Luis Guilherme Mansor Basso

Effects of the antimalarial drug primaquine on the dynamic structure of lipid model membranes.

Primaquine (PQ) is a potent therapeutic agent used in the treatment of malaria and its mechanism of action still lacks a more detailed understanding at a molecular level. In this context, we used differential scanning calorimetry (DSC), pressure perturbation calorimetry (PPC), and electron spin resonance (ESR) to investigate the effects of PQ on the lipid phase transition, acyl chain dynamics, and on volumetric properties of lipid model membranes. DSC thermograms revealed that PQ stabilizes the fluid phase of the lipid model membranes and interacts mainly with the lipid headgroups. This result was revealed by the great effect on the pretransition of phosphatidylcholines and the destabilization of the inverted hexagonal phase of a phosphatidylethanolamine bilayer. Spin probes located at different positions along the lipid chain were used to monitor different membrane regions. ESR results indicated that PQ is effective in changing the acyl chain ordering and dynamics of the whole chain of dimyristoylphosphatidylcholine (DMPC) phospholipid in the rippled gel phase. The combined ESR and PPC results revealed that the slight DMPC volume changes at the main phase transition induced by the presence of PQ is probably due to a less dense lipid gel phase. At physiological pH, the cationic amphiphilic PQ strongly interacts with the lipid headgroup region of the bilayers, causing considerable disorganization in the hydrophobic core. These results shed light on the molecular mechanism of primaquine-lipid interaction, which may be useful in the understanding of the complex mechanism of action and/or the adverse effects of this antimalarial drug.

Dynamics and Conformational Studies of TOAC Spin Labeled Analogues of Ctx(Ile21)-Ha Peptide from Hypsiboas albopunctatus

Antimicrobial peptides (AMPs) isolated from several organisms have been receiving much attention due to some specific features that allow them to interact with, bind to, and disrupt cell membranes. The aim of this paper was to study the interactions between a membrane mimetic and the cationic AMP Ctx(Ile21)-Ha as well as analogues containing the paramagnetic amino acid 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) incorporated at residue positions n = 0, 2, and 13. Circular dichroism studies showed that the peptides, except for [TOAC13]Ctx(Ile21)-Ha, are unstructured in aqueous solution but acquire different amounts of α-helical secondary structure in the presence of trifluorethanol and lysophosphocholine micelles. Fluorescence experiments indicated that all peptides were able to interact with LPC micelles. In addition, Ctx(Ile21)-Ha and [TOAC13]Ctx(Ile21)-Ha peptides presented similar water accessibility for the Trp residue located near the N-terminal sequence. Electron spin resonance experiments showed two spectral components for [TOAC0]Ctx(Ile21)-Ha, which are most likely due to two membrane-bound peptide conformations. In contrast, TOAC2 and TOAC13 derivatives presented a single spectral component corresponding to a strong immobilization of the probe. Thus, our findings allowed the description of the peptide topology in the membrane mimetic, where the N-terminal region is in dynamic equilibrium between an ordered, membrane-bound conformation and a disordered, mobile conformation; position 2 is most likely situated in the lipid polar head group region, and residue 13 is fully inserted into the hydrophobic core of the membrane.

SARS-CoV fusion peptides induce membrane surface ordering and curvature

Viral membrane fusion is an orchestrated process triggered by membrane-anchored viral fusion glycoproteins. The S2 subunit of the spike glycoprotein from severe acute respiratory syndrome (SARS) coronavirus (CoV) contains internal domains called fusion peptides (FP) that play essential roles in virus entry. Although membrane fusion has been broadly studied, there are still major gaps in the molecular details of lipid rearrangements in the bilayer during fusion peptide-membrane interactions. Here we employed differential scanning calorimetry (DSC) and electron spin resonance (ESR) to gather information on the membrane fusion mechanism promoted by two putative SARS FPs. DSC data showed the peptides strongly perturb the structural integrity of anionic vesicles and support the hypothesis that the peptides generate opposing curvature stresses on phosphatidylethanolamine membranes. ESR showed that both FPs increase lipid packing and head group ordering as well as reduce the intramembrane water content for anionic membranes. Therefore, bending moment in the bilayer could be generated, promoting negative curvature. The significance of the ordering effect, membrane dehydration, changes in the curvature properties and the possible role of negatively charged phospholipids in helping to overcome the high kinetic barrier involved in the different stages of the SARS-CoV-mediated membrane fusion are discussed.

