Human Diacylglycerol Kinase ε N-Terminal Segment Regulates the Phosphatidylinositol Cycle, Controlling the Rate but Not the Acyl Chain Composition of Its Lipid Intermediates.

Diacylglycerol kinase ε (DGKε), an enzyme of the phosphatidylinositol (PI) cycle, bears a highly conserved hydrophobic N-terminal segment, which was proposed to anchor the enzyme into the membrane. However, the importance of this segment to the DGKε function remains to be determined. To address this question, it is here reported an in silico and in vitro combined research strategy. Capitalizing on the AlphaFold 2.0 predicted structure of human DGKε, it is shown that its hydrophobic N-terminal segment anchors it into the membrane via a transmembrane α-helix. Coarse-grained based elastic network model studies showed that a conformational change in the hydrophobic N-terminal segment determines the proximity between the active site of DGKε and the membrane-water interface, likely regulating its kinase activity. In vitro studies with a purified DGKε construct lacking the hydrophobic N-terminal segment (His-SUMO*-Δ50-DGKε) corroborated the role of the N-terminus in regulating DGKε enzymatic properties. The comparison between the enzymatic properties of DGKε and His-SUMO*-Δ50-DGKε showed that the conserved N-terminal segment markedly inhibits the enzyme activity and its sensitivity to membrane intrinsic negative curvature, while also playing a role in the modulation of the enzyme by phosphatidylserine. On the other hand, this segment did not strongly affect its diacylglycerol acyl chain specificity, the modulation of the enzyme by membrane morphological changes, or the activation by phosphatidic acid-rich lipid domains. Hence, these results suggest that the conservation of the hydrophobic N-terminal segment of DGKε throughout evolution guaranteed not only membrane anchorage but also an efficient and elegant manner to regulate the rate of the PI cycle.

Rondonin: antimicrobial properties and mechanism of action.

Infectious diseases are among the major causes of death in the human population. A wide variety of organisms produce antimicrobial peptides (AMPs) as part of their first line of defense. A peptide from Acanthoscurria rondoniae plasma, rondonin-with antifungal activity, a molecular mass of 1236 Da and primary sequence IIIQYEGHKH-was previously studied (UniProt accession number B3EWP8). It showed identity with the C terminus of subunit 'D' of the hemocyanin of the Aphonopelma hentzi spider. This result led us to propose a new pathway of the immune system of arachnids that suggests a new function to hemocyanin: production of antimicrobial peptides. Rondonin does not interact with model membranes and was able to bind to yeast nucleic acids but not bacteria. It was not cytotoxic against mammalian cells. The antifungal activity of rondonin is pH-dependent and peaks at pH ˜ 4-5. The peptide presents synergism with gomesin (spider hemocyte antimicrobial peptide-UniProtKB-P82358) against human yeast pathogens, suggesting a new potential alternative treatment option. Antiviral activity was detected against RNA viruses, measles, H1N1, and encephalomyocarditis. This is the first report of an arthropod hemocyanin fragment with activity against human viruses. Currently, it is vital to invest in the search for natural and synthetic antimicrobial compounds that, above all, present alternative mechanisms of action to first-choice antimicrobials.

Rondonin: antimicrobial properties and mechanism of action

Infectious diseases are among the major causes of death in the human population. A wide variety of organisms produce antimicrobial peptides (AMPs) as part of their first line of defense. A peptide from Acanthoscurria rondoniae plasma, rondonin – with antifungal activity, a molecular mass of 1,236 Da and primary sequence IIIQYEGHKH – was previously studied (Uniprot accession number B3EWP8). It showed identity with the C-terminus of subunit "D" of the hemocyanin of the Aphonopelma hentzi spider. This result led us to propose a new pathway of the immune system of arachnids that suggests a new function to hemocyanin: production of antimicrobial peptides. Rondonin does not interact with model membranes and was able to bind to yeast nucleic acids but not bacteria. It was not cytotoxic against mammalian cells. The antifungal activity of rondonin is pH dependent and peaks at pH ~4-5. The peptide presents synergism with Gomesin (spider hemocyte antimicrobial peptide - UniProtKB - P82358) against human yeast pathogens, suggesting a new potential alternative treatment option. Antiviral activity was detected against RNA viruses, measles, H1N1 and encephalomyocarditis. This is the first report of an arthropod hemocyanin fragment with activity against human viruses. Currently, it is vital to invest in the search for natural and synthetic antimicrobial compounds that, above all, present alternative mechanisms of action to first-choice antimicrobials.

