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Cytoglobin is a hemoprotein widely expressed in fibroblasts and related cell lineages with yet undefined physiological function. Cytoglobin, as other heme proteins, can reduce nitrite to nitric oxide (NO) providing a route to generate NO in vivo in low oxygen conditions. In addition, cytoglobin can also bind lipids such as oleic acid and cardiolipin with high affinity. These two processes are potentially relevant to cytoglobin function. Little is known about how specific amino acids contribute to nitrite reduction and lipid binding. Here we investigate the role of the distal histidine His81 (E7) and several surface residues on the regulation of nitrite reduction and lipid binding. We observe that the replacement of His81 (E7) greatly increases heme reactivity towards nitrite, with nitrite reduction rate constants of up to 1100 M−1s−1 for the His81Ala mutant. His81 (E7) mutation causes a small decrease in lipid binding affinity, however experiments on the presence of imidazole indicate that His81 (E7) does not compete with the lipid for the binding site. Mutations of the surface residues Arg84 and Lys116 largely impair lipid binding. Our results suggest that dissociation of His81 (E7) from the heme mediates the formation of a hydrophobic cavity in the proximal heme side that can accommodate the lipid, with important contributions of the hydrophobic patch around residues Thr91, Val105, and Leu108, whereas the positive charges from Arg84 and Lys116 stabilize the carboxyl group of the fatty acid. Gain and loss-of-function mutations described here can serve as tools to study in vivo the physiological role of these putative cytoglobin functions. • Replacement of cytoglobin distal histidine increases reactivity towards nitrite. • Mutations of the cytoglobin surface residues Arg84 and Lys116 impair lipid binding. • Dissociation of His81 (E7) from the heme is required for lipid binding. • The hydrophobic patch formed by Thr91, Val105, and Leu108 mediates lipid binding. [ABSTRACT FROM AUTHOR]

• Hybrid resolution molecular dynamics simulation method is described. • Hybrid resolution molecular dynamics simulations allow characterization of interactions of amyloid proteins with lipid membranes on long time scales. • The hybrid resolution method was applied to computer modeling of the interactions of α-synuclein protein with a mixed lipid bilayer. • Interaction of α -syn with the lipid bilayer leads to dramatic changes in the conformation of the protein and to a smaller degree its structure. A broad range of human diseases, including Alzheimer's and Parkinson's diseases, arise from or have as key players intrinsically disordered proteins. The aggregation of these amyloid proteins into fibrillar aggregates are the key events of such diseases. Characterizing the conformation dynamics of the proteins involved is crucial for understanding the molecular mechanisms of aggregation, which in turn is important for drug development efforts against these diseases. Computational approaches have provided extensive detail about some steps of the aggregation process, however the biologically relevant elements responsible for the aggregation and or aggregation propagation have not been fully characterized. Here we describe a hybrid resolution molecular dynamics simulation method that can be employed to investigate the interaction of amyloid proteins with lipid membranes, shown to dramatically accelerate the aggregation propensity of amyloid proteins. The hybrid resolution method enables routine and accurate simulation of multi-protein and complex membrane systems, mimicking biologically relevant lipid membranes, on microsecond time scales. The hybrid resolution method was applied to computer modeling of the interactions of α -synuclein protein with a mixed lipid bilayer. [ABSTRACT FROM AUTHOR]

Desmin, an intermediate filament protein expressed in muscle cells, plays a key role in the integrity and regulation of the contractile system. Furthermore, the distribution of desmin in cells and its interplay with plasma and organelle membranes are crucial for cell functions; however, the fundamental properties of lipid-desmin interactions remain unknown. Using a water-in-oil method for a limited space system in vitro , we examined the distribution of desmin in three types of phospholipid droplets: 1,2-dioleoyl- sn -glycero-3-phosphocholine (DOPC), 1,2-dioleoyl- sn -glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl- sn -glycero-3-phosphoserine (DOPS). When fluorescent-labeled desmin was observed for 60 min after desmin assembly was initiated by adding 25 mM KCl, desmin accumulated on both the DOPE and DOPS layers; however, it did not accumulate on the DOPC layer of droplets. An increase in salt concentration did not moderate the accumulation. The initial form of either oligomer or mature filament affected the accumulation on each lipid layer. When liposomes were included in the droplets, desmin was associated with DOPE but not on DOPC liposomes. These results suggest that desmin has the potential for association with phospholipids concerning desmin form and lipid shape. The behavior and composition of living membranes may affect the distribution of desmin networks. [Display omitted] • Desmin was associated with DOPE and DOPS layers. • Maturation of desmin filaments weakened the affinity for phospholipid layers. • Desmin could accumulate around hybrid DOPE liposomes. [ABSTRACT FROM AUTHOR]

Lipid droplets (LDs), or oil bodies in plants, are specialized organelles that primarily serve as hubs of cellular metabolic energy storage and consumption. These ubiquitous cytoplasmic organelles are derived from the endoplasmic reticulum (ER) and consist of a hydrophobic neutral lipid core - mainly consisting of triglycerides and sterol esters - that is encircled by a phospholipid monolayer. The dynamic metabolic functions of the LDs are mainly executed and regulated by proteins on the monolayer surface. However, its unique architecture puts some structural constraints on the types of proteins that can associate with LDs. The lipid monolayer is decorated with either peripheral proteins or with integral membrane proteins that adopt a monotopic topology. Due to its oil-water interface, which is energetically costly, the LD surface happens to be favorable to the recruitment of many proteins involved in metabolic but also non-metabolic functions. We only started very recently to understand biophysical and biochemical principles controlling protein targeting to LDs. This review aims to summarize the most recent findings regarding this topic and proposes directions that will potentially lead to a better understanding of LD surface characteristics, as compared to bilayer membranes, and how that impacts protein-LD interactions. [ABSTRACT FROM AUTHOR]

Recently, Zika virus (ZIKV) emerged as a global public health concern and is distinct from other flaviviruses in many aspects, for example, causing transplacental infection, fetal abnormalities and vector-independent transmission through body fluids in humans. The capsid (C) protein is a multifunctional protein, since it binds to viral RNA in the process of nucleocapsid assembly and plays important roles in virus infection processes by interacting with cellular proteins, modulating cellular metabolism, apoptosis and immune response. Here we solved the crystal structure of ZIKV C protein at a resolution of 1.9 Å. The ZIKV C protein structure contains four α helices with a long pre-α1 loop and forms dimers. The unique long pre-α1 loop in ZIKV C contributes to the tighter association of dimeric assembly and renders a divergent hydrophobic feature at the lipid bilayer interface in comparison with the known C structures of West Nile and dengue viruses. We reported the interaction between the ZIKV C protein and lipid droplets through confocal microscopy analysis. Substitutions of key amino acids in the pre-α1 loop of ZIKV C disrupted the interaction with lipid droplets, indicating that the loop is critical for membrane association. We also recognized that ZIKV C protein possesses broad binding capability to different nucleotide types, including single-stranded and double-stranded RNAs or DNAs. Furthermore, the highly positively charged interface, mainly formed by α4 helix, is proposed to be responsible for nucleotide binding. These findings will greatly enhance our understanding of ZIKV C protein, providing information for anti-ZIKV drug design targeting the C protein. [ABSTRACT FROM AUTHOR]

Sterols are essential components of cellular membranes and shape their biophysical properties. The recently discovered family of Lipid transfer proteins Anchored at Membrane contact sites (LAMs) has been suggested to carry out intracellular sterol traffic using StART-like domains. Here, we studied the second StART-like domain of Lam4p from S. cerevisiae by NMR. We show that NMR data are consistent with the StART-like domain structure, and that several functionally important regions within the domain exhibit significant conformational dynamics. NMR titration experiments confirm sterol binding to the canonical sterol-binding site and suggest a role of membrane interactions on the thermodynamics and kinetics of sterol binding. [ABSTRACT FROM AUTHOR]

Neuroglobin (Ngb) and cytoglobin (Cygb) are two six-coordinate heme proteins of unknown physiological function. Although studies on the mammalian proteins have elucidated aspects of Ngb and Cygb biophysics and indicated potential functions, the properties of non-mammalian Ngbs and Cygbs are largely uncharacterized. We have expressed the recombinant zebrafish proteins Ngb, Cygb1, and Cygb2 in Escherichia coli and characterized their nitrite reduction rates, spectral properties, autoxidation rate constants, redox potentials and lipid binding properties. The three zebrafish proteins can catalyze the reduction of nitrite to nitric oxide with a broad range of reaction rate constants. (Ngb, 0.68 ± 0.04 M −1 s −1 ; Cygb1, 28.6 ± 3.1 M −1 s −1 ; Cygb2, 0.94 ± 0.18 M −1 s −1 ). We observe that zebrafish Ngb and Cygb2 have comparable spectral features to those of human Ngb and Cygb, consistent with a six-coordinate heme, whereas unexpectedly Cygb1 has a five-coordinate heme, a slower autoxidation and in general has properties more akin to oxygen transport proteins. In agreement with a possible oxygen carrier and nitrite reductase role, we detect mRNA transcript for Cygb1 but not Cygb2 or Ngb in zebrafish blood. Unlike human Cygb, neither of the zebrafish globins binds oleic acid with high affinity. This finding suggests that lipid binding may be a trait acquired later during evolution and not an ancestral property of cytoglobins. Altogether, our results uncover unexpected properties of zebrafish globins and reveal the pivotal role of cytoglobins in the transition of heme globins from six-coordinate to five-coordinate oxygen carriers and nitrite reductases. [ABSTRACT FROM AUTHOR]

[Display omitted] • Ca2+ and Mg2+ modulate membrane partitioning of polypeptides. • Effects of Ca2+ and Mg2+ cannot be explained by charge screening. • Ca2+ and Mg2+ can increase interfacial partitioning by 15 kcal/mole. • Ca2+ and Mg2+ can increase transmembrane insertion by 5 kcal/mole. Accurate quantitative estimates of protein-membrane interactions are critical to studies of membrane proteins. Here, we demonstrate that thermodynamic analyses based on current hydropathy scales do not account for the significant and experimentally determined effects that Ca2+ or Mg2+ have on protein-membrane interactions. We examined distinct modes of interaction (interfacial partitioning and folding and transmembrane insertion) by studying three highly divergent peptides: Bid-BH3 (derived from apoptotic regulator Bid), peripherin-2-derived prph2-CTER, and the cancer-targeting pH-Low-Insertion-Peptide (pHLIP). Fluorescence experiments demonstrate that adding 1–2 mM of divalent cations led to a substantially more favorable bilayer partitioning and insertion, with free energy differences of 5–15 kcal/mol. [ABSTRACT FROM AUTHOR]

Pmel17 is an important protein for pigmentation in human skin and eyes. Proteolytic fragments from Pmel17 form fibrils upon which melanin is deposited in melanosomes. The repeat domain (RPT) derived from Pmel17 only forms fibrils under acidic melanosomal conditions. Here, we examined the effects of lipids on RPT aggregation to explore whether intramelanosomal vesicles can facilitate fibrillogenesis. Using transmission electron microscopy, circular dichroism, and fluorescence spectroscopy, we monitored fibril formation at the ultrastructural, secondary conformational, and local levels, respectively. Phospholipid vesicles and lysophospholipid (lysolipid) micelles were employed as membrane mimics. The surfactant-like lysolipids are particularly pertinent due to their high content in melanosomal membranes. Interestingly, RPT aggregation kinetics were influenced only by lysolipid-containing phospholipid vesicles. While both vesicles containing either anionic lysophosphatidylglycerol (LPG) or zwitterionic lysophosphatidylcholine (LPC) stimulate aggregation, LPG exerted a greater effect on reducing the apparent nucleation time. A detailed comparison showed distinct behaviors of LPG versus LPC monomers and micelles plausibly originating from their headgroup hydrogen bonding capabilities. Acceleration and retardation of aggregation were observed for LPG monomers and micelles, respectively. Because a specific interaction between LPG and RPT was identified by intrinsic W423 fluorescence and induced α-helical structure, it is inferred that binding of LPG near the C-terminal amyloid core initiates intermolecular association, whereas stabilization of α-helical conformation inhibits β-sheet formation. Contrastingly, LPC promotes RPT aggregation at both submicellar and micellar concentrations via non-specific binding with undetectable secondary structural change. Our findings suggest that protein–lysolipid interactions within melanosomes may regulate amyloid formation in vivo . [ABSTRACT FROM AUTHOR]

Abstract: Moesin and calmodulin (CaM) jointly associate with the cytoplasmic domain of l-selectin in the cell to modulate the function and ectodomain shedding of l-selectin. Using fluorescence spectroscopy, we have examined the association of moesin FERM domain with the recombinant transmembrane and cytoplasmic domains of l-selectin (CLS) reconstituted in model phospholipid liposomes. The dissociation constant of moesin FERM domain to CLS in the phosphatidylcholine liposome is about 300nM. In contrast to disrupting the CaM association with CLS, inclusion of anionic phosphatidylserine lipids in the phosphatidylcholine liposome increased the apparent binding affinity of moesin FERM domain for CLS. Using the environmentally sensitive fluorescent probe attached to the cytoplasmic domain of CLS and the nitroxide quencher attached to the lipid bilayer, we showed that the association of moesin FERM domain induced the desorption of the basic-rich cytoplasmic domain of CLS from the anionic membrane surface, which enabled subsequent association of CaM to the cytoplasmic domain of CLS. These results have elucidated the molecular basis for the moesin/l-selectin/CaM ternary complex and suggested an important role of phospholipids in modulating l-selectin function and shedding. [Copyright &y& Elsevier]

