Low Peptide Concentrations Research Articles

A Novel Functional Class of Pore-Forming Peptides

Peptides that self-assemble into membrane spanning pores, at low concentration, to allow the passage of macromolecules would be beneficial in multiple areas in biotechnology, including biosensor design and drug delivery. However, there are few, if any, natural or designed peptides have this property. Here we show that the 26-residue peptide “MelP5”, a gain-of-function variant of the bee venom lytic peptide melittin, identified in a high-throughput screen for small molecule leakage, enables the passage of macromolecules across bilayers. In surface supported bilayers, MelP5 forms unusually high conductance, equilibrium pores at low peptide concentration, and also increases the capacitance of the supported bilayers, suggesting that it forms large pores that affect a significant fraction of the membrane surface. The increase in bilayer conductance due to MelP5 is much higher than the decrease due to either melittin or by the potent equilibrium pore-forming peptide, alamethicin. This result prompted us to develop two novel assays for macromolecule leakage from vesicles and use them to characterize MelP5, melittin and alamethicin. Under conditions where osmotic lysis does not occur, MelP5 allows the passage of macromolecules across vesicle membranes at peptide to lipid ratios as low as 1:100. Neither the melittin nor alamethicin release macromolecules significantly under these conditions. Therefore, the macromolecule-sized, equilibrium pores forms by MelP5 are unique, and MelP5 appears to belong to a novel functional class of peptide with many potential biotechnological applications.

Process of Inducing Pores in Membranes by Melittin

Melittin is a prototype of the ubiquitous antimicrobial peptides that induce pores in membranes. It is commonly used as a molecular device for membrane permeabilization. Even at concentrations in the nanomolar range, melittin can induce transient pores that allow transmembrane conduction of atomic ions but not leakage of glucose or larger molecules. At micromolar concentrations, melittin induces stable pores allowing transmembrane leakage of molecules up to tens of kDa, corresponding to its antimicrobial activities. Despite extensive studies, aspects of molecular mechanism for pore formation remain unclear. To clarify the mechanism, one must know the states of melittin-bound membrane before and after the process. By correlating experiment of giant unilamellar vesicles with that of peptide-lipid multilayers, we found that melittin bound on the vesicle translocated and redistributed to both sides of the membrane before the formation of stable pores. Furthermore, stable pores are formed only above a critical peptide-to-lipid ratio. The initial states for transient and stable pores are different, which implies different mechanisms at low and high peptide concentrations. To determine the lipidic structure of the pore, the pores in peptide-lipid multilayers were induced to form a lattice and examined by anomalous X-ray diffraction. The electron density distribution of lipid labels shows that the pore is formed by merging of two interfaces through a hole. The molecular property of melittin is such that it adsorbs strongly to the bilayer interface. Pore formation can be viewed as the bilayer adopting a lipid configuration to accommodate its excessive interfacial area.

Solid-State NMR Structure Analysis of the Short Multifunctional Peptide BP100 in Membranes

The 11-residue peptide BP100 (KKLFKKILKYL-amide) is a short antimicrobial peptide that has been optimized against plant pathogens, and it is also able to act as a cell-penetrating agent. To address the conformation, orientation and dynamics of BP100 in the membrane-bound state, we have labeled all of the six hydrophobic amino acids individually with CF3-L-Bpg for solid-state 19F-NMR analysis, and a 15N-label was incorporated at Leu8. Circular dichroism (CD) analysis showed that the substitutions did not perturb the overall structure of the α-helical peptide, and the antimicrobial activity also remained unaffected. Using highly sensitive 19F-NMR, we found that the orientation of BP100 in macroscopically oriented DMPC/PG membranes remains unchanged as a function of peptide concentration over a wide range of peptide-to-lipid molar ratios from 1:10 to 1:3000, accessible only by fluorine NMR. The 19F-NMR data analysis was compatible with a large family of possible helix tilt angles, but these could by narrowed down by including peptide dynamics in the structure determination process. These results were complemented by 15N-NMR and oriented CD spectroscopy, both showing that the amphiphilic BP100 helix is oriented parallel to the membrane surface. In summary, our results show that the short BP100 molecule assumes a surface bound state under different concentration regimes and remains highly mobile. We suggest that at low peptide concentration BP100 may be able to permeate cellular membranes in a transient way, but at high concentration it perturbs the membrane via a carpet mechanism.

The Structure of Integrin α1I Domain in Complex with a Collagen-mimetic Peptide

We have determined the structure of the human integrin α1I domain bound to a triple-helical collagen peptide. The structure of the α1I-peptide complex was investigated using data from NMR, small angle x-ray scattering, and size exclusion chromatography that were used to generate and validate a model of the complex using the data-driven docking program, HADDOCK (High Ambiguity Driven Biomolecular Docking). The structure revealed that the α1I domain undergoes a major conformational change upon binding of the collagen peptide. This involves a large movement in the C-terminal helix of the αI domain that has been suggested to be the mechanism by which signals are propagated in the intact integrin receptor. The structure suggests a basis for the different binding selectivity observed for the α1I and α2I domains. Mutational data identify residues that contribute to the conformational change observed. Furthermore, small angle x-ray scattering data suggest that at low collagen peptide concentrations the complex exists in equilibrium between a 1:1 and 2:1 α1I-peptide complex.

Long-Time-Scale Interaction Dynamics between a Model Antimicrobial Peptide and Giant Unilamellar Vesicles

The interaction dynamics between a lytic peptide and a biomembrane was studied using time-lapse fluorescence lifetime imaging microscopy. The model membrane was 1,2-dipalmitoyl-sn-glycero-3-phosphochloine giant unilamellar vesicles (GUVs), and the peptide was the K14 derivative of melittin, to which the polarity-sensitive fluorescent probe AlexaFluor 430 was grafted. The interaction of the peptide with the GUVs resulted in a progressive quenching of the fluorescence lifetime over a period of minutes. From previous photophysics characterization of the peptide, we were able to deconvolve the contribution of three distinct peptide states to the lifetime trajectory and use this data to develop a kinetics model for the interaction process. It was found that the peptide-membrane interaction was well described by a two-step mechanism: peptide monomer adsorption followed by membrane surface migration, assembly, and insertion to form membrane pores. There was an equilibrium exchange between pore and surface monomers at all lipid/peptide (L/P) concentration ratios, suggesting that the fully inserted phase was reached, even at low peptide concentrations. In contrast to previous studies, there was no evidence of critical behavior; irrespective of L/P ratio, lytic pores were the dominant peptide state at equilibrium and were formed even at very low peptide concentrations. We suggest that this behavior is seen in GUVs because their low curvature means low Laplace pressure. Membrane elasticity is therefore relatively ineffective at damping the thermal fluctuations of lipid molecules that lead to random molecular-level lipid protrusions and membrane undulations. The transient local membrane deformations that result from these thermal fluctuations create the conditions necessary for facile peptide insertion.

Chromogranin A is a T cell antigen in human type 1 diabetes

Chromogranin A (ChgA) is a beta cell secretory granule protein and a peptide of ChgA, WE14, was recently identified as a ligand for diabetogenic CD4 T cell clones derived from the NOD mouse. In this study we compared responses of human CD4 T cells from recent onset type 1 diabetic (T1D) and control subjects to WE14 and to an enzymatically modified version of this peptide. T cell responders to antigens were detected in PBMCs from study subjects by an indirect CD4 ELISPOT assay for IFN-γ. T1D patients (n = 27) were recent onset patients within one year of diagnosis, typed for HLA-DQ8. Controls (n = 31) were either 1st degree relatives with no antibodies or from the HLA-matched general population cohort of DAISY/TEDDY. A second cohort of patients (n = 11) and control subjects (n = 11) was tested at lower peptide concentrations. We found that WE14 is recognized by T cells from diabetic subjects vs. controls in a dose dependent manner. Treatment of WE14 with transglutaminase increased reactivity to the peptide in some patients. This work suggests that ChgA is an important target antigen in human T1D subjects and that post-translational modification may play a role in its reactivity and relationship to disease.