Lipid asymmetry of a model mitochondrial outer membrane affects Bax-dependent permeabilization.

The presence of an asymmetric distribution of lipids in biological membranes was first described ca. 50 years ago. While various studies had reported the role of loss of lipid asymmetry on signaling processes, its effect on membrane physical properties and membrane-protein interactions lacks further understanding. The recent description of new technologies for the preparation of asymmetric model membranes has helped to fill part of this gap. However, the major effort so far has been on plasma membrane models. Here we describe the preparation of liposomes mimicking the mitochondria outer membrane (MOM) in regard to its lipid composition and asymmetry. By employing the methyl-ß-cyclodextrin-catalyzed lipid exchange technology and accurate quantification of lipid asymmetry with head group-specific probes we showed the successful preparation of a MOM model bearing a physiologically relevant lipid composition and asymmetry. In addition, by a direct comparison with its lipid symmetrical counterpart it is shown that asymmetric models were more resistant to tBid-promoted Bax-permeabilization, suggesting a role played by MOM lipid asymmetry on the mitochondria pathway of apoptosis. The barrier imposed by lipid asymmetry on membrane permeabilization was in part due to a decrease in the concentration of membrane-bound proteins, which was likely a consequence of the two mutually-dependent properties; i.e., the lower electrostatic surface potential and the higher molecular packing imposed by lipid asymmetry. It is proposed that MOM lipid asymmetry imparts different physical properties on the membrane and might add an additional component of regulation in intricate mitochondrial processes.

A comparison of activity, toxicity, and conformation of tritrpticin and two TOAC-labeled analogues. Effects on the mechanism of action.

A strategy that has been gaining increased application for the study of the conformation, dynamics, orientation, and physicochemical properties of peptides is labeling with the paramagnetic amino acid TOAC. This approach was used to gain a deeper understanding on the mechanism of action of the antimicrobial peptide tritrpticin (TRP3). TRP3 was labeled with TOAC at the N-terminus (prior to V1, TOAC0-TRP3) or internally (replacing P5, TOAC5-TRP3). Functional studies showed that labeling led to peptides with higher activity against Gram-positive bacteria and lower hemolytic activity with respect to TRP3. Peptide-induced model membranes permeabilization and ion channel-like activity studies corroborated the functional assays qualitatively, showing higher activity of the peptides against negatively charged membranes, which had the purpose of mimicking bacterial membranes. TOAC presented a greater freedom of motion at the N-terminus than at the internal position, as evinced by EPR spectra. EPR and fluorescence spectra reported on the peptides conformational properties, showing acquisition of a more packed conformation in the presence of the secondary structure-inducing solvent, TFE. CD studies showed that TOAC0-TRP3 acquires a conformation similar to that of TRP3, both in aqueous solution and in TFE, while TOAC5-TRP3 presents a different conformation in all environments. While the mechanism of action of TRP3 was impacted to some extent by TOAC labeling at the N-terminus, it did change upon replacement of P5 by TOAC. The results demonstrated that TOAC-labeling could be used to modulate TRP3 activity and mechanism of action and, more importantly, the critical role of P5 for TRP3 pore formation.

