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1.
The prevalence of antibiotic-resistant pathogens is a major medical concern, prompting increased interest in the development of novel antimicrobial compounds. One such set of naturally occurring compounds, known as antimicrobial peptides (AMPs), have broad-spectrum activity, but come with many limitations for clinical use. Recent work has resulted in a set of antimicrobial lipopeptides (AMLPs) with micromolar minimum inhibitory concentrations and excellent selectivity for bacterial membranes. To characterize a potent, synthetic lipopeptide, C16-KGGK, we used multi-microsecond coarse-grained simulations with the MARTINI forcefield, with a total simulation time of nearly 46μs. These simulations show rapid binding of C16-KGGK, which forms micelles in solution, to model bacterial lipid bilayers. Furthermore, upon binding to the surface of the bilayer, these lipopeptides alter the local lipid organization by recruiting negatively charged POPG lipids to the site of binding. It is likely that this drastic reorganization of the bilayer has major effects on bilayer dynamics and cellular processes that depend on specific bilayer compositions. By contrast, the simulations revealed no association between the lipopeptides and model mammalian bilayers. These simulations provide biophysical insights into lipopeptide selectivity and suggest a possible mechanism for antimicrobial action. This article is part of a Special Issue entitled: Membrane protein structure and function. 相似文献
2.
Interactions of bioactive lipopeptides, iturin A and surfactin from Bacillus subtilis. 总被引:4,自引:0,他引:4
L Thimon F Peypoux R Maget-Dana B Roux G Michel 《Biotechnology and applied biochemistry》1992,16(2):144-151
The antifungal activity of iturin A and its interaction with erythrocyte membranes were enhanced in the presence of surfactin. The modification of the properties of iturin A was explained by the formation of mixed iturin A-surfactin micelles. Such mixed micelles were easily generated when both lipopeptides were in aqueous solutions in the absence of mineral salts but the formation of these micelles did not occur when the solutions contained a high molarity of mineral cations. 相似文献
3.
We have used sedimentation equilibrium analytical ultracentrifugation to measure the free energy change for the glycophorin A transmembrane helix-helix dimerization in C14 betaine micelles. By varying the amount of micellar C14 betaine, we show that the protein association reaction in the micellar C14 phase behaves as an ideal-dilute solution. In this hydrophobic environment, the mole-fraction standard state free energy change for self-association of the SNGpA99 glycophorin A construct is -5.7 (+/-0.3, N=5) kcal mol(-1) at 25 degrees C. Compared with previous results carried out in C(8)E(5) micellar solutions, the free energy of dimerization is 1.3 kcal mol(-1) less favorable in C14 betaine micelles. In contrast, when considered on a per-interface basis, the formation of the glycophorin A transmembrane dimer in C14 betaine micelles may be more favorable than the association of several designed transmembrane peptides. 相似文献
4.
B Uhl V Speth B Wolf G Jung W G Bessler S Hauschildt 《European journal of cell biology》1992,58(1):90-98
Synthetic lipopeptide analogues of the N-terminal region of bacterial lipoprotein are potent activators of macrophages. In a previous study we showed that within minutes after their addition to macrophage cultures, lipopeptides were found attached to the plasma membranes and within different compartments of the cells. Their rapid interaction with the plasma membrane is thought to occur via the insertion of their three fatty acids. We used the freeze-fracture technique to study the influence of lipopeptides on the architecture of plasma membranes. Fifteen to thirty seconds after addition of the lipopeptides, the freeze-fractured plasma membranes show a rapid decrease in the particle density. This effect is not due to a loss of proteins, but is caused by lateral diffusion of single particles, which subsequently aggregate. These alterations are transient, temperature-sensitive and disappear 20 min after stimulation. At 4 degrees C, no change is found in the architecture of the plasma membranes. Using electron energy loss spectroscopy (EELS), lipopeptides can neither be detected on the membrane nor within the cells when incubated at this temperature. Our findings suggest that membrane protein aggregation is involved in the rapid uptake of lipopeptides into macrophages after their interaction with the plasma membranes. 相似文献
5.
《Journal of molecular biology》2021,433(8):166886
CLC-ec1 is a Cl−/H+ antiporter that forms stable homodimers in lipid bilayers, with a free energy of −10.9 kcal/mol in 2:1 POPE/POPG lipid bilayers. The dimerization interface is formed by four transmembrane helices: H, I, P and Q, that are lined by non-polar side-chains that come in close contact, yet it is unclear as to whether their interactions drive dimerization. To investigate whether non-polar side-chains are required for dimer assembly, we designed a series of constructs where side-chain packing in the dimer state is significantly reduced by making 4–5 alanine substitutions along each helix (H-ala, I-ala, P-ala, Q-ala). All constructs are functional and three purify as stable dimers in detergent micelles despite the removal of significant side-chain interactions. On the other hand, H-ala shows the unique behavior of purifying as a mixture of monomers and dimers, followed by a rapid and complete conversion to monomers. In lipid bilayers, all four constructs are monomeric as examined by single-molecule photobleaching analysis. Further study of the H-helix shows that the single mutation L194A is sufficient to yield monomeric CLC-ec1 in detergent micelles and lipid bilayers. X-ray crystal structures of L194A reveal the protein re-assembles to form dimers, with a structure that is identical to wild-type. Altogether, these results demonstrate that non-polar membrane embedded side-chains play an important role in defining dimer stability, but the stoichiometry is highly contextual to the solvent environment. Furthermore, we discovered that L194 is a molecular hot-spot for defining dimerization of CLC-ec1. 相似文献
6.
“Charged” amino acids play countless important roles in protein structure and function. Yet when these side chains come into contact with membranes we do not fully understand their behavior. This is highlighted by a recent model of voltage-gated ion channel activity and translocon-based experiments that suggest small penalties to expose these side chains to lipids, opposing the prevailing view in membrane biophysics. Here we employ a side chain analog as well as a transmembrane helix model to determine the free energy as a function of protonation state and position for a lipid-exposed arginine (Arg) residue across a membrane. We observe high free energy barriers for both the charged and neutral states. Due to the stabilizing influence of membrane deformations for the protonated form, the Arg side chain experiences a pKa shift of ≤4.5 units and remains mostly protonated. The cost for exposing Arg to lipid hydrocarbon is prohibitively high with implications for many membrane translocating processes and the activation mechanisms of voltage-gated ion channels. 相似文献
8.
Katarzyna E. Greber Joanna Zielińska Łukasz Nierzwicki Krzesimir Ciura Piotr Kawczak Joanna Nowakowska Tomasz Bączek Wiesław Sawicki 《生物化学与生物物理学报:生物膜》2019,1861(1):93-99
Short cationic lipopeptides are amphiphilic molecules that exhibit antimicrobial activity mainly against Gram-positives. These compounds bind to bacterial membranes and disrupt their integrity. Here we examine the structure-activity relation (SAR) of lysine-based lipopeptides, with a prospect to rationally design more active compounds. The presented study aims to explain how antimicrobial activity of lipopeptides is affected by the charge of lipopeptide headgroup and the length of lipopeptide acyl chain. The obtained SAR models suggest that the lipophilicity of short synthetic cationic lipopeptides is the major factor that determines their antimicrobial activities. In order to link the differences in antimicrobial activity to the mechanism of action of lipopeptides containing one and two hydrophobic chains, we additionally performed molecular dynamic (MD) simulations. By using combined coarse-grained and all-atom simulations we also show that these compounds neither affect the organization of the membrane lipids nor aggregate to form separate phases. These results, along with the onset of antimicrobial activity of lipopeptides well below the critical micelle concentration (CMC), indicate that lipopeptides do not act in a simple detergent-like manner. 相似文献
9.
To investigate the susceptibilities of fungal and mammalian cells to amphotericin B (AmB), AmB-loaded lysophosphatidylcholine (LPC)micelles as drug delivery vehicles were incubated at 37 degrees C with phosphatidylcholine vesicles containing different sterols as model systems for fungal and mammalian cells. The binding and kinetics of AmB to sterols in the membranes were judged by UV-visible spectroscopy. In the 91% monomeric form, AmB interacted rapidly with ergosterol and slowly with 7-dehydrocholesterol (7-DHC), while it did not interact with cholesterol. In the 50% monomeric form, AmB formed complexes more rapidly with ergosterol or 7-DHC than in the monomeric form, whereas it did not still interact with cholesterol. The interaction was also characterized by resonance energy transfer between the fluorescent probe trimethylammonium diphenylhexatriene (TMA-DPH) and AmB. In the 91% monomeric form, AmB caused initial fluorescence quenching in bilayer membranes containing any sterol as well as sterol-free bilayer membranes due to the release of AmB and its incorporation within the membranes. However, a second phase of increasing fluorescence was found in the case of ergosterol alone. On the other hand, in the 47% monomeric form, AmB gave a biphasic intensity profile in membranes containing any sterol as well as sterol-free membranes. However, the extent of the second phase of increasing fluorescence intensity was markedly dependent upon sterol composition. Studies using sterol-containing vesicles provide important insights into the role of the aggregation state of AmB in its effects on cells. 相似文献
10.