Effects of rabbit anti-α-melanocyte-stimulating hormone (α-MSH) immunoglobulins on α-MSH signaling related to food intake control

Anti-α-melanocyte-stimulating hormone (α-MSH) polyclonal antibodies have been used for α-MSH neutralization in functional studies, but the results are sometime inconsistent with the antibody expected blocking properties. The present study aimed to determine if rabbit (Rb) anti-α-MSH immunoglobulins (Ig) may inhibit or enhance α-MSH signaling on melanocortin receptor type 4 (MC4R) and α-MSH-induced anorexigenic effect if presented as immune complexes with α-MSH. Polyclonal Rb anti-α-MSH IgG were commercially available and their ability to bind α-MSH has been confirmed by the immunohistochemical detection of α-MSH neurons in the rat hypothalamus. In vitro assay of the cyclic-adenosine mono-phosphate (cAMP) secreted by cells transfected with MC4R was performed to analyze effect of Rb IgG on α-MSH-induced cAMP production. We found that adding Rb IgG to α-MSH resulted in stimulation of cAMP detected at lower peptide concentrations as compared to α-MSH alone. To determine effects of Rb IgG on food intake, rats were injected into the arcuate hypothalamic nucleus with either α-MSH, Rb IgG alone or Rb IgG preincubated with α-MSH. During 2days after injections, food intake was increased in both groups of rats receiving Rb IgG. However, during following 4days when food was restricted to 1h/day, only the Rb IgG group displayed higher food intake. Furthermore, after the refeeding, 24h food intake was lower in rats receiving Rb IgG - α-MSH immune complexes. This group of rats was also characterized by higher number of immunopositive neurons in the arcuate nucleus expressing α-MSH and agouti-related protein but not tyrosine hydroxylase. Taken together, these results show that Rb anti-α-MSH antisera, although efficient for immunohistochemical detection of α-MSH, does not always display α-MSH blocking properties but, in contrast, may enhance α-MSH binding to MC4R and increase α-MSH anorexigenic effects when presented as immune complexes with the peptide.

Protocolo diagnóstico y diagnóstico diferencial de la pericarditis constrictiva

Constrictive pericarditis should be considered in any patient with an unexplained elevated jugular venous pressure and fluid overload. Complementary test findings as relatively low plasma concentrations of brain natriuretic peptide (BNP), electrocardiogram with low voltage and nonspecific T-wave repolarization abnormalities may support the clinical suspicion. Pericardial calcifications may be noted on the chest radiograph, although absence of calcification does not exclude constrictive pericarditis. The diagnosis is confirmed with: constrictive physiology proved by echocardiography or invasive hemodynamic study, or pericardial thickening with or without calcification by cardiac computed tomography and cardiac magnetic resonance imaging. Results of both imaging modalities will help plan surgery.

Process of inducing pores in membranes by melittin

Melittin is a prototype of the ubiquitous antimicrobial peptides that induce pores in membranes. It is commonly used as a molecular device for membrane permeabilization. Even at concentrations in the nanomolar range, melittin can induce transient pores that allow transmembrane conduction of atomic ions but not leakage of glucose or larger molecules. At micromolar concentrations, melittin induces stable pores allowing transmembrane leakage of molecules up to tens of kilodaltons, corresponding to its antimicrobial activities. Despite extensive studies, aspects of the molecular mechanism for pore formation remain unclear. To clarify the mechanism, one must know the states of the melittin-bound membrane before and after the process. By correlating experiments using giant unilamellar vesicles with those of peptide-lipid multilayers, we found that melittin bound on the vesicle translocated and redistributed to both sides of the membrane before the formation of stable pores. Furthermore, stable pores are formed only above a critical peptide-to-lipid ratio. The initial states for transient and stable pores are different, which implies different mechanisms at low and high peptide concentrations. To determine the lipidic structure of the pore, the pores in peptide-lipid multilayers were induced to form a lattice and examined by anomalous X-ray diffraction. The electron density distribution of lipid labels shows that the pore is formed by merging of two interfaces through a hole. The molecular property of melittin is such that it adsorbs strongly to the bilayer interface. Pore formation can be viewed as the bilayer adopting a lipid configuration to accommodate its excessive interfacial area.

Prokaryotic Selectivity, Bactericidal Mechanism and LPS-neutralizing Activity of Lys-linked Dimeric Peptide of Indolicidin C-terminal Hexapeptide