A comparison of activity, toxicity, and conformation of tritrpticin and two TOAC-labeled analogues. Effects on the mechanism of action

A strategy that has been gaining increased application for the study of the conformation, dynamics, orientation, and physicochemical properties of peptides is labeling with the paramagnetic amino acid TOAC. This approach was used to gain a deeper understanding on the mechanism of action of the antimicrobial peptide tritrpticin (TRP3). TRP3 was labeled with TOAC at the N-terminus (prior to V1, TOAC0-TRP3) or internally (replacing P5, TOAC5-TRP3). Functional studies showed that labeling led to peptides with higher activity against Gram-positive bacteria and lower hemolytic activity with respect to TRP3. Peptide-induced model membranes permeabilization and ion channel-like activity studies corroborated the functional assays qualitatively, showing higher activity of the peptides against negatively charged membranes, which had the purpose of mimicking bacterial membranes. TOAC presented a greater freedom of motion at the N-terminus than at the internal position, as evinced by EPR spectra. EPR and fluorescence spectra reported on the peptides conformational properties, showing acquisition of a more packed conformation in the presence of the secondary structure-inducing solvent, TFE. CD studies showed that TOAC0-TRP3 acquires a conformation similar to that of TRP3, both in aqueous solution and in TFE, while TOAC5-TRP3 presents a different conformation in all environments. While the mechanism of action of TRP3 was impacted to some extent by TOAC labeling at the N-terminus, it did change upon replacement of P5 by TOAC. The results demonstrated that TOAC-labeling could be used to modulate TRP3 activity and mechanism of action and, more importantly, the critical role of P5 for TRP3 pore formation.

A comparison of activity, toxicity, and conformation of tritrpticin and two TOAC-labeled analogues. Effects on the mechanism of action

A strategy that has been gaining increased application for the study of the conformation, dynamics, orientation, and physicochemical properties of peptides is labeling with the paramagnetic amino acid TOAC. This approach was used to gain a deeper understanding on the mechanism of action of the antimicrobial peptide tritrpticin (TRP3). TRP3 was labeled with TOAC at the N-terminus (prior to V1, TOAC0-TRP3) or internally (replacing P5, TOAC5-TRP3). Functional studies showed that labeling led to peptides with higher activity against Gram-positive bacteria and lower hemolytic activity with respect to TRP3. Peptide-induced model membranes permeabilization and ion channel-like activity studies corroborated the functional assays qualitatively, showing higher activity of the peptides against negatively charged membranes, which had the purpose of mimicking bacterial membranes. TOAC presented a greater freedom of motion at the N-terminus than at the internal position, as evinced by EPR spectra. EPR and fluorescence spectra reported on the peptides conformational properties, showing acquisition of a more packed conformation in the presence of the secondary structure-inducing solvent, TFE. CD studies showed that TOAC0-TRP3 acquires a conformation similar to that of TRP3, both in aqueous solution and in TFE, while TOAC5-TRP3 presents a different conformation in all environments. While the mechanism of action of TRP3 was impacted to some extent by TOAC labeling at the N-terminus, it did change upon replacement of P5 by TOAC. The results demonstrated that TOAC-labeling could be used to modulate TRP3 activity and mechanism of action and, more importantly, the critical role of P5 for TRP3 pore formation.

Characterization of phospholipid vesicles containing lauric acid: physicochemical basis for process and product development.

Lauric acid (LAH) strongly inhibits the growth of acne-causing bacteria. LAH is essentially water-insoluble and the solubility of laurate (LA) salts are medium and temperature dependent. Hence, LAH/LA preparations are difficult to formulate. Here we fully characterized phospholipid vesicles containing up to 50 mol% LAH. Vesicles of dipalmitoylphosphatidylcholine (DPPC) containing LAH, at pHs 7.4 and 5.0, were characterized measuring size, charge, bilayer phase transition temperature (Tm) and permeability of water-soluble probes. Small angle X-ray scattering and cryotransmission electron microscopy showed multilamellar vesicles at low LAH %. Increasing LAH % had a negligible effect on particle size. An internal aqueous compartment in all vesicle's preparations, even at equimolar DPPC: LAH fractions, was demonstrated using water-soluble probes. At pH 5.0, the interaction between DPPC and LAH increased the Tm and phase transition cooperativity showing a single lipid phase formed by hydrogen-bonded DPPC: LAH complexes. At pH 7.4, vesicles containing 50 mol% LAH exhibited distinct phases, ascribed to complex formation between LAH and LA or LAH and DPPC. LAH incorporated in the vesicles minimally permeated a skin preparation at both pHs, indicating that the primary sites of LAH solubilization were the skin layers. These results provide the foundations for developing processes and products containing DPPC: LAH.