The two sides of a lipid-protein story

Protein–membrane interactions play essential roles in a variety of cell functions such as signaling, membrane trafficking, and transport. Membrane-recruited cytosolic proteins that interact transiently and interfacially with lipid bilayers perform several of those functions. Experimental techniques capable of probing changes on the structural dynamics of this weak association are surprisingly limited. Among such techniques, electron spin resonance (ESR) has the enormous advantage of providing valuable local information from both membrane and protein perspectives by using intrinsic paramagnetic probes in metalloproteins or by attaching nitroxide spin labels to proteins and lipids. In this review, we discuss the power of ESR to unravel relevant structural and functional details of lipid–peripheral membrane protein interactions with special emphasis on local changes of specific regions of the protein and/or the lipids. First, we show how ESR can be used to investigate the direct interaction between a protein and a particular lipid, illustrating the case of lipid binding into a hydrophobic pocket of chlorocatechol 1,2-dioxygenase, a non-heme iron enzyme responsible for catabolism of aromatic compounds that are industrially released in the environment. In the second case, we show the effects of GPI-anchored tissue-nonspecific alkaline phosphatase, a protein that plays a crucial role in skeletal mineralization, and on the ordering and dynamics of lipid acyl chains. Then, switching to the protein perspective, we analyze the interaction with model membranes of the brain fatty acid binding protein, the major actor in the reversible binding and transport of hydrophobic ligands such as long-chain, saturated, or unsaturated fatty acids. Finally, we conclude by discussing how both lipid and protein views can be associated to address a common question regarding the molecular mechanism by which dihydroorotate dehydrogenase, an essential enzyme for the de novo synthesis of pyrimidine nucleotides, and how it fishes out membrane-embedded quinones to perform its function.

HsDHODH Microdomain–Membrane Interactions Influenced by the Lipid Composition

Human dihydroorotate dehydrogenase (HsDHODH) enzyme has been studied as selective target for inhibitors to block the enzyme activity, intending to prevent proliferative diseases. The N-terminal microdomain seems to play an important role in the enzyme function. However, the molecular mechanism of action and dynamics of this region are not totally understood yet. This study analyzes the interaction and conformation in model membranes of HsDHODH microdomain using peptide analogues containing the paramagnetic amino acid TOAC at strategic positions. In buffer solution, the analogues presented a disordered conformation, but acquired a high content of α-helical structure in membrane mimetics, which was found to be lipid dependent. The microdomain peptide structure in micelles showed a very different peptide conformation when compared to the reported crystal structure, displaying a conformational flexibility of its helices, promoted by the connecting loop, which might be functionally relevant. Electron spin resonance in membrane compositions containing POPC, POPE, and cardiolipin showed that interaction of the analogues was enhanced by the presence of cardiolipin, indicating that the microdomain preferentially interacts with cardiolipin-containing membranes. Therefore, the great flexibility of the microdomain and the cardiolipin affinity should be considered in further studies aimed at finding new inhibitory compounds to fight proliferative diseases.

Biophysical characterization and antitumor activity of synthetic Pantinin peptides from scorpion's venom

Antimicrobial peptides have been extensively described as bioactive agents, mainly considering their selective toxicity towards bacteria but not to healthy mammalian cells. In past years, this class of compounds has been classified as an attractive and novel family of anticancer agents. Pantinin peptides isolated from scorpion Pandinus imperator presented antimicrobial activity. In this study, we have explored the in vitro antitumor activity of antimicrobial pantinin peptides against the tumor cell lines MDA-MB-231 (breast adenocarcinoma) and DU - 145 (prostate adenocarcinoma) and healthy fibroblasts HGF - 1. To further improve our mechanistic understanding for this class of compounds, we have also performed a biophysical characterization of these peptides in lipid model membranes. Cell viability assays revealed that all peptides were more effective on tumor cells when compared to fibroblasts, indicating selectivity towards cancer cells. Furthermore, flow cytometry analysis revealed that all peptides induced apoptosis in cancer cells in a different way from fibroblasts. Circular dichroism spectroscopy showed that all peptides adopted an α-helical structure and an evaluation of the binding constant indicates a higher affinity of the peptides to negatively charged phospholipids. Additionally, permeabilization assays showed that POPG and POPS anionic vesicles were more susceptible to peptide-induced lysis than POPC:Chol and POPC:POPE vesicles. Moreover, we have observed that increasing concentrations of cholesterol inhibits peptide binding process. Therefore, our findings suggest that Pantinin peptides may have chemotherapeutic potential for cancer treatment.

Ordering Effect Induced by SARS-CoV Fusion Peptides on Membranes Containing Negatively Charged Lipids Might be Important to Membrane Fusion

The S2 subunit of the spike glycoprotein from SARS coronavirus (CoV) contains internal membranotropic domains that play important roles to the viral and host cell membrane fusion. These functional domains, which include a so-called fusion peptide (FP) and an internal FP, are exposed to membrane interactions upon a specific trigger. Although membrane fusion has been broadly studied in recent years, many aspects of the molecular mechanism behind the virus-host cell membrane fusion remain unknown, including conformational changes of the lipid bilayers during peptide-membrane interactions. Here we employed spin-labeling Electron Spin Resonance (ESR), 31P oriented sample solid state (OSSS) Nuclear Magnetic Resonance (NMR), and Differential Scanning Calorimetry (DSC) to address these questions of fusion peptide - membrane interactions from the membrane perspective. DSC results showed that the peptides strongly perturb the thermotropic behavