulated lipid patches.
... doi: bioRxiv preprint first posted online Sep. 20, 2019; of the distance from the protein complex, and we found that interactions between the TG molecules and the seipin complex raise the local concentration of TG in the proximity of the protein to 4%, twice the initial value in the bulk membrane ( Figure 4C,D) in our MD simulations. This increase in local concentration is due to a decrease in TG diffusion in proximity of the protein (Figure E,F), analogously to what has been shown for diffusion of lipids around proteins 29 . (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. ...
Cells store energy in the form of neutral lipids packaged into micrometer-sized organelles named lipid droplets (LDs). These structures emerge from the endoplasmic reticulum (ER), but their biogenesis remains poorly understood. Using molecular simulations, we found that LD formation proceeds via a liquid-liquid phase separation process that is modulated by the physical properties and lipid composition of the ER membrane. LD formation is promoted at ER sites characterized by high membrane curvature and by the presence of the ER-associated protein seipin, that cause accumulation of triglycerides by slowing down their diffusion in the membrane. Our data indicate how a combination of membrane physical properties and protein scaffolding is used by the cell to regulate a broad and energetically-efficient biophysical process such as liquid/liquid phase separation to achieve LD biogenesis.
... Membrane lipid packing is critical to warrant proper protein sorting. MD simulations of simplified lipid membranes have suggested that prokaryotic membrane proteins form with their adjacent lipids dynamic protein-lipid complexes with up to 50 to 100 lipids that diffuse laterally together [278]. Considering that lipid diffusion rates are significantly reduced within these shells and accepting that membranes are "more mosaic than fluid" [279], it becomes difficult to tell apart an actively recruited annulus from lipids from preexisting Lo domains, in which the lateral mobility of lipids is reduced. ...
Calcium ions (Ca2+) are major messengers in cell signaling, impacting nearly every aspect of cellular life. Those signals are generated within a wide spatial and temporal range through a large variety of Ca2+ channels, pumps, and exchangers. More and more evidences suggest that Ca2+ exchanges are regulated by their surrounding lipid environment. In this review, we point out the technical challenges that are currently being overcome and those that still need to be defeated to analyze the Ca2+ transport protein–lipid interactions. We then provide evidences for the modulation of Ca2+ transport proteins by lipids, including cholesterol, acidic phospholipids, sphingolipids, and their metabolites. We also integrate documented mechanisms involved in the regulation of Ca2+ transport proteins by the lipid environment. Those include: (i) Direct interaction inside the protein with non-annular lipids; (ii) close interaction with the first shell of annular lipids; (iii) regulation of membrane biophysical properties (e.g., membrane lipid packing, thickness, and curvature) directly around the protein through annular lipids; and (iv) gathering and downstream signaling of several proteins inside lipid domains. We finally discuss recent reports supporting the related alteration of Ca2+ and lipids in different pathophysiological events and the possibility to target lipids in Ca2+-related diseases.
... This effect is difficult to observe through experimental procedures due to spatial and temporal limitations(51). MPs, in turn, affect membrane properties such as lipid diffusion(52), membrane thickness (53), and membrane curvature or distortion(54). Such effects of MPs on shaping the membrane are exemplified by molecular dynamics (MD) simulations of the voltage-gated potassium channels (KvAP K + channel) S4 transmembrane helix. ...
... 21 To this end, we calculated the mean in-plane displacements of lipids over a 1 ns interval as a function of distance from the probe lipid. Using an analogy to the diffusion of lipids in the vicinity of a transmembrane protein, 23 we expect the lipids next to the sAv-bound probe lipid to move slowly and the movement of lipids to speed up as their distance from the probe lipid increases. This is also what we observe ( Figure 3B). ...
Single particle tracking (SPT) is an experimental technique, which allows one to follow the dynamics of individual molecules in biological membranes with unprecedented precision. Given the importance of lipid and membrane protein diffusion in the formation of nanoscale functional complexes, it is critical to understand what exactly is measured in SPT experiments. To clarify this issue, we employed nanoscale computer simulations designed to match SPT experiments that exploit streptavidin-functionalized Au nanoparticles (AuNPs). The results show that lipid labeling interferes critically the diffusion process, thus the diffusion measured in SPT is a far more complex process than what has been assumed. It turns out that the influence of AuNP-based labels on the dynamics of probe lipids includes not only the AuNP-induced viscous drag that is the more significant the larger the NP is, but more importantly also the effects related to the interactions of the streptavidin linker with membrane lipids. Due to these effects, the probe lipid moves in a concerted manner as a complex with the linker protein and numerous unlabeled lipids, which can slow down the motion of the probe by almost an order of magnitude. Furthermore, our simulations show that non-linker streptavidin tetramers on the AuNP surface are able to interact with the membrane lipids, which could potentially lead to multivalent labeling of the NPs by the probe lipids. Our results further demonstrate that in the sub-microsecond time domain the motion of the probe lipid is uncorrelated with the motion of the AuNP, showing that there is a microsecond limit for the temporal resolution of the SPT technique. However, this limit for the temporal resolution depends on the nanoparticle size and increases rapidly with growing AuNPs. Overall, the results provide a molecular-scale framework to accurately interpret SPT data and to design protocols that minimize label-induced artifacts.
... Furthermore, MD simulations are frequently used to study nonspecific lipid binding. Key papers in this area are from Vattulainen and co-workers, 430,431 in which they show strong correlations between the lateral diffusion of membrane proteins and a shell of 50−100 annular lipids. This kind of protein−lipid binding is observed even in lipid membranes composed of a single lipid type and therefore clearly nonspecific. ...
Cell membranes contain a large variety of lipid types and are crowded with proteins, endowing them with the plasticity needed to fulfill their key roles in cell functioning. The compositional complexity of cellular membranes gives rise to a heterogeneous lateral organization, which is still poorly understood. Computational models, in particular molecular dynamics simulations and related techniques, have provided important insight into the organizational principles of cell membranes over the past decades. Now, we are witnessing a transition from simulations of simpler membrane models to multicomponent systems, culminating in realistic models of an increasing variety of cell types and organelles. Here, we review the state of the art in the field of realistic membrane simulations and discuss the current limitations and challenges ahead.
... This is illustrated by the so-called "lipid shell" formed by lipids surrounding the transmembrane segment of a protein. It is assumed that such lipid shells behave like individual thermodynamically stable structures ( (Niemela et al., 2010) and for review (Anderson and Jacobson, 2002)). ...
The plasma membrane is organized at numerous levels as a result of its large variety of molecular constituents and of selective interactions between them. Lateral diffusion, a direct physical consequence of the Brownian agitation, plays a key organizational role by constantly redistributing the membrane constituents among the possible molecular associations. In this context, we will first review the physical mechanisms contributing to the creation of inhomogeneity. We will then describe the current methodological approaches allowing us to measure diffusion in living cells. The different levels of membrane organization will be discussed before illustrating the impact of the dynamic organization of the membrane on cellular functions.
The lateral mobility of molecules within the cell membrane is ultimately governed by the local environment of the membrane. Confined regions induced by membrane structures, such as protein aggregates or the actin meshwork, occur over a wide range of length scales and can impede or steer the diffusion of membrane components. However, a detailed picture of the origins and nature of these confinement effects remains elusive. Here, we prepare model lipid systems on substrates patterned with confined domains of varying geometries constructed with different materials to explore the influences of physical boundary conditions and specific molecular interactions on diffusion. We demonstrate a platform that is capable of significantly altering and steering the long-range diffusion of lipids by using simple oxide deposition approaches, enabling us to systematically explore how confinement size and shape impact diffusion over multiple length scales. While we find that a “boundary condition” description of the system captures underlying trends in some cases, we are also able to directly compare our systems to analytical models, revealing the unexpected breakdown of several approximate solutions. Our results highlight the importance of considering the length scale dependence when discussing properties such as diffusion.
Lipid bilayers—the main matrix of cell membranes—are a paradigm of soft molecular assemblies whose properties have been evolutionarily optimized to satisfy the functional requirements of cells. For instance, lipid bilayers must be rigid enough to serve as the protective barrier between cells and their environment, yet fluid enough to enable the diffusion of proteins and molecular clusters necessary for biological functions. Inspired by their biological multifunctionality, lipid membranes have also been used as a central design element in many practical applications including artificial cells, drug nanocarriers, and biosensors. Whether biological or synthetic, lipid membranes often involve molecular or nanoscopic additives that modulate the membrane properties through various mechanisms. Hence, how lipid membranes respond to additives has justifiably drawn much attention in recent years. This review summarizes findings and observations on different classes of additives and their effects on structural, thermodynamic, elastic, and dynamical membrane properties that are central to biological function or synthetic membrane performance. The review primarily focuses on phospholipids as a major component of cell membranes and a widely used lipid type in synthetic membrane designs.
Structural and mechanical properties of membranes such as thickness, tail order, bending modulus and curvature energetics play crucial role in controlling various cellular functions that depend on the local lipid organization and membrane reshaping. While behavior of these biophysical properties are well understood in single component membranes, very little is known about how do they change in the mixed lipid membranes. Often various properties of the mixed lipid bilayers are assumed to change linearly with the mole fractions of the constituent lipids which, however, is true for “ideal” mixing only. In this study, using molecular dynamics simulations, we show that structural and mechanical properties of binary lipid mixture change nonlinearly with the lipid mole fractions, and the strength of the nonlinearity depends on two factors - spontaneous curvature difference and locally inhomogeneous interactions between the lipid components.
The lateral diffusion of cell membrane inclusions, such as integral membrane proteins and bound receptors, drives critical biological processes, including the formation of complexes, cell-cell signaling, and membrane trafficking. These diffusive processes are complicated by how concentrated, or "crowded", the inclusions are, which can occupy between 30-50% of the area fraction of the membrane. In this work, we elucidate the effects of increasing concentration of model membrane inclusions in a free-standing artificial cell membrane on inclusion diffusivity and the apparent viscosity of the membrane. By multiple particle tracking of fluorescent microparticles covalently tethered to the bilayer, we show the transition from expected Brownian dynamics, which accurately measure the membrane viscosity, to subdiffusive behavior with decreased diffusion coefficient as the particle area fraction increases from 1% to around 30%, approaching physiological levels of crowding. At high crowding, the onset of non-Gaussian behavior is observed. Using hydrodynamic models relating the 2D diffusion coefficient to the viscosity of a membrane, we determine the apparent viscosity of the bilayer from the particle diffusivity and show an increase in the apparent membrane viscosity with increasing particle area fraction. However, the scaling of this increase is in contrast with the behavior of monolayer inclusion diffusion and bulk suspension rheology. These results demonstrate that physiological levels of model membrane crowding nontrivially alter the dynamics and apparent viscosity of the system, which has implications for understanding membrane protein interactions and particle-membrane transport processes.
Withdrawal Statement
The authors have withdrawn this manuscript owing to its merge with BIORXIV/2023/540959. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author. The merged preprint can be found at doi.org/10.1101/2023.05.16.540959
The tetrameric water channel aquaporin-0 (AQP0) forms square arrays in lens membranes through an as yet unknown mechanism. Lens membranes are enriched in cholesterol and sphingomyelin, suggesting that these raft lipids may play a role in AQP0 array formation. We produced two-dimensional crystals of AQP0 in sphingomyelin membranes with low and high cholesterol content and determined their structures by electron crystallography. At the higher cholesterol concentration, cholesterol associates with AQP0 and shifts lipids in the cytoplasmic leaflet away from the bilayer center, increasing the hydrophobic thickness of the annular lipid shell. AQP0 tetramers may thus. CC-BY-NC 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 18, 2023. ; https://doi.org/10.1101/2023.05.16.540959 doi: bioRxiv preprint 2 cluster to mitigate the resulting hydrophobic mismatch. Moreover, neighboring AQP0 tetramers sandwich a cholesterol in the center of the membrane. Since each tetramer interacts with four such 'glue' cholesterols, avidity effects may stabilize larger arrays. The principles proposed to drive AQP0 array formation could also underlie protein clustering in lipid rafts.