Fragments of the second transmembrane helix of three G-protein-coupled receptors: comparative synthetic, structural and conformational studies.

We compared the synthesis and structural/conformational details of the (66-97) segments of the second transmembrane helix of AT1, MAS and B2, all of which belong to the class of G-protein-coupled receptors (GPCR). Step-by-step monitoring of the coupling reactions during the growth of these transmembrane peptides revealed that the increase in the level of difficulty started at the 6-10 regions of the sequence. Possibly due to their long and hydrophobic sequences, the final estimated synthesis yields decreased progressively by up to 20-25%. Analytical high pressure liquid chromatography showed that the hydrophobicity indexes of each TM-8, -16, -24 and -32 segments correlated linearly with their retention time. Microscopic measurements of peptide-resin beads indicated that, in general, dichloromethane and dimethylsulfoxide were the best solvents for solvating resin beads in the initial and final stages of the synthesis, respectively. Results from electron paramagnetic resonance experiments with Toac (2, 2, 6, 6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) spin-labeled peptide resins revealed that the level of peptide chain mobility throughout the polymer network was in agreement with their swelling data measured in different solvents. Initial results regarding conformational features determined by circular dichroism (CD) spectra revealed typical α-helicoidally structures for MAS and B2 TM32 fragments when in more than roughly 30% (v/v) trifluoroethanol (TFE). In contrast, the AT1-TM32 segment revealed CD spectra, more representatives of a mixture of other secondary helical conformers, regardless of the amount of TFE. These findings observed in different aspects of these receptors' fragments support further investigations of GPCR-type macromolecules.

Self-association and folding in membrane determine the mode of action of peptides from the lytic segment of sticholysins.

Sticholysin I and II (Sts: St I and St II) are proteins of biomedical interest that form pores upon the insertion of their N-terminus in the plasma membrane. Peptides spanning the N-terminal residues of StI (StI1-31) or StII (StII1-30) can mimic the permeabilizing ability of these toxins, emerging as candidates to rationalize their potential biomedical applications. These peptides have different activities that correlate with their hydrophobicity. However, it is not clear how this property contributes to peptide folding in solution or upon binding to membranes. Here we compared the conformational properties of these peptides and shorter versions lacking the hydrophobic segment 1-11 of StI (StI12-31) or 1-10 of StII (StII11-30). Folding of peptides was assessed in solution and in membrane mimetic systems and related with their ability to bind to membranes and to permeabilize lipid vesicles. Our results suggest that the differences in activity among peptides could be ascribed to their different folding propensity and different membrane binding properties. In solution, StII1-30 tends to acquire α-helical conformation coexisting with self-associated structures, while StI1-31 remains structureless. Both peptides fold as α-helix in membrane; but StII1-30 also self-associates in the lipid environment, a process that is favored by its higher affinity for membrane. We stress the contribution of the non-polar/polar balance of the 1-10 amino acid sequence of the peptides as a determining factor for different self-association capabilities. Such difference in hydrophobicity seems to determine the molecular path of peptides folding upon binding to membranes, with an impact in their permeabilizing activity. This study contributes to a better understanding of the molecular mechanisms underlying the permeabilizing activity of Sts N-terminal derived peptides, with connotation for the exploitation of these small molecules as alternative of the full-length toxins in clinical settings.

Dissecting the mechanism of action of actinoporins. Role of the N-terminal amphipathic α-helix in membrane binding and pore activity of sticholysins I and II.

Actinoporins sticholysin I and sticholysin II (St I, St II) are proposed to lyse model and biomembranes via toroidal pore formation by their N-terminal domain. Based on the hypothesis that peptide fragments can reproduce the structure and function of this domain, the behavior of peptides containing St I residues 12-31 (StI12-31), St II residues 11-30 (StII11-30), and its TOAC-labeled analogue (N-TOAC-StII11-30) was examined. Molecular modeling showed a good match with experimental structures, indicating amphipathic α-helices in the same regions as in the toxins. CD spectra revealed that the peptides were essentially unstructured in aqueous solution, acquiring α-helical conformation upon interaction with micelles and large unilamellar vesicles (LUV) of variable lipid composition. Fluorescence quenching studies with NBD-containing lipids indicated that N-TOAC-StII11-30's nitroxide moiety is located in the membranes polar head group region. Pyrene-labeled phospholipid inter-leaflet redistribution suggested that the peptides form toroidal pores, according to the mechanism of action proposed for the toxins. Binding occurred only to negatively charged LUV, indicating the importance of electrostatic interactions; in contrast the peptides bound to both negatively charged and zwitterionic micelles, pointing to a lesser influence of these interactions. In addition, differences between bilayers and micelles in head group packing and in curvature led to differences in peptide-membrane interaction. We propose that the peptides topography in micelles resembles that of the toxins in the toroidal pore. The peptides mimicked the toxins permeabilizing activity, St II peptides being more effective than StI12-31. To our knowledge, this is the first demonstration that differences in the toxins N-terminal amphipathic α-helix play a role in the difference between St I and St II activities.

Synthesis, biophysical and functional studies of two BP100 analogues modified by a hydrophobic chain and a cyclic peptide.

Antimicrobial peptides (AMPs) work as a primary defense against pathogenic microorganisms. BP100, (KKLFKKILKYL-NH2), a rationally designed short, highly cationic AMP, acts against many bacteria, displaying low toxicity to eukaryotic cells. Previously we found that its mechanism of action depends on membrane surface charge and on peptide-to-lipid ratio. Here we present the synthesis of two BP100 analogs: BP100­alanyl­hexadecyl­1­amine (BP100-Ala-NH-C16H33) and cyclo(1­4)­d­Cys1, Ile2, Leu3, Cys4-BP100 (Cyclo(1­4)­cILC-BP100). We examined their binding to large unilamellar vesicles (LUV), conformational and functional properties, and compared with those of BP100. The analogs bound to membranes with higher affinity and a lesser dependence on electrostatic forces than BP100. In the presence of LUV, BP100 and BP100-Ala-NH-C16H33 acquired α-helical conformation, while Cyclo(1­4)­cILC-BP100) was partly α-helical and partly ß-turn. Taking in conjunction: 1. particle sizes and zeta potential, 2. effects on lipid flip-flop, 3. leakage of LUVs internal contents, and 4. optical microscopy of giant unilamellar vesicles, we concluded that at high concentrations, all three peptides acted by a carpet mechanism, while at low concentrations the peptides acted by disorganizing the lipid bilayer, probably causing membrane thinning. The higher activity and lesser membrane surface charge dependence of the analogs was probably due to their greater hydrophobicity. The MIC values of both analogs towards Gram-positive and Gram-negative bacteria were similar to those of BP100 but both analogues were more hemolytic. Confocal microscopy showed Gram-positive B. subtilis killing with concomitant extensive membrane damage suggestive of lipid clustering, or peptide-lipid aggregation. These results were in agreement with those found in model membranes.

Acemetacin-phosphatidylcholine interactions are determined by the drug ionization state.

Gastrointestinal (GI) toxicity is a major drawback of the chronic use of nonsteroidal anti-inflammatory drugs (NSAIDs). The NSAIDs topical actions on the protective phospholipid layers of the GI mucosa seem to be a central toxicity mechanism of these pharmaceuticals. This work describes the interactions of acemetacin, a commercialized NSAID, with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers at pH 3.0, 5.0, and 7.4. This pH range was chosen to mimic the pH gradient found in the gastric mucosa, and to ultimately gain insights into the mechanisms underlying the acemetacin-induced gastric toxicity. Various experimental techniques were combined to characterize the partitioning of acemetacin in DMPC bilayers, and its effects on the phase transition behavior, as well as the structure and dynamics of DMPC bilayers. The acemetacin-DMPC interactions were clearly pH-dependent. The neutral (protonated) form of acemetacin had more affinity for the DMPC bilayer than the negatively charged form. Due to the higher affinity of neutral acemetacin, the drug effects on the phase transition and the structure and dynamics of the DMPC bilayer were more pronounced at lower pH values. In general, acemetacin decreased the temperature and the cooperativity of the lipid phase transition and induced changes in the packing and dynamics of the DMPC bilayer. These results support the hypothesis that acemetacin-induced gastric toxicity may be related to its effects on the protective phospholipid layers of the mucosal barrier.