Indolicidin (Ind) is a cationic antimicrobial peptide (AMP) derived from the granules of bovine neutrophils. It is a short, 13 amino acid peptide (ILPWKWPWWPWRR-NH2) that is amidated at the C-terminus and contains the highest proportion of Trp and Pro residues. Like many other AMPs, it exhibits a broad spectrum of antimicrobial activity against bacteria, fungi and protozoa. It has been shown that Ind adopts a disordered conformation in solution and a membrane-interacting structure containing coils and turns. However, Ind exhibits some toxicity to human erythrocytes and mouse T lymphocytes. In the present study, to investigate the effect of Lys-linked dimerization of Ind C-terminal hexapeptide (WWPWRRNH2) on prokaryotic selectivity, bactericidal mechanism and LPS-neutralizing activity and to develop a novel short AMP having prokaryotic selectivity and LPS-neutralizing activity, we synthesized a Lys-linked dimeric peptide (di-Ind-6) of Ind C-terminal hexapeptide (Ind-6) (Table 1). We examined the antimicrobial activity of the peptides against a representative set of bacterial strains, including three Gram-negative bacteria (Escherichia coli [KCTC 1682], Pseudomonas aeruginosa [KCTC 1637] and Salmonella typhimurium [KCTC 1926]) and three Gram-positive bacteria (Bacillus subtilis [KCTC 3068], Staphylococcus epidermidis [KCTC 1917] and Staphylococcus aureus [KCTC 1621]). The MIC values of the peptides are shown in Table 2. A Lys-linked dimeric peptide (di-Ind-6) displayed 2to 4-fold increased antimicrobial activity compared to monomeric peptide (Ind6). Furthermore, Di-Ind-6 displayed a 2-fold enhanced antimicrobial activity against Gram-negative bacteria as compare to Ind. Next, the cytotoxicity of the peptides to mammalian cells was determined by measuring their hemolytic activity toward human red blood cells (h-RBCs) Concentrationresponse curves for the hemolytic activity of the peptides are shown in Figure 1. For a quantitative measure of the hemolytic activity of the peptides, we introduced the hemolytic concentration (HC50) defined as the lowest peptide concentration that produces 50% hemolysis (Table 3). The HC50 value for Ind and di-Ind-6 was 160 μg/mL and 192 μg/mL, respectively. Di-Ind-6 was somewhat less hemolytic than Ind. In contrast, Ind-6 did not induce hemolysis even at the highest peptide concentration tested (200 μg/mL). The therapeutic potential of peptide antimicrobial drugs lies in their prokaryotic selectivity to effectively kill bacterial cells without exhibiting significant cytotoxicity toward mammalian cells such as human red blood cells. To determine prokaryotic selectivity of the peptides, we calculated their therapeutic index (TI) (Table 3). The TI is a widely employed parameter to represent the specificity of antimicrobial agents. The TI of the peptides is calculated as the ratio of the HC50 to GM (geometric mean of MICs against six selected Table 1. Amino acid sequences and calculated and observed molecular masses of the peptides

Structural characterization of the model amphipathic peptide Ac-LKKLLKLLKKLLKL-NH2 in aqueous solution and with 2,2,2-trifluoroethanol and 1,1,1,3,3,3-hexafluoroisopropanol

Short-chain amphipathic peptides are promising components in the new generation of engineered biomaterials. The model 14-residue leucine–lysine peptide Ac-LKKLLKLLKKLLKL-NH2 (LKα) is one such amphipathic peptide. In dilute aqueous solution (<0.05 mmol/L), it was previously proposed, using CD spectroscopic data, that LKα existed in a cooperative monomeric (unstructured) – tetrameric (α-helical) equilibrium that shifted towards the tetramer at high NaCl and peptide concentrations. Here, at similar peptide concentrations, CD spectroscopy shows that LKα readily adopts α-helical structure in the presence of 2,2,2-trifluoroethanol (TFE) and 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) with maximal helical character in 20% TFE and ∼10% HFIP (v/v). The helical character in fluorinated alcohols suggested by the CD data at low peptide concentrations (0.06 mmol/L) is corroborated at high peptide concentrations (1.5 mmol/L) by NMR NOE data that also show that 1.5 mmol/L LKα is helical in 100% water. Size exclusion chromatography and estimations of rotational correlation times (τc) showed that the self-assembled LKα complexes contained three to five peptides. Removing the N-terminal acetyl group prevents LKα from forming helices and self-associating at high NaCl and peptide concentrations. This more detailed characterization of the structural and physical properties of LKα over a greater range of peptide concentrations and in the presence of fluorinated alcohols will assist the design of biomaterials containing amphipathic peptides and guide the ability to control self-assembly.

Physiologically-relevant modes of membrane interactions by the human antimicrobial peptide, LL-37, revealed by SFG experiments.