Integral membrane proteins are embedded into cell membranes by spanning the width of the lipid bilayer. They play an essential role in important biological functions for the survival of living organisms. Their functions include the transportation of ions and molecules across the cell membrane and initiating signaling pathways. The dynamic behavior of integral membrane proteins is very important for their function. Due to the complex behavior of integral membrane proteins in the cell membrane, studying their structural dynamics using biophysical approaches is challenging. Here, we concisely discuss challenges and recent advances in technical and methodological aspects of biophysical approaches for gleaning dynamic properties of integral membrane proteins to answer pertinent biological questions associated with these proteins.
Theoretical work suggests that collective spatiotemporal behaviour of integral membrane proteins (IMPs) should be modulated by boundary lipids sheathing their membrane anchors. Here, we show evidence for this prediction whilst investigating the mechanism for maintaining a steady amount of the active form of IMP Lck kinase (LckA) by Lck trans-autophosphorylation regulated by the phosphatase CD45. We used super-resolution microscopy, flow cytometry, and pharmacological and genetic perturbation to gain insight into the spatiotemporal context of this process. We found that LckA is generated exclusively at the plasma membrane, where CD45 maintains it in a ceaseless dynamic equilibrium with its unphosphorylated precursor. Steady LckA shows linear dependence, after an initial threshold, over a considerable range of Lck expression levels. This behaviour fits a phenomenological model of trans-autophosphorylation that becomes more efficient with increasing LckA. We then challenged steady LckA formation by genetically swapping the Lck membrane anchor with structurally divergent ones, such as that of Src or the transmembrane domains of LAT, CD4, palmitoylation-defective CD4 and CD45 that were expected to drastically modify Lck boundary lipids. We observed small but significant changes in LckA generation, except for the CD45 transmembrane domain that drastically reduced LckA due to its excessive lateral proximity to CD45. Comprehensively, LckA formation and maintenance can be best explained by lipid bilayer critical density fluctuations rather than liquid-ordered phase-separated nanodomains, as previously thought, with “like/unlike” boundary lipids driving dynamical proximity and remoteness of Lck with itself and with CD45.
Sticholysins are α-pore-forming toxins produced by the sea-anemone Stichodactyla helianthus. These toxins exert their activity by forming pores on sphingomyelin-containing membranes. Recognition of sphingomyelin by sticholysins is required to start the process of pore formation. Sphingomyelin recognition is coupled with membrane binding and followed by membrane penetration and oligomerization. Many features of these processes are known. However, the extent of contact with each of the different kinds of lipids present in the membrane has received little attention. To delve into this question, we have used a phosphatidylcholine analogue labeled at one of its acyl chains with a doxyl moiety, a known quencher of tryptophan emission. Here we present evidence for the contact of sticholysins with phosphatidylcholine lipids in the sticholysin oligomer, and for how each sticholysin isotoxin is affected differently by the inclusion of cholesterol in the membrane. Furthermore, using phosphatidylcholine analogs that were labeled at different positions of their structure (acyl chains and headgroup) in combination with a variety of sticholysin mutants, we also investigated the depth of the tryptophan residues of sticholysins in the bilayer. Our results indicate that the position of the tryptophan residues relative to the membrane normal is deeper when cholesterol is absent from the membrane.
Numerous key biological processes rely on the concept of multivalency, where ligands achieve stable binding only upon engaging multiple receptors. These processes, like viral entry or immune synapse formation, occur on the diffusive cellular membrane. One crucial, yet underexplored aspect of multivalent binding is the mobility of coupled receptors. Here, we discuss the consequences of mobility in multivalent processes from four perspectives: (I) The facilitation of receptor recruitment by the multivalent ligand due to their diffusivity prior to binding. (II) The effects of receptor preassembly, which allows their local accumulation. (III) The consequences of changes in mobility upon the formation of receptor/ligand complex. (IV) The changes in the diffusivity of lipid environment surrounding engaged receptors. We demonstrate how understanding mobility is essential for fully unravelling the principles of multivalent membrane processes, leading to further development in studies on receptor interactions, and guide the design of new generations of multivalent ligands.
The dynamic behavior of plasma membrane proteins mediates various cellular processes such as cellular motility, communication, and signaling. It is widely accepted that the dynamics of the membrane proteins is determined either by the interactions of the transmembrane domain with the surrounding lipids or by the interactions of the intracellular domain with cytosolic components such as cortical actin. Although initiation of different cellular signaling events at the plasma membrane has been attributed to the extracellular domain (ECD) properties recently, the impact of ECDs on the dynamic behavior of membrane proteins is rather unexplored. Here, we investigate how the ECD properties influence protein dynamics in the lipid bilayer by reconstituting ECDs of different sizes or glycosylation in model membrane systems and analyzing ECD-driven protein sorting in lipid domains as well as protein mobility. Our data shows that increasing the ECD mass or glycosylation leads to a decrease in ordered domain partitioning and diffusivity. Our data reconciles different mechanisms proposed for the initiation of cellular signaling by linking the ECD size of membrane proteins with their localization and diffusion dynamics in the plasma membrane.