Fersht and co-workers have applied a linear free energy relation (Br?nsted equation) to analyze site-directed mutagenesis experiments involving the enzyme tyrosyl-tRNA synthetase and have suggested that the Br?nsted exponent is linearly correlated with the value of the reaction coordinate at the transition state. We point out that when the mutants differ solely through the formation or deletion of a hydrogen bond away from the reaction center, a linear free energy relation is expected only in limiting cases for which the Br?nsted relation exponent is 0, 1 or infinity. The results may be correlated with a conformational coordinate but not with the development of the reaction coordinate per se. 相似文献
11.
Fleming KG 《Journal of molecular biology》2002,323(3):563-571
Side-to-side associations of transmembrane alpha-helices are integral components of the structure and function of helical membrane proteins. A fundamental unknown in the understanding of the chemical principles driving the lateral interactions between transmembrane alpha-helices is the balance of forces arising from the polypeptide sequence versus the hydrophobic solvent. To begin to address this question, a consideration of basic thermodynamic principles has been applied to assess the experimental free energy change associated with transmembrane helix dimerization in micelles. This analysis demonstrates the ability to partition the apparent free energy of transmembrane helix-helix association into two components. The first component is a statistical energy term, which arises from the fact that there are an unequal number of reactants and products. The second component is a standard state free energy change, which informs on the molecular details of the transmembrane helix self-association reaction. The advantage of separating these two energy terms arises from the fact that extrapolation to the standard state free energy change normalizes the statistical energy term so that it applies equivalently in all experimental systems. Accompanying experimental results for the glycophorin A transmembrane alpha-helix dimer measured in micelles are well described by these theoretical components assuming an ideal-dilute solution. 相似文献
12.
We propose a testable general mechanism by which ligand binding energy can be used to drive a catalytic step in an enzyme catalyzed reaction or to do other forms of work involving protein molecules. This energy transduction theory is based on our finding of the widespread occurrence of ligand binding-induced protein macrostate interconversions each having a large invariant delta H0 accompanied by a small but highly variable delta G0. This phenomenon, which can be recognized by the large delta Cp0's it generates, can provide the necessary energy input step but is not in itself sufficient to constitute a workable transduction mechanism. A viable mechanism requires the additional presence of an 'energy transmission step' which is terminated to trigger the 'power' stroke at a precise location on the reaction coordinate, followed by an energetically inexpensive 'return' step to restore the machine to its initial conditions. In the model we propose here, these additional steps are provided by the existence of ligand inducible 2-state transitions in the free enzyme and in each of the enzyme complexes that occur along the reaction coordinate, and by the selective blocking of certain of these interconversions by high energetic barriers. We provide direct experimental evidence supporting the facts that these additional mechanistic components do exist and that the liver glutamate dehydrogenase reaction is indeed driven by just such machinery. We describe some aspects of the chemical nature of these transitions, and evidence for their occurrence in other systems. 相似文献
13.
《Biophysical journal》2021,120(17):3718-3731
The collective behavior of lipids with diverse chemical and physical features determines a membrane’s thermodynamic properties. Yet, the influence of lipid physicochemical properties on lipid dynamics, in particular interbilayer transport, remains underexplored. Here, we systematically investigate how the activation free energy of passive lipid transport depends on lipid chemistry and membrane phase. Through all-atom molecular dynamics simulations of 11 chemically distinct glycerophospholipids, we determine how lipid acyl chain length, unsaturation, and headgroup influence the free energy barriers for two elementary steps of lipid transport: lipid desorption, which is rate limiting, and lipid insertion into a membrane. Consistent with previous experimental measurements, we find that lipids with longer, saturated acyl chains have increased activation free energies compared to lipids with shorter, unsaturated chains. Lipids with different headgroups exhibit a range of activation free energies; however, no clear trend based solely on chemical structure can be identified, mirroring difficulties in the interpretation of previous experimental results. Compared to liquid-crystalline phase membranes, gel phase membranes exhibit substantially increased free energy barriers. Overall, we find that the activation free energy depends on a lipid’s local hydrophobic environment in a membrane and that the free energy barrier for lipid insertion depends on a membrane’s interfacial hydrophobicity. Both of these properties can be altered through changes in lipid acyl chain length, lipid headgroup, and membrane phase. Thus, the rate of lipid transport can be tuned through subtle changes in local membrane composition and order, suggesting an unappreciated role for nanoscale membrane domains in regulating cellular lipid dynamics. 相似文献
14.