Antimicrobial peptides (AMPs) could become the next generation antibiotic compounds which can overcome bacterial resistance by disrupting cell membranes and it is essential to determine the factors underlying its mechanism of action. Although high-resolution NMR and other biological studies have provided valuable insights, it has been a major challenge to follow the AMP-membrane interactions at physiologically-relevant low peptide concentrations. In this study, we demonstrate a novel approach to overcome this major limitation by performing Sum Frequency Generation (SFG) vibrational spectroscopic experiments on lipid bilayers containing an AMP, LL-37. Our results demonstrate the power of SFG to study non-linear helical peptides and also infer that lipid-peptide interaction and the peptide orientation depend on the lipid membrane composition. The observed SFG signal changes capture the aggregating process of LL-37 on membrane. In addition, our SFG results on cholesterol-containing lipid bilayers indicate the inhibition effect of cholesterol on peptide-induced membrane permeation process.

Observing the translocation of a mitochondria-penetrating peptide with solid-state NMR

A new class of penetrating peptides that can target the mitochondria with high specificity was recently discovered. In this work, we developed a model inner mitochondrial membrane, equipped with a transmembrane gradient, suitable for solid-state NMR experiments. Using solid-state NMR, we observed a mitochondria-penetrating peptide interacting with the model inner mitochondrial membrane to gain insight into the mechanism of translocation. The paramagnetic relaxation effect due to Mn2+ ions on 13C magic angle spinning NMR was used to measure the insertion depth of the peptide and its distribution in each monolayer of the membrane. We found that at low peptide concentration the peptide binds to the outer leaflet and at high concentration, it crosses the hydrophobic bilayer core and is distributed in both leaflets. In both concentration regimes, the peptide binds at the C2 position on the lipid acyl chain. The mitochondria-penetrating peptide crossed to the inner leaflet of the model membranes without disrupting the lamellarity. These results provide evidence that supports the electroporation model of translocation. We estimated the energy associated with crossing the inner mitochondrial membrane. We found that the transmembrane potential provides sufficient energy for the peptide to cross the hydrophobic core, which is the most unfavorable step in translocation.

Dependence of Alamethicin Membrane Orientation on the Solution Concentration

Alamethicin has been extensively studied as an antimicrobial peptide and is widely used as a simple model for ion channel proteins. It has been shown that the antimicrobial activity of peptides is related to their membrane orientation. In this study, we determined the relationship between the solution concentration of alamethicin and its membrane orientation in lipid bilayers using sum frequency generation (SFG) vibrational spectroscopy. Our SFG results indicated that the alamethicin molecules more or less lay down on the surface of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayers at a low peptide concentration of 0.84 μM; the α-helix segment tilts at about 88°, and 310-helix segment tilts at about 58° versus the surface normal. However, when the peptide concentration was increased to 15.6 μM, we observed that alamethicin molecules further inserted into the lipid bilayers: the α-helical component changes its orientation to make a 37° tilt from the lipid bilayer normal, and the 310-helical component tilts at about 50° versus the surface normal. This is in agreement with the barrel-stave mode for the alamethicin-cell membrane interaction as reported previously. Additionally, we have also studied membrane orientation of alamethicin as a function of peptide concentration with SFG. Our results showed that the membrane orientation of the alamethicin α-helical component changed substantially with the increase of the alamethicin concentration, while the membrane orientation of the 310-helical component remained more or less the same.

Hierarchical ordering of amyloid fibrils on the mica surface

The aggregation of amyloid peptides into ordered fibrils is closely associated with many neurodegenerative diseases. The surfaces of cell membranes and biomolecules are believed to play important roles in modulation of peptide aggregation under physiological conditions. Experimental studies of fibrillogenesis at the molecular level in vivo, however, are inherently challenging, and the molecular mechanisms of how surface affects the structure and ordering of amyloid fibrils still remain elusive. Herein we have investigated the aggregation behavior of insulin peptides within water films adsorbed on the mica surface. AFM measurements revealed that the structure and orientation of fibrils were significantly affected by the mica lattice and the peptide concentration. At low peptide concentration (~0.05 mg mL(-1)), there appeared a single layer of short and well oriented fibrils with a mean height of 1.6 nm. With an increase of concentration to a range of 0.2-2.0 mg mL(-1), a different type of fibrils with a mean height of 3.8 nm was present. Interestingly, when the concentration was above 2.0 mg mL(-1), the thicker fibrils exhibited two-dimensional liquid-crystal-like ordering probably caused by the combination of entropic and electrostatic forces. These results could help us gain better insight into the effects of the substrate on amyloid fibrillation.