SIGNIFICANCE STATEMENT
We studied how the size and glycosylation of the proteins influences their dynamic behavior in a lipid bilayer by reconstituting the ECDs of different sizes or glycosylation in model membrane systems and analyzing their sorting into lipid domains as well as their mobility. We observe that increasing the ECD apparent mass leads to a decrease in membrane ordered domain partitioning and diffusivity. Our data reconciles multiple mechanisms proposed for the initiation of cellular signaling by linking the ECD properties of membrane proteins with their localization and diffusion dynamics in the plasma membrane.
Over the years, my colleagues and I have come to realise that the likelihood of pharmaceutical drugs being able to diffuse through whatever unhindered phospholipid bilayer may exist in intact biological membranes in vivo is vanishingly low. This is because (i) most real biomembranes are mostly protein, not lipid, (ii) unlike purely lipid bilayers that can form transient aqueous channels, the high concentrations of proteins serve to stop such activity, (iii) natural evolution long ago selected against transport methods that just let any undesirable products enter a cell, (iv) transporters have now been identified for all kinds of molecules (even water) that were once thought not to require them, (v) many experiments show a massive variation in the uptake of drugs between different cells, tissues, and organisms, that cannot be explained if lipid bilayer transport is significant or if efflux were the only differentiator, and (vi) many experiments that manipulate the expression level of individual transporters as an independent variable demonstrate their role in drug and nutrient uptake (including in cytotoxicity or adverse drug reactions). This makes such transporters valuable both as a means of targeting drugs (not least anti-infectives) to selected cells or tissues and also as drug targets. The same considerations apply to the exploitation of substrate uptake and product efflux transporters in biotechnology. We are also beginning to recognise that transporters are more promiscuous, and antiporter activity is much more widespread, than had been realised, and that such processes are adaptive (i.e., were selected by natural evolution). The purpose of the present review is to summarise the above, and to rehearse and update readers on recent developments. These developments lead us to retain and indeed to strengthen our contention that for transmembrane pharmaceutical drug transport “phospholipid bilayer transport is negligible”.
Cellular life relies on membranes, which provide a resilient and adaptive cell boundary. Many essential processes depend upon the ease with which the membrane is able to deform and bend; features that can be characterized by the bending rigidity. Quantitative investigations of such mechanical properties of biological membranes have primarily been undertaken in solely lipid bilayers and frequently in the absence of buffers. In contrast, much less is known about the influence of integral membrane proteins on bending rigidity under physiological conditions. We focus on an exemplar member of the ubiquitous major facilitator superfamily of transporters and assess the influence of lactose permease on the bending rigidity of lipid bilayers. Fluctuation analysis of giant unilamellar vesicles (GUVs) is a useful means to measure bending rigidity. We find that using a hydrogel substrate produces GUVs that are well suited to fluctuation analysis. Moreover, the hydrogel method is amenable to both physiological salt concentrations and anionic lipids, which are important to mimic key aspects of the native lactose permease membrane. Varying the fraction of the anionic lipid in the lipid mixture DOPC:DOPE:DOPG allows us to assess the dependence of membrane bending rigidity on the topology and concentration of an integral membrane protein in the lipid bilayer of GUVs. The bending rigidity gradually increases with the incorporation of lactose permease, but there is no further increase with greater amounts of the protein in the membrane.
Textbooks of biochemistry will explain that the otherwise endergonic reactions of ATP synthesis can be driven by the exergonic reactions of respiratory electron transport, and that these two half-reactions are catalyzed by protein complexes embedded in the same, closed membrane. These views are correct. The textbooks also state that, according to the chemiosmotic coupling hypothesis, a (or the) kinetically and thermodynamically competent intermediate linking the two half-reactions is the electrochemical difference of protons that is in equilibrium with that between the two bulk phases that the coupling membrane serves to separate. This gradient consists of a membrane potential term Δψ and a pH gradient term ΔpH, and is known colloquially as the protonmotive force or pmf. Artificial imposition of a pmf can drive phosphorylation, but only if the pmf exceeds some 150–170 mV; to achieve in vivo rates the imposed pmf must reach 200 mV. The key question then is ‘does the pmf generated by electron transport exceed 200 mV, or even 170 mV?’ The possibly surprising answer, from a great many kinds of experiment and sources of evidence, including direct measurements with microelectrodes, indicates it that it does not. Observable pH changes driven by electron transport are real, and they control various processes; however, compensating ion movements restrict the Δψ component to low values. A protet-based model, that I outline here, can account for all the necessary observations, including all of those inconsistent with chemiosmotic coupling, and provides for a variety of testable hypotheses by which it might be refined.
The metabotropic glutamate receptor (mGluR) 2 plays a key role in the central nervous system. mGluR2 has been shown to be regulated by its surrounding lipid environment, especially by cholesterol, by an unknown mechanism. Here, using a combination of biochemical approaches, photocrosslinking experiments, and molecular dynamics simulations we show the interaction of cholesterol with at least two, but potentially five more, preferential sites on the mGluR2 transmembrane domain. Our simulations demonstrate that surface matching, rather than electrostatic interactions with specific amino acids, is the main factor defining cholesterol localization. Moreover, the cholesterol localization observed here is similar to the sterol-binding pattern previously described in silico for other members of the mGluR family. Biochemical assays suggest little influence of cholesterol on trafficking or dimerization of mGluR2. Nevertheless, simulations revealed a significant reduction of residue-residue contacts together with an alteration in the internal mechanical stress at the cytoplasmic side of the helical bundle when cholesterol was present in the membrane. These alterations may be related to destabilization of the basal state of mGluR2. Due to the high sequence conservation of the transmembrane domains of mGluRs, the molecular interaction of cholesterol and mGluR2 described here is also likely to be relevant for other members of the mGLuR family.
Infections in many virulent bacterial strains are triggered by the release of pore forming toxins (PFTs), which form oligomeric transmembrane pore complexes on the target plasma membrane. The spatial extent...
Polycystin-2 (PC2) is a transient receptor potential (TRP) channel present in ciliary membranes of the kidney. PC2 shares a transmembrane fold with other TRP channels, in addition to an extracellular domain found in TRPP and TRPML channels. Using molecular dynamics (MD) simulations and cryoelectron microscopy we identify and characterize PIP2 and cholesterol interactions with PC2. PC2 is revealed to have a PIP binding site close to the equivalent vanilloid/lipid binding site in the TRPV1 channel. A 3.0-Å structure reveals a binding site for cholesterol on PC2. Cholesterol interactions with the channel at this site are characterized by MD simulations. The two classes of lipid binding sites are compared with sites observed in other TRPs and in Kv channels. These findings suggest PC2, in common with other ion channels, may be modulated by both PIPs and cholesterol, and position PC2 within an emerging model of the roles of lipids in the regulation and organization of ciliary membranes.
Our current knowledge of the structural dynamics and complexity of lipid bilayers is still developing. Computational techniques, especially molecular dynamics simulations, have increased our understanding significantly as they allow us to model functions that cannot currently be experimentally resolved. Here we review available computational tools and techniques, the role of the major lipid species, insights gained into lipid bilayer structure and function from molecular dynamics simulations, and recent progress towards the computational modelling of the physiological complexity of eukaryotic lipid bilayers.
The plasma membranes of cells are thin viscous sheets in which some transmembrane proteins have two-dimensional mobility and some are immobilized. Previous studies have shown that immobile proteins retard the short-time diffusivity of mobile particles through hydrodynamic interactions and that steric effects of immobile proteins reduce the long-time diffusivity in a model that neglects hydrodynamic interactions. We present a rigorous derivation of the long-time diffusivity of a single mobile protein interacting hydrodynamically and thermodynamically with an array of immobile proteins subject to periodic boundary conditions. This method is based on a finite element method (FEM) solution of the probability density of the mobile protein diffusing with a position-dependent mobility determined through a multipole solution of Stokes equations. The simulated long-time diffusivity in square arrays decreases as the spacing in the array approaches the particle size in a manner consistent with a lubrication analysis. In random arrays, steric effects lead to a percolation threshold volume fraction above which long-time diffusion is arrested. The FEM/multipole approach is used to compute the long-time diffusivity far away from this threshold. An approximate analysis of mobile protein diffusion through a network of pores connected by bonds with resistances determined by the FEM/multipole calculations is then used to explore higher immobile area fractions and to evaluate the finite simulation cell size scaling behaviour of diffusion near the percolation threshold. Surprisingly, the ratio of the long-time diffusivity to the spatially averaged short-time diffusivity in these two-dimensional fixed arrays is higher in the presence of hydrodynamic interactions than in their absence. Finally, the implications of this work are discussed, including the possibility of using the methods developed here to investigate more complex diffusive phenomena observed in cell membranes.
Peptide appended pillar[5]arene (PAP) is an artificial water channel resembling biological water channel proteins, which has shown a significant potential for designing bioinspired water purification systems. Given that PAP channels need to be incorporated at a high density in membrane matrices, it is critical to examine the role of channel-channel and channel-membrane interactions in governing the structural and functional characteristics of channels. To resolve the atomic-scale details of these interactions, we have carried out atomistic molecular dynamics (MD) simulations of multiple PAP channels inserted in a lipid or a block-copolymer (BCP) membrane matrix. Classical MD simulations on a sub-microsecond timescale showed clustering of channels only in the lipid membrane, but enhanced sampling MD simulations showed thermodynamically-favorable dimerized states of channels in both lipid and BCP membranes. The dimerized configurations of channels, with an extensive buried surface area, were stabilized via interactions between the aromatic groups in the peptide arms of neighboring channels. The conformational metrics characterizing the orientational and structural changes in channels revealed a higher flexibility in the lipid membrane as opposed to the BCP membrane although hydrogen bonds between the channel and the membrane molecules were not a major contributor to the stability of channels in the BCP membrane. We also found that the channels undergo wetting/dewetting transitions in both lipid and BCP membranes with a marginally higher probability of undergoing a dewetting transition in the BCP membrane. Collectively, these results highlight the role of channel dynamics in governing channel-channel and channel-membrane interfacial interactions, and provide atomic-scale insights needed to design stable and functional biomimetic membranes for efficient separations.
Polycystin-2 (PC2) is a member of the TRPP subfamily of TRP channels and is present in ciliary membranes of the kidney. PC2 can be either homo-tetrameric, or heterotetrameric with PC1. PC2 shares a common transmembrane fold with other TRP channels, in addition to having a novel extracellular domain. Several TRP channels have been suggested to be regulated by lipids, including phosphatidylinositol phosphates (PIPs). We have combined molecular dynamics simulations with cryoelectron microscopy to explore possible lipid interactions sites on PC2. We propose that PC2 has a PIP-binding site close to the equivalent vanilloid/lipid-binding site in the TRPV1 channel. A 3.0 Å cryoelectron microscopy map reveals a binding site for cholesterol on PC2. Cholesterol interactions with the channel at this site are further characterized by MD simulations. These results help to position PC2 within an emerging model of the complex roles of lipids in the regulation and organization of ciliary membranes.
Biological membranes are tricky to investigate. They are complex in terms of molecular composition and structure, functional over a wide range of time scales, and characterized by nonequilibrium conditions. Because of all of these features, simulations are a great technique to study biomembrane behavior. A significant part of the functional processes in biological membranes takes place at the molecular level; thus computer simulations are the method of choice to explore how their properties emerge from specific molecular features and how the interplay among the numerous molecules gives rise to function over spatial and time scales larger than the molecular ones. In this review, we focus on this broad theme. We discuss the current state-of-the-art of biomembrane simulations that, until now, have largely focused on a rather narrow picture of the complexity of the membranes. Given this, we also discuss the challenges that we should unravel in the foreseeable future. Numerous features such as the actin-cytoskeleton network, the glycocalyx network, and nonequilibrium transport under ATP-driven conditions have so far received very little attention; however, the potential of simulations to solve them would be exceptionally high. A major milestone for this research would be that one day we could say that computer simulations genuinely research biological membranes, not just lipid bilayers.