The development of novel antibiotic drugs is one of the most pressing biomedical problems due to the increasing number of antibiotic-resistant pathogens. Antimicrobial peptides and lipopeptides are a promising category of candidates, but the molecular origins of their antimembrane activity is unclear. Here we explore a series of recently developed antimicrobial lipopeptides, using coarse-grained molecular-dynamics simulations and free energy methods to uncover the thermodynamics governing their binding to membranes. Specifically, we quantify C16-KGGK’s binding affinity to the two types of membrane by umbrella sampling. We also examined the origin of C16-KGGK’s selectivity for bacterial versus mammalian membranes by systematically varying the peptide sequence and salt concentration. Our data showed that the C16 hydrophobic tail is the main contributor to its affinity to lipid membrane, whereas the peptide portion is mainly responsible for its selectivity. Furthermore, the electrostatic interaction between the cationic peptide and anionic bacterial membrane plays a significant role in the selectivity. 相似文献
15.
Sapra KT Balasubramanian GP Labudde D Bowie JU Muller DJ 《Journal of molecular biology》2008,376(4):1076-1090
Using single-molecule force spectroscopy, we investigated the effect of single point mutations on the energy landscape and unfolding pathways of the transmembrane protein bacteriorhodopsin. We show that the unfolding energy barriers in the energy landscape of the membrane protein followed a simple two-state behavior and represent a manifestation of many converging unfolding pathways. Although the unfolding pathways of wild-type and mutant bacteriorhodopsin did not change, indicating the presence of same ensemble of structural unfolding intermediates, the free energies of the rate-limiting transition states of the bacteriorhodopsin mutants decreased as the distance of those transition states to the folded intermediate states decreased. Thus, all mutants exhibited Hammond behavior and a change in the free energies of the intermediates along the unfolding reaction coordinate and, consequently, their relative occupancies. This is the first experimental proof showing that point mutations can reshape the free energy landscape of a membrane protein and force single proteins to populate certain unfolding pathways over others. 相似文献
16.
All-or-none membrane permeabilization by fengycin-type lipopeptides from Bacillus subtilis QST713 总被引:2,自引:0,他引:2
Patel H Tscheka C Edwards K Karlsson G Heerklotz H 《Biochimica et biophysica acta》2011,1808(8):2000-2008
The fungicidal activity of Bacillus subtilis QST713 has been utilized for the highly effective and environmentally safe protection of crops against a variety of pathogens. It is based mainly on the production of cyclic lipopeptides of the fengycin (FEs), surfactin, and iturin families. The mixed population of native FEs forms micelles which solubilize individual FEs such as agrastatin 1 (AS1) that are otherwise rather insoluble on their own. Fluorescence lifetime-based calcein efflux measurements and cryo transmission electron microscopy show that these FEs show a unique scenario of membrane permeabilization. Poor miscibility of FEs with lipid probably promotes the formation of pores in 10% of the vesicles at only≈1μM free FE and in 15% of the vesicles at 10 μM. We explain why this limited, all-or-none leakage could nevertheless account for the killing of virtually all fungi whereas the same extent of graded vesicle leakage may be biologically irrelevant. Then, crystallization of AS1 and micellization of plipastatins cause a cut-off in leakage at 15% that might regulate the biological activity of FEs, protecting Bacillus and plant membranes. The fact that FE micelles solubilize only about 10 mol-% fluid lipid resembles the behavior of detergent resistance. 相似文献
17.
Yi D Guoming L Gao L Wei L 《Biochemical and biophysical research communications》2007,359(4):1024-1029
Short oligomers of arginine (R8) have been shown to cross readily a variety of biological barriers. A hypothesis was put forward that inverted micelles form in biological membranes in the presence of arginine oligomer peptides, facilitating their transfer through the membranes. In order to define the role of peptide-lipid interaction in this mechanism, we prepared liposomes as the model membrane to study the ability of R8 inducing calcein release from liposomes, the fusion of liposomes, R8 binding to liposomes and membrane disturbing activity of the bound R8. The results show that R8 binding to liposome membrane depends on lipid compositions, negative surface charge density and interior water phase pH values of liposomes. R8 has no activity to induce the leakage of calcein from liposomes or improve liposome fusion. R8 does not permeabilize through the membrane spontaneously. These peptides delivering drugs through membranes may depend on receptors and energy. 相似文献
18.