Membrane Destabilization by Alzheimer's Amyloid β Peptide

Alzheimer's Amyloid-β peptide (Aβ) is a 39-42 amino acid fragment of the amyloid precursor protein. Accumulation of the neurotoxic Aβ in the brains of Alzheimer's patients forms detrimental plaques. Another mechanism of neuronal damage is membrane destabilization by insertion of the peptide into brain cell membranes and disruption of ionic homeostasis. The molecular mechanism of membrane permeabilization by Aβ remains largely uncharacterized. Experiments with live cells and lipid membranes have identified ion channels formed by Aβ. The channel formed by an 11 amino acid residue fragment, Aβ25-35, has been modeled by Molecular Dynamics simulations as an 8-stranded β-barrel, but this structure has not been supported by direct experimental data. The objective of this work was to study the mechanism of membrane permeabilization by Aβ using biophysical techniques. Unilamellar lipid vesicles were prepared in a buffer containing 30 mM CaCl2, followed by removal of calcium from the external medium via a desalting column and addition of 0.1 mM Quin-2, a fluorescent calcium indicator. Before peptide addition, the vesicle sample showed little fluorescence because of spatial sequestration of calcium and Quin-2. Addition of submicromolar concentration of the Aβ25-35 peptide resulted in gradual increase in Quin-2 fluorescence, indicating formation of Ca2+-permeable pores in vesicle membranes. Addition of higher concentrations of the peptide resulted in instantaneous increase in fluorescence, comparable to the effect of detergents like Triton X-100, indicating rupture of vesicle membranes. Thus, the data suggest membrane pore formation at low peptide concentrations and membrane disruption at higher concentrations. These data are consistent with a mechanism of detergent-like action of the Aβ peptide; at concentrations below the aggregation threshold (similar to critical micelle concentration) it forms membrane pores, and at higher concentration it acts like a detergent and solubilizes the membranes of surrounding cells.

Concentration Dependent Transition of Membrane-Bound Beta-Stranded KIGAKI Peptides from Unstructured Monomers into Immobilized Amyloid Fibrils Observed by Solid-State 19F-NMR

The structure, membrane alignment, flexibility, and aggregation behavior of the β-stranded antimicrobial peptide KIGAKI [with sequence (KIGAKI)3-NH2] has been determined in oriented lipid bilayers using circular dichroism (CD), oriented CD (OCD), and solid state NMR spectroscopy. Several IIe or Ala residues were replaced one at a time with CF3-L/D-Bpg or Ala-d3. At high peptide-to-lipid molar ratios (P/L) of 1:200 or above, the 19F dipolar coupling of all labeled positions exhibits the maximum possible value of +16 kHz, indicating that at high concentration KIGAKI self-assembles into immobilized β-sheets, which lie flat on the membrane surface, which is also supported by CD and OCD spectra. Transmission electron microscopy images reveal that the aggregated KIGAKI forms amyloid-like fibrils, with less propensity for the CF3-D-Bpg labeled peptides to aggregate, compared with the CF3-L-Bpg labeled peptides. This aggregation difference is also reflected in the biological activity of the differently labeled peptides. At low peptide concentrations, on the other hand, all labeled positions show dipolar couplings of +8 kHz, indicating that their time-averaged alignment is still parallel to the bilayer normal, but the mobility of the peptides increases drastically, with monomeric peptides being the most likely state. Thus, flexible β-strands float on the membrane surface and undergo motional averaging in the membrane plane, similar to intrinsically unstructured proteins in solution. This is the first example of a concentration dependent transition of a flexible β-strand to an amyloid-like fibril on membrane surfaces that has been directly observed by solid state NMR.

Stereochemical effects on the aggregation and biological properties of the fibril-forming peptide [KIGAKI]3 in membranes

Single D-amino acid substitutions can be used to suppress or slow down the aggregation of peptides into β-sheeted assemblies compared to the respective L-amino acids. Here, we investigate the influence of local stereochemistry in the model peptide [KIGAKI]3-NH2, which is known to form amyloid-like fibrils. To find out whether aggregation plays a role in various biologically relevant functions that involve peptide-lipid interactions, we studied the antimicrobial, hemolytic and fusogenic activities of this amphiphilic membrane-active molecule. The stiff and sterically constrained amino acid CF3-Bpg [3-(trifluoromethyl)-bicyclopent-[1,1,1]-1-ylglycine] was incorporated either as an L- or a D-enantiomer at different hydrophobic positions of the KIGAKI sequence. D-Epimers have a higher aggregation threshold than the L-epimers, yet the aggregation of both was confirmed using electron microscopy and circular dichroism. Solid-state (19)F-NMR analysis showed that the peptide aggregated in native membranes from human erythrocytes and bacterial protoplasts in the same way as in synthetic lipid bilayers. We then monitored the effect of the single L- or D-CF3-Bpg substitutions in KIGAKI on its distinct biological activities, which have to be measured at low peptide concentrations where the aggregation threshold cannot be directly assessed. These functional assays showed that the aggregation propensity of KIGAKI does not play a role in its antimicrobial action, but an increased tendency to aggregate promotes other undesirable effects such as hemolysis and membrane fusion. These results confirm the membranolytic and thereby toxic nature of amyloidogenic peptides, and emphasize the unpredictable role of peptide aggregation in the different assays used to study biological activities.

Dynamic Interaction of the Escherichia coli Cell Division ZipA and FtsZ Proteins Evidenced in Nanodiscs

The full-length ZipA protein from Escherichia coli, one of the essential components of the division proto-ring that provides membrane tethering to the septation FtsZ protein, has been incorporated in single copy into nanodiscs formed by a membrane scaffold protein encircling an E. coli phospholipid mixture. This is an acellular system that reproduces the assembly of part of the cell division components. ZipA contained in nanodiscs (Nd-ZipA) retains the ability to interact with FtsZ oligomers and with FtsZ polymers. Interactions with FtsZ occur at similar strengths as those involved in the binding of the soluble form of ZipA, lacking the transmembrane region, suggesting that the transmembrane region of ZipA has little influence on the formation of the ZipA·FtsZ complex. Peptides containing partial sequences of the C terminus of FtsZ compete with FtsZ polymers for binding to Nd-ZipA. The affinity of Nd-ZipA for the FtsZ polymer formed with GTP or GMPCPP (a slowly hydrolyzable analog of GTP) is moderate (micromolar range) and of similar magnitude as for FtsZ-GDP oligomers. Polymerization does not stabilize the binding of FtsZ to ZipA. This supports the role of ZipA as a passive anchoring device for the proto-ring with little implication, if any, in the regulation of its assembly. Furthermore, it indicates that the tethering of FtsZ to the membrane shows sufficient plasticity to allow for its release from noncentral regions of the cytoplasmic membrane and its subsequent relocation to midcell when demanded by the assembly of a division ring.

Probing the Self-Assembly Mechanism of Diphenylalanine-Based Peptide Nanovesicles and Nanotubes

Nanostructures, particularly those from peptide self-assemblies, have attracted great attention lately due to their potential applications in nanotemplating and nanotechnology. Recent experimental studies reported that diphenylalanine-based peptides can self-assemble into highly ordered nanostructures such as nanovesicles and nanotubes. However, the molecular mechanism of the self-organization of such well-defined nanoarchitectures remains elusive. In this study, we investigate the assembly pathway of 600 diphenylalanine (FF) peptides at different peptide concentrations by performing extensive coarse-grained molecular dynamics (MD) simulations. Based on forty 0.6-1.8 μs trajectories at 310 K starting from random configurations, we find that FF dipeptides not only spontaneously assemble into spherical vesicles and nanotubes, consistent with previous experiments, but also form new ordered nanoarchitectures, namely, planar bilayers and a rich variety of other shapes of vesicle-like structures including toroid, ellipsoid, discoid, and pot-shaped vesicles. The assembly pathways are concentration-dependent. At low peptide concentrations, the self-assembly involves the fusion of small vesicles and bilayers, whereas at high concentrations, it occurs through the formation of a bilayer first, followed by the bending and closure of the bilayer. Energetic analysis suggests that the formation of different nanostructures is a result of the delicate balance between peptide-peptide and peptide-water interactions. Our all-atom MD simulation shows that FF nanostructures are stabilized by a combination of T-shaped aromatic stacking, interpeptide head-to-tail hydrogen-bonding, and peptide-water hydrogen-bonding interactions. This study provides, for the first time to our knowledge, the self-assembly mechanism and the molecular organization of FF dipeptide nanostructures.