Currently, there is no comprehensive model for the dynamics of cellular membranes. The understanding of even the basic dynamic processes, such as lateral diffusion of lipids, is still quite limited. Recent studies of one-component membrane systems have shown that instead of single-particle motions, the lateral diffusion is driven by a more complex, concerted mechanism for lipid diffusion (E. Falck et al., J. Am. Chem. Soc., 2008, 130, 44-45), where a lipid and its neighbors move in unison in terms of loosely defined clusters. In this work, we extend the previous study by considering the concerted lipid diffusion phenomena in many-component raft-like membranes. This nature of diffusion phenomena emerge in all the cases we have considered, including both atom-scale simulations of lateral diffusion within rafts and coarse-grained MARTINI simulations of diffusion in membranes characterized by coexistence of raft and non-raft domains. The data allows us to identify characteristic time scales for the concerted lipid motions, which turn out to range from hundreds of nanoseconds to several microseconds. Further, we characterize typical length scales associated with the correlated lipid diffusion patterns and find them to be about 10 nm, or even larger if weak correlations are taken into account. Finally, the concerted nature of lipid motions is also found in dissipative particle dynamics simulations of lipid membranes, clarifying the role of hydrodynamics (local momentum conservation) in membrane diffusion phenomena.
Cell membranes display a tremendous complexity of lipids and proteins designed to perform the functions cells require. To
coordinate these functions, the membrane is able to laterally segregate its constituents. This capability is based on dynamic
liquid-liquid immiscibility and underlies the raft concept of membrane subcompartmentalization. Lipid rafts are fluctuating
nanoscale assemblies of sphingolipid, cholesterol, and proteins that can be stabilized to coalesce, forming platforms that
function in membrane signaling and trafficking. Here we review the evidence for how this principle combines the potential
for sphingolipid-cholesterol self-assembly with protein specificity to selectively focus membrane bioactivity.
Structure and dynamics of voltage-gated ion channels, in particular the motion of
the S4 helix, is a highly interesting and hotly debated topic in current
membrane protein research. It has critical implications for insertion and
stabilization of membrane proteins as well as for finding how transitions occur
in membrane proteins—not to mention numerous applications in drug
design. Here, we present a full 1 µs atomic-detail molecular dynamics
simulation of an integral Kv1.2 ion channel, comprising 120,000 atoms. By
applying 0.052 V/nm of hyperpolarization, we observe structural rearrangements,
including up to 120° rotation of the S4 segment, changes in
hydrogen-bonding patterns, but only low amounts of translation. A smaller
rotation (∼35°) of the extracellular end of all S4 segments is
present also in a reference 0.5 µs simulation without applied field,
which indicates that the crystal structure might be slightly different from the
natural state of the voltage sensor. The conformation change upon
hyperpolarization is closely coupled to an increase in 310 helix
contents in S4, starting from the intracellular side. This could support a model
for transition from the crystal structure where the hyperpolarization
destabilizes S4–lipid hydrogen bonds, which leads to the helix
rotating to keep the arginine side chains away from the hydrophobic phase, and
the driving force for final relaxation by downward translation is partly
entropic, which would explain the slow process. The coordinates of the
transmembrane part of the simulated channel actually stay closer to the recently
determined higher-resolution Kv1.2 chimera channel than the starting structure
for the entire second half of the simulation (0.5–1 µs).
Together with lipids binding in matching positions and significant thinning of
the membrane also observed in experiments, this provides additional support for
the predictive power of microsecond-scale membrane protein simulations.
A new aspect of cell membrane structure is presented, based on the dynamic clustering of sphingolipids and cholesterol to form rafts that move within the fluid bilayer. It is proposed that these rafts function as platforms for the attachment of proteins when membranes are moved around inside the cell and during signal transduction.
The biological function of transmembrane proteins is closely related to their insertion, which has most often been studied through their lateral mobility. For >30 years, it has been thought that hardly any information on the size of the diffusing object can be extracted from such experiments. Indeed, the hydrodynamic model developed by Saffman and Delbrück predicts a weak, logarithmic dependence of the diffusion coefficient D with the radius R of the protein. Despite widespread use, its validity has never been thoroughly investigated. To check this model, we measured the diffusion coefficients of various peptides and transmembrane proteins, incorporated into giant unilamellar vesicles of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) or in model bilayers of tunable thickness. We show in this work that, for several integral proteins spanning a large range of sizes, the diffusion coefficient is strongly linked to the protein dimensions. A heuristic model results in a Stokes-like expression for D, (D ∝ 1/R), which fits literature data as well as ours. Diffusion measurement is then a fast and fruitful method; it allows determining the oligomerization degree of proteins or studying lipid–protein and protein–protein interactions within bilayers.
• bilayers
• transmembrane proteins
• diffusion
• peptides
• sponge phase
Neurons transmit information through electrical signals generated by voltage-gated ion channels. These channels consist of a large superfamily of proteins that form channels selective for potassium, sodium, or calcium ions. In this review we focus on the molecular mechanisms by which these channels convert changes in membrane voltage into the opening and closing of "gates" that turn ion conductance on and off. An explosion of new studies in the last year, including the first X-ray crystal structure of a mammalian voltage-gated potassium channel, has led to radically different interpretations of the structure and molecular motion of the voltage sensor. The interpretations are as distinct as the techniques employed for the studies: crystallography, fluorescence, accessibility analysis, and electrophysiology. We discuss the likely causes of the discrepant results in an attempt to identify the missing information that will help resolve the controversy and reveal the mechanism by which a voltage sensor controls the channel's gates.
The paradigm of biological membranes has recently gone through a major update. Instead of being fluid and homogeneous, recent studies suggest that membranes are characterized by transient domains with varying fluidity. In particular, a number of experimental studies have revealed the existence of highly ordered lateral domains rich in sphingomyelin and cholesterol (CHOL). These domains, called functional lipid rafts, have been suggested to take part in a variety of dynamic cellular processes such as membrane trafficking, signal transduction, and regulation of the activity of membrane proteins. However, despite the proposed importance of these domains, their properties, and even the precise nature of the lipid phases, have remained open issues mainly because the associated short time and length scales have posed a major challenge to experiments. In this work, we employ extensive atom-scale simulations to elucidate the properties of ternary raft mixtures with CHOL, palmitoylsphingomyelin (PSM), and palmitoyloleoylphosphatidylcholine. We simulate two bilayers of 1,024 lipids for 100 ns in the liquid-ordered phase and one system of the same size in the liquid-disordered phase. The studies provide evidence that the presence of PSM and CHOL in raft-like membranes leads to strongly packed and rigid bilayers. We also find that the simulated raft bilayers are characterized by nanoscale lateral heterogeneity, though the slow lateral diffusion renders the interpretation of the observed lateral heterogeneity more difficult. The findings reveal aspects of the role of favored (specific) lipid-lipid interactions within rafts and clarify the prominent role of CHOL in altering the properties of the membrane locally in its neighborhood. Also, we show that the presence of PSM and CHOL in rafts leads to intriguing lateral pressure profiles that are distinctly different from corresponding profiles in nonraft-like membranes. The results propose that the functioning of certain classes of membrane proteins is regulated by changes in the lateral pressure profile, which can be altered by a change in lipid content.
A large-scale molecular dynamics simulation is performed on a glass-forming Lennard-Jones mixture to determine the nature of dynamical heterogeneities which arise in this model fragile liquid. We observe that the most mobile particles exhibit a cooperative motion in the form of string-like paths (``strings'') whose mean length and radius of gyration increase as the liquid is cooled. The length distribution of the strings is found to be similar to that expected for the equilibrium polymerization of linear polymer chains. Comment: 6 pages of RevTex, 6 postscript figures, uses epsf.sty
The effect of the inhomogeneous environment upon solvent molecules close to a macromolecular surface is evaluated from a molecular-dynamics simulation of a protein, myoglobin, in water solution. The simulation is analyzed in terms of a mean-field potential from the protein upon the water molecules and spatially varying translational diffusion coefficients for solvent molecules in directions parallel and perpendicular to the protein surface. The diffusion coefficients can be obtained from the slope of the average-square displacements vs time, as well as from the integral of the velocity autocorrelation functions. It is shown that the former procedure gives a lot of ambiguities due to the variation of the slope of the curve with time. The latter, however, after analytic correction for the contribution from algebraic long-time tails, furnish a much more reliable alternative.
The motion of phospholipids has previously been studied on many time scales due to the significance for living cells and technological applications. The motions on a pico- to nanosecond time scale were determined by quasielastic neutron scattering (QENS) to be much faster than the ones on the microsecond scale covered by fluorescence recovery after photobleaching (FRAP). This was explained by assuming that the molecules rattle fast in a cage of neighbors (observed with QENS) from which they escape once in a while; this escape was then the primary step of the slower diffusion measured by FRAP. However, nanosecond MD simulation studies could not observe any escape events; recent findings even suggested that the long-range motion in phospholipid membranes on short time scales is not diffusive but has flow-like characteristics. To check this novel view, we have repeated the QENS experiments with today's significantly improved instrumentation. By using the advantage of QENS that allows tuning of the observation time in the pico- to nanosecond range, it was possible to study the evolution of motions in this time frame. Localized motions, e.g., of the head and tail groups, appear separated from the long-range motion and do not obfuscate the analysis as they do in a mean squared displacement plot. The results for the long-range motion are indeed compatible with flow patterns, whereas the localized motions can account for the fast motions interpreted as motions in a cage before. Hereby, we give experimental evidence for a completely different mechanism of long-range motion on short time scales in phospholipid membranes.
Many biologically interesting phenomena occur on a time scale that is too long to be studied by atomistic simulations. These phenomena include the dynamics of large proteins and self-assembly of biological materials. Coarse-grained (CG) molecular modeling allows computer simulations to be run on length and time scales that are 2–3 orders of magnitude larger compared to atomistic simulations, providing a bridge between the atomistic and the mesoscopic scale. We developed a new CG model for proteins as an extension of the MARTINI force field. Here, we validate the model for its use in peptide-bilayer systems. In order to validate the model, we calculated the potential of mean force for each amino acid as a function of its distance from the center of a dioleoylphosphatidylcholine (DOPC) lipid bilayer. We then compared amino acid association constants, the partitioning of a series of model pentapeptides, the partitioning and orientation of WALP23 in DOPC lipid bilayers and a series of KALP peptides in dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine (DPPC) bilayers. A comparison with results obtained from atomistic models shows good agreement in all of the tests performed. We also performed a systematic investigation of the partitioning of five series of polyalanine-leucine peptides (with different lengths and compositions) in DPPC bilayers. As expected, the fraction of peptides partitioned at the interface increased with decreasing peptide length and decreasing leucine content, demonstrating that the CG model is capable of discriminating partitioning behavior arising from subtle differences in the amino acid composition. Finally, we simulated the concentration-dependent formation of transmembrane pores by magainin, an antimicrobial peptide. In line with atomistic simulation studies, disordered toroidal pores are formed. In conclusion, the model is computationally efficient and effectively reproduces peptide-lipid interactions and the partitioning of amino acids and peptides in lipid bilayers.
We measured the lateral mobility of integral membrane proteins reconstituted in giant unilamellar vesicles (GUVs), using fluorescence correlation spectroscopy. Receptor, channel, and transporter proteins with 1-36 transmembrane segments (lateral radii ranging from 0.5 to 4 nm) and a alpha-helical peptide (radius of 0.5 nm) were fluorescently labeled and incorporated into GUVs. At low protein-to-lipid ratios (i.e., 10-100 proteins per microm(2) of membrane surface), the diffusion coefficient D displayed a weak dependence on the hydrodynamic radius (R) of the proteins [D scaled with ln(1/R)], consistent with the Saffman-Delbruck model. At higher protein-to lipid ratios (up to 3000 microm(-2)), the lateral diffusion coefficient of the molecules decreased linearly with increasing the protein concentration in the membrane. The implications of our findings for protein mobility in biological membranes (protein crowding of approximately 25,000 microm(-2)) and use of diffusion measurements for protein geometry (size, oligomerization) determinations are discussed.
Advances in optical microscopy techniques and single-molecule detection have paved the way to exploring new approaches for investigating membrane dynamics and organization, thereby revealing details on the processing of signals, complex association/dissociation, chemical reactions and transport at and around the membrane. These events rely on a tight regulation of lipid-protein and protein-protein interactions in space and time. Fluorescence Correlation Spectroscopy (FCS) provides exquisite sensitivity in measuring local concentrations, association/dissociation constants, chemical rate constants and, in general, in probing the chemical environment of the species of interest and its interactions with potential partners. Here, we review some applications of FCS to lipid and protein organization in biomimetic membranes with lateral heterogeneities, which share some physico-chemical properties with cellular rafts. What we learn from investigations of lipid-lipid and lipid-protein interactions in simple model membranes can be regarded as an essential basic lecture for studies in more complex cellular membranes.
Membrane lateral heterogeneity is accepted as a requirement for the function of biological membranes and the notion of lipid rafts gives specificity to this broad concept. However, the lipid raft field is now at a technical impasse because the physical tools to study biological membranes as a liquid that is ordered in space and time are still being developed. This has lead to a disconnection between the concept of lipid rafts as derived from biochemical and biophysical assays and their existence in the cell. Here, we compare the concept of lipid rafts as it has emerged from the study of synthetic membranes with the reality of lateral heterogeneity in biological membranes. Further application of existing tools and the development of new tools are needed to understand the dynamic heterogeneity of biological membranes.
There is no comprehensive model for the dynamics of cellular membranes. Even mechanisms of basic dynamic processes, such as lateral diffusion of lipids, are poorly understood. Our atomic-scale molecular dynamics simulations support a novel, concerted mechanism for lipid diffusion. We find that a lipid and its nearest neighbors move in unison, forming loosely defined clusters. What is more, the motions of lipids are correlated over tens of nanometers: the lateral displacements of lipids in a given monolayer produce striking two-dimensional flow patterns. These flow patterns should have wide implications, affecting, for example, the formation of membrane domains, protein functionality, and action of lipases and drugs on membranes.
In the Fluid Mosaic Model for biological membrane structure, proposed by Singer and Nicolson in 1972, the lipid bilayer is represented as a neutral two-dimensional solvent in which the proteins of the membrane are dispersed and distributed randomly. The model portrays the membrane as dominated by a membrane lipid bilayer, directly exposed to the aqueous environment, and only occasionally interrupted by transmembrane proteins. This view is reproduced in virtually every textbook in biochemistry and cell biology, yet some critical features have yet to be closely examined, including the key parameter of the relative occupancy of protein and lipid at the center of a natural membrane. Here we show that the area occupied by protein and lipid at the center of the human red blood cell (RBC) plasma membrane is at least ≈23% protein and less than ≈77% lipid. This measurement is in close agreement with previous estimates for the RBC plasma membrane and the recently published measurements for the synaptic vesicle. Given that transmembrane proteins are surrounded by phospholipids that are perturbed by their presence, the occupancy by protein of more than ≈20% of the RBC plasma membrane and the synaptic vesicle plasma membrane implies that natural membrane bilayers may be more rigid and less fluid than has been thought for the past several decades, and that studies of pure lipid bilayers do not fully reveal the properties of lipids in membranes. Thus, it appears to be the case that membranes may be more mosaic than fluid, with little unperturbed phospholipid bilayer.
• fluid mosaic model
• membrane lipid bilayer
Membrane proteins do not work alone. The interaction of proteins with membrane lipids can be highly specific and is often important for full functional and structural integrity of the protein. Providing the appropriate lipid environment is of great importance for the purification and crystallisation of membrane proteins. The lipid content can be modulated by adjusting purification protocols or by adding back native or non-native lipids. Lipids can facilitate crystallisation by stabilising the protein and by providing lattice contacts. Of special interest is the crystallisation in lipidic cubic phase and with bicelles, as they appear to provide a membrane-like environment. These strategies have been instrumental for recent successful structure determinations of a human G-protein-coupled receptor, the beta(2)-adrenergic receptor. Lipid supplementation can also help to obtain membrane protein structures in a native conformation, as shown for voltage-gated potassium channels. Membrane protein structures, especially those derived from lipid-enriched preparations, contain bound lipid molecules. Specific protein-lipid interactions not only require careful evaluation and interpretation, but also permit a directed approach to elucidate the structural and/or functional role of these interactions.
The observation of membrane domains in vivo and in vitro has triggered a renewed interest in the size-dependent diffusion of membrane inclusions (e.g., clusters of transmembrane proteins and lipid rafts). Here, we have used coarse-grained membrane simulations to quantify the influence of a hydrophobic mismatch between the inclusion's transmembrane portion and the surrounding lipid bilayer on the diffusive mobility of the inclusion. Our data indicate only slight changes in the mobility (<30%) when altering the hydrophobic mismatch, and the scaling of the diffusion coefficient D is most consistent with previous hydrodynamic predictions, i.e., with the Saffman-Delbruck relation and the edgewise motion of a thin disk in the limit of small and large radii, respectively.
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