The dramatically increased frequency of opportunistic fungal infections has prompted research to diversify the arsenal of antifungal agents. Antimicrobial peptides constitute a promising family for future antibiotics with a new mode of action. However, only a few are effective against fungal pathogens because of their ability to self-assemble. Recently, we showed that the conjugation of fatty acids to the potent antibacterial peptide magainin endowed it with antifungal activity concomitant with an increase in its oligomeric state in solution. To investigate whether a high potency of the parental peptide is prerequisite for antifungal activity, we conjugated undecanoic acid (UA) and palmitic acid (PA) to inactive diastereomers of magainin containing four d-amino acids ([D]-4-magainin), as well as to a weakly active diastereomeric lytic peptide containing Lys and Leu ([D]-K(5)L(7)). All lipopeptides gained potent activity toward Cryptococcus neoformans. Most importantly, [D]-K(5)L(7)-UA was highly potent against all microorganisms tested, including bacteria, yeast, and opportunistic fungi. All lipopeptides increased the permeability of Escherichia coli spheroplasts and intact C. neoformans, as well as their corresponding membranes, phosphatidylethanol (PE)/phosphatidylglycerol (PG) and phosphatidylcholine (PC)/PE/phosphatidylinositol (PI)/ergosterol, respectively. The extent of membrane-permeating activity correlated with their biological function, suggesting that the plasma membrane was one of their major targets. Circular dichroism (CD) and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy revealed that their mode of oligomerization in solution, structure, and organization in membranes have important roles regarding their antibacterial and antifungal activities. Together with the advantage of using diastereomers versus all l-amino acid peptides, this study paves the way to the design of a new group of potent antifungal peptides urgently needed to combat opportunistic fungal infection. 相似文献
19.
Structural distortions of DNA are essential for its biological function due to the genetic information of DNA not being physically accessible in the duplex state. Base flipping is one of the simplest structural distortions of DNA and may represent an initial event in strand separation required to access the genetic code. Flipping is also utilized by DNA-modifying and repair enzymes to access specific bases. It is typically thought that base flipping (or base-pair opening) occurs via the major groove whereas minor groove flipping is only possible when mediated by DNA-binding proteins. Here, umbrella sampling with a novel center-of-mass pseudodihedral reaction coordinate was used to calculate the individual potentials of mean force (PMF) for flipping of the Watson-Crick (WC) paired C and G bases in the CCATGCGCTGAC DNA dodecamer. The novel reaction coordinate allowed explicit investigation of the complete flipping process via both the minor and major groove pathways. The minor and major groove barriers to flipping are similar for C base flipping while the major groove barrier is slightly lower for G base flipping. Minor groove flipping requires distortion of the WC partner while the flipping base pulls away from its partner during major groove flipping. The flipped states are represented by relatively flat free energy surfaces, with a small, local minimum observed for the flipped G base. Conserved patterns of phosphodiester backbone dihedral distortions during flipping indicate their essential role in the flipping process. During flipping, the target base tracks along the respective grooves, leading to hydrogen-bonding interactions with neighboring base-pairs. Such hydrogen-bonding interactions with the neighboring sequence suggest a novel mechanism of sequence dependence in DNA dynamics. 相似文献
20.
Molecular interactions between receptors and ligands can be characterized by their free energy landscape. In its simplest representation, the energy landscape is described by a barrier of certain height and width that determines the dissociation rate of the complex, as well as its dynamic strength. Some interactions, however, require a more complex landscape with additional barriers and roughness along the reaction coordinate. This roughness slows down the dissociation kinetics of the interaction and contributes to its dynamic strength. The streptavidin-biotin complex has been extensively studied due to its remarkably low dissociation kinetics. However, single molecule measurements from independent experiments showed scattered and disparate results. In this work, the energy landscape roughness of the streptavidin-biotin interaction was estimated to be in the range of 5-8kBT using dynamic force spectroscopy (DFS) measurements at three different temperatures. These results can be used to explain both its slow dissociation kinetics and the discrepancies in the reported force measurements. 相似文献