Can NO-indomethacin counteract the topical gastric toxicity induced by indomethacin interactions with phospholipid bilayers?

Nitric oxide (NO)-releasing nonsteroidal anti-inflammatory drugs (NSAIDs) have been developed to overcome the gastrointestinal and cardiovascular toxicity of NSAIDs, by chemically associating a NO-releasing moiety with commercial NSAIDs. Since increasing evidence supports that NSAIDs toxicity is related to their topical actions in membrane lipids, this work aims to evaluate the impact of adding a NO-releasing moiety to parent NSAIDs regarding their effect on lipid bilayers. Thus, the interactions of NO-indomethacin and indomethacin (parent drug) with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers were described herein at pH 3.0 and 7.4. Diverse experimental techniques were combined to characterize the partitioning and location of drugs in DMPC bilayers, and to analyze their effect on the lipid phase transition and the bilayer structure and dynamics. The partitioning of NO-indomethacin into DMPC bilayers was similar to that of charged indomethacin and smaller than that of neutral indomethacin. Both drugs were found to insert the DMPC bilayer and the membrane location of indomethacin was pH-dependent. NO-indomethacin and indomethacin induced a decrease of the main phase transition temperature of DMPC. The effect of these drugs on the bilayer structure and dynamics was dependent on diverse factors, namely drug ionization state, drug:lipid molar ratio, temperature and lipid phase. It is noteworthy that NO-indomethacin induced more pronounced alterations in the biophysical properties of DMPC bilayers than indomethacin, considering equivalent membrane concentrations. Such modifications may have in vivo implications, particularly in the gastric mucosa, where NO-NSAIDs-induced changes in the protective properties of phospholipid layers may contribute to the occurrence of adverse effects.

Insights on the structure-activity relationship of peptides derived from Sticholysin II.

Sticholysin II (StII) is a pore-forming actinoporin from the sea anemone Stichodactyla helianthus. A mechanistic model of its action has been proposed: proteins bind to cell membrane, insert their N-termini into the lipid core and assemble into homo-tetramer pores responsible for host-cell death. Because very likely the first 10 residues of StII N-terminus are critical for membrane penetration, to dissect the molecular details of that functionality, we studied two synthetic peptides: StII1-30 and StII16-35 . They show diverse haemolytic and candidacidal activity that correlate with distinct orientations in SDS micelles. NMR shows that StII1-30 partly inserts into the micelle, while StII16-35 lays on the micelle surface. These results justify the diverse concentration dependence of their candidacidal activity supposing a different mechanism of action and providing new hints on StII lytic activity at molecular level. Biotechnological application of these peptides, focused on the development of therapeutic immunocomplexes, may be envisaged.

Structural and Dynamic Insights of the Interaction between Tritrpticin and Micelles: An NMR Study.

A large number of antimicrobial peptides (AMPs) acts with high selectivity and specificity through interactions with membrane lipid components. These peptides undergo complex conformational changes in solution; upon binding to an interface, one major conformation is stabilized. Here we describe a study of the interaction between tritrpticin (TRP3), a cathelicidin AMP, and micelles of different chemical composition. The peptide's structure and dynamics were examined using one-dimensional and two-dimensional NMR. Our data showed that the interaction occurred by conformational selection and the peptide acquired similar structures in all systems studied, despite differences in detergent headgroup charge or dipole orientation. Fluorescence and paramagnetic relaxation enhancement experiments showed that the peptide is located in the interface region and is slightly more deeply inserted in 1-myristoyl-2-hydroxy-sn-glycero-3-phospho-1'-rac-glycerol (LMPG, anionic) than in 1-lauroyl-2-hydroxy-sn-glycero-3-phosphocholine (LLPC, zwitterionic) micelles. Moreover, the tilt angle of an assumed helical portion of the peptide is similar in both systems. In previous work we proposed that TRP3 acts by a toroidal pore mechanism. In view of the high hydrophobic core exposure, hydration, and curvature presented by micelles, the conformation of TRP3 in these systems could be related to the peptide's conformation in the toroidal pore.