Abstract: Many membrane proteins are functional as stable oligomers. An understanding of the conditions that elicit and enhance oligomerization is important in many therapeutics. In this regard, protein–protein and protein–lipid interactions play crucial roles in the assembly and stability of oligomeric complexes. Recent years have seen a rapid increase in the mechanistic information on the importance of cytoplasmic termini in determining subunit assembly and stability of oligomeric complexes. In addition, the role of specific protein–lipid interaction between anionic phospholipids and “hot spots” on the protein surface has also become evident in stabilizing oligomeric assemblies. This review focuses on several contemporary developments of membrane proteins that stabilize oligomers by taking the potassium channel KcsA as an exemplary ion channel. [Copyright &y& Elsevier]

Abstract: Adsorption of small chain alcohols into lipid membranes significantly changes the conformational states of intrinsic membrane proteins. In this study, the effects of membrane-active strong cosolvent hexafluoroisopropanol (HFIP) on the intrinsic tetrameric stability of potassium channel KcsA were investigated. Presence of acidic phosphatidylglycerol (PG) in non-bilayer phosphatidylethanolamine (PE) or bilayer phosphatidylcholine (PC) significantly increased the tetrameric stability compared to zwitterionic pure PC bilayers. The stabilizing effect of PG in both lipid bilayers was completely abolished upon deletion of the membrane-anchored N-terminus. Tryptophan fluorescence and circular dichroism experiments indicated that HFIP destabilizes the tetramer possibly via drastic changes in the lateral pressure profile close to the membrane–water interface. The data suggest that HFIP disturbs the ionic, H-bonding and hydrophobic interactions among KcsA subunits where N-terminus presumably plays a crucial role in determining the channel proper folding and tetrameric structure via ionic/H-bond interactions between the helix dipole and the membrane lipids. [Copyright &y& Elsevier]

Abstract: α-Synuclein (α-syn) is a 140-residue protein of unknown function, involved in several neurodegenerative disorders, such as Parkinson''s disease. Recently, the possible interaction between α-syn and polyunsaturated fatty acids has attracted a strong interest. Indeed, lipids are able to trigger the multimerization of the protein in vitro and in cultured cells. Docosahexaenoic acid (DHA) is one of the main fatty acids (FAs) in cerebral gray matter and is dynamically released following phospholipid hydrolysis. Moreover, it has been found in high levels in brain areas containing α-syn inclusions in patients affected by Parkinson''s disease. Debated and unsolved questions regard the nature of the molecular interaction between α-syn and DHA and the effect exerted by the protein on the aggregated state of the FA. Here, we show that α-syn is able to strongly interact with DHA and that a mutual effect on the structure of the protein and on the physical state of the lipid derives from this interaction. α-Syn acquires an α-helical conformation in a simple two-state transition. The binding of the protein to the FA leads to a reduction of the size of the spontaneously formed aggregated species of DHA as well as of the critical aggregate concentration of the lipid. Specifically, biophysical methods and electron microscopy observations indicated that the FA forms oil droplets in the presence of α-syn. Limited proteolysis experiments showed that, when the protein is bound to the FA oil droplets, it is initially cleaved in the 89–102 region, suggesting that this chain segment is sufficiently flexible or unfolded to be protease-sensitive. Subsequent proteolytic events produce fragments corresponding to the first 70–80 residues that remain structured and show high affinity for the lipid. The fact that a region of the polypeptide chain remains accessible to proteases, when interacting with the lipid, suggests that this region could be involved in other interactions, justifying the ambivalent propensity of α-syn towards folding or aggregation in the presence of FAs. [Copyright &y& Elsevier]

Abstract: Protein targeting by the bacterial signal recognition particle requires the specific interaction of the signal recognition particle (SRP)–ribosome–nascent chain complex with FtsY, the bacterial SRP receptor. Although FtsY in Escherichia coli lacks a transmembrane domain, the membrane-bound FtsY displays many features of an integral membrane protein. Our data reveal that it is the cooperative action of two lipid-binding helices that allows this unusually strong membrane contact. Helix I comprises the first 14 amino acids of FtsY and the second is located at the interface between the A- and the N-domain of FtsY. We show by site-directed cross-linking and binding assays that both helices bind to negatively charged phospholipids, with a preference for phosphatidyl glycerol. Despite the strong lipid binding, helix I does not seem to be completely inserted into the lipid phase, but appears to be oriented parallel with the membrane surface. The two helices together with the connecting linker constitute an independently folded domain, which maintains its lipid binding even in the absence of the conserved NG-core of FtsY. In summary, our data reveal that the two consecutive lipid-binding helices of FtsY can provide a membrane contact that does not differ significantly in stability from that provided by a transmembrane domain. This explains why the bacterial SRP receptor does not require an integral β-subunit for membrane binding. [Copyright &y& Elsevier]

Abstract: Ribotoxins are a family of toxic extracellular fungal RNases that first enter into the cells and then exert a highly specific ribonucleolytic activity on the larger rRNA molecule, leading to protein synthesis inhibition and cell death by apoptosis. α-Sarcin is the best characterized ribotoxin. Previous characterization of a deletion variant of this protein showed that its long NH2-terminal β-hairpin is essential for its cytotoxicity. Docking, enzymatic, and lipid–protein interaction studies suggested that this β-hairpin establishes specific interactions with ribosomal proteins and that it is a region involved in the interaction with cell membranes. Consequently, in order to assess the influence of the basic character of this NH2-terminal β-hairpin (there are 1 arginine and 4 lysines along its 16 residues) on the ribotoxins cytotoxic ability, five individual mutants substituting these five basic residues by glutamic acid were produced, purified to homogeneity, and characterized. Regarding ribosomal recognition, all mutants showed a diminished activity in a cell-free reticulocyte lysate, whereas the activity against an oligoribonucleotide mimicking the sarcin/ricin loop rRNA (SRL) or the homopolymer poly(A) remained unaffected, confirming that the mutated basic residues participate in electrostatic interactions with other ribosomal elements apart from this SRL. The study of the interaction with phospholipid vesicles showed that Lys 17, Arg 22, and, most importantly, Lys 14 and Lys 21, are crucial residues in the first stages of the aggregation phenomenon, where protein–vesicle and protein–protein interactions are required. The data obtained reveal that electrostatic interactions involving basic residues of the β-hairpin are required not only for establishing specific interactions with ribosomal regions other than the SRL but also to explain the ability of the protein to interact with acid phospholipid bilayers. [Copyright &y& Elsevier]

Abstract: The dynamics of the binding of human coagulation factor Xa (FXa) and prothrombin to small unilamellar vesicles (25% phosphatidylserine, 75% phosphatidylcholine) were compared and quantified by Biacore, using two immobilization techniques. The vesicles were either tagged with different molar ratios of cholesterol–DNA and attached on Au chips or fused directly on L1 chips. The diameter in solution was 145nm, but the more DNA tags/vesicle the more compressed the immobilized vesicles became; with 30 DNA tags the calculated thickness was 88nm and with 1 DNA tag it was 138nm. In both models the affinity for the vesicles was higher for the activated coagulation factors than for the corresponding zymogens. FXa and prothrombin had the highest affinities. The affinity was dependent on the vesicle preparation since overall K D values were up to 10 times lower for N2-dried than for vacuum-dried phospholipids, although with apparently fewer binding sites. However, compression of the vesicles had no effect on the K D. In contrast, the rate constants were dependent on the number of DNA tags; thus deformation of the vesicles was observed. The k a and k d for FXa were similar for vesicles attached with 30 DNA tags or fused on the L1 chip but higher with fewer tags and approximately 10 times higher if attached with 1 tag. Thus for controlled kinetic studies immobilized DNA-tagged vesicles should be used. [Copyright &y& Elsevier]

Abstract: Toxicity mechanisms of Bacillus thuringiensis Cry insecticidal proteins involve membrane insertion and lytic pore formation in lipid bilayers of the target larval midgut cell membranes. The B. thuringiensis Cry4Ba mosquito-larvicidal protein has been shown to be capable of permeabilizing liposome vesicles and of forming ion channels in planar lipid bilayers. Here, the membrane interaction of the 65-kDa activated Cry4Ba protein with the lipid monolayers, comprising dipalmitoyl phosphatidylcholine, dioleoyl phosphatidylethanolamine, and cholesterol (Chol), was studied using Langmuir–Blodgett technique. The interactions of the Cry4Ba protein with the lipid monolayers were measured from the surface pressure versus area isotherms of the protein–lipid monolayers. The increase in the mean molecular area was demonstrated as an incorporation of the protein into lipid monolayers. The insertion of the Cry4Ba protein was monitored by measuring as an increase of the surface pressure at constant molecular area. For a given monolayer, the membrane insertion of the Cry4Ba reduced as the initial surface pressure increased. The Cry4Ba protein showed a strong preference of an insertion towards a Chol monolayer. In addition, the mixed monolayers of Chol showed an enhanced effect on the insertion kinetics of Cry4Ba into lipid films, suggesting its involvement in the modulation of the protein insertion. These findings provide the first evidence that the Cry4Ba protein is capable of inserting itself into lipid monolayers, depending on the packing density of the monolayers. Our results also indicate that only a limited part of the protein is likely to be involved in the insertion. [Copyright &y& Elsevier]

Abstract: Non-specific adsorption and specific interaction between a chimeric green fluorescent protein (GFP) carrying metal-binding region and the immobilized zinc ions on artificial solid-supported lipid membranes was investigated using the quartz crystal microbalance technique and the atomic force microscopy (AFM). Supported lipid bilayer, composed of octanethiol and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-[N-(5-amino-1-carboxypentyl iminodiacetic acid)succinyl] (NTA-DOGS)-Zn2+, was formed on the gold electrode of quartz resonator (5MHz). Binding of the chimeric GFP to zinc ions resulted in a rapid decrease of resonance frequency. Reversibility of the process was demonstrated via the removal of metal ions by EDTA. Nanoscale structural orientation of the chimeric GFP on the membrane was imaged by AFM. Association constant of the specific binding to metal ions was 2- to 3-fold higher than that of the non-specific adsorption, which was caused by the fluidization effect of the metal-chelating lipid molecules as well as the steric hindrance effect. This infers a possibility for a further development of biofunctionalized membrane. However, maximization is needed in order to attain closer advancement to a membrane-based sensor device. [Copyright &y& Elsevier]

Previous results [J. Biol. Chem. 276 (2001) 16978] indicated that an apolipoprotein A-I (apoAI) central region swings away from lipid contact in discoidal high density lipoproteins (HDL), but it is able to penetrate into the bilayer of lipid vesicles. In this work, we have studied the interaction with lipid membranes of a synthetic peptide with the sequence of apoAI region between residues 77 and 120 (AI 77–120). Like apoAI, AI 77–120 binds to phospholipid vesicles and shows selectivity for cholesterol-containing membranes. Moreover, AI 77–120 promotes cholesterol desorption from membranes in a similar fashion as apoAI and can stimulate cholesterol efflux from Chinese hamster ovary cells. AI 77–120 has a considerable α-helical content in water solution, and its secondary structure is not largely modified after binding to membranes. Both apoA-I and AI 77–120 are oligomeric in the lipid-bound state, suggesting that dimerization of the central domain could be required for the membrane binding activity of apoA-I in HDL. [Copyright &y& Elsevier]

The synapsins (I, II, and III) comprise a family of peripheral membrane proteins that are involved in both regulation of neurotransmitter release and synaptogenesis. Synapsins are concentrated at presynaptic nerve terminals and are associated with the cytoplasmic surface of synaptic vesicles. Membrane-binding of synapsins involves interaction with both protein and lipid components of synaptic vesicles. Synapsin I binds rapidly and with high affinity to liposomes containing anionic lipids. The binding of bovine synapsin I to liposomes was studied using fluoresceinphosphatidyl-ethanolamine (FPE) to measure membrane electrostatic potential. Synapsin binding to liposomes caused a rapid increase in FPE fluorescence, indicating an increase in positive charge at the membrane surface. Synapsin I binding to monolayers resulted in a substantial increase in monolayer surface pressure. At higher initial surface pressures, the synapsin-induced increase in monolayer surface pressure is dependent on the presence of anionic lipids in the monolayer. Synapsin I also induced rapid aggregation of liposomes, but did not induce leakage of entrapped carboxyfluorescein, while other aggregation-inducing agents promoted extensive leakage. These results are in agreement with the presence of amphipathic stretches of amino acids in synapsin I that exhibit both electrostatic and hydrophobic interactions with membranes, and offer a molecular explanation for the high affinity binding of synapsin I to liposomes and for stabilization of membranes by synapsin I. [Copyright &y& Elsevier]