Paramagnetic bradykinin analogues as substrates for angiotensin I-converting enzyme: Pharmacological and conformation studies.

This study uses EPR, CD, and fluorescence spectroscopy to examine the structure of bradykinin (BK) analogues attaching the paramagnetic amino acid-type Toac (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) at positions 0, 3, 7, and 9. The data were correlated with the potencies in muscle contractile experiments and the substrate properties towards the angiotensin I-converting enzyme (ACE). A study of the biological activities in guinea pig ileum and rat uterus indicated that only Toac0-BK partially maintained its native biological potency among the tested peptides. This and its counterpart, Toac3-BK, maintained the ability to act as ACE substrates. These results indicate that peptides bearing Toac probe far from the ACE cleavage sites were more susceptible to hydrolysis by ACE. The results also emphasize the existence of a finer control for BK-receptor interaction than for BK binding at the catalytic site of this metallodipetidase. The kinetic kcat/Km values decreased from 202.7 to 38.9µM-1min-1 for BK and Toac3-BK, respectively. EPR, CD, and fluorescence experiments reveal a direct relationship between the structure and activity of these paramagnetic peptides. In contrast to the turn-folded structures of the Toac-internally labeled peptides, more extended conformations were displayed by N- or C-terminally Toac-labeled analogues. Lastly, this work supports the feasibility of monitoring the progress of the ACE-hydrolytic process of Toac-attached peptides by examining time-dependent EPR spectral variations.

Adsorption of the antimicrobial peptide tritrpticin onto solid and liquid surfaces: Ion-specific effects.

Developing functional biointerfaces is important for technological applications. We investigated the interaction and adsorption of the antimicrobial peptide tritrpticin (VRRFPWWWPFLRR, TRP3) onto solid and liquid surfaces and the influence of ions on these processes by several techniques. Surface tension measurements showed that salt addition to TRP3 solution causes a high decrease of surface tension due to the adsorption of TRP3 at air-liquid surface. Ellipsometry studies show the TRP3 adsorption on silicon surfaces forming nanometric films that are able to further interact with liposomes. Contact angle measurements gave insight on the nature of thin film and its roughness. AFM shows the topology of the film on the solid substrates. In addition, those techniques also showed that anions can act as modulators on adsorption phenomena and are correlated with the Hofmeister series. The findings of the current work are relevant for the development of functional interfaces such as biocidal surfaces.

Unfolding and folding pathway of lysozyme induced by sodium dodecyl sulfate.

Proteins may exhibit an unfolding or folding state in the presence of a surfactant. In the present study, the unfolding and folding pathway of hen egg white lysozyme (HEWL) induced by sodium dodecyl sulfate (SDS) is studied. The stoichiometry obtained from isothermal titration calorimetry (ITC) provides guidelines for other techniques. The fluorescence spectra and circular dichroism show that the fluorescence properties and secondary structure of proteins undergo a two-step change upon binding with SDS, in which the intensity decreases, the emission blue shifts and the helical conformation decreases at low ratios of SDS to HEWL, while all of them return to the native-like state upon the addition of SDS at higher ratios. At the end of the binding, HEWL presents a higher α-helical content but its tertiary structure is lost compared to its native state, which is namely a molten globule state. Small angle X-ray scattering (SAXS) analysis and the derived model reveal that the complexes possess a decorated core-shell structure, with the core composed of dodecyl chains and the shell consisting of SDS head groups with a protein in molten globule state. Five binding steps, including the individual details involved in the denaturation, were obtained to describe the unfolding and folding pathway of HEWL induced by SDS. The results of this study not only present details about the denaturation of protein induced by SDS and the structure of the complexes involved in each binding step, but also provide molecular insights into the mechanism of the higher helical conformation of proteins in the presence of surfactant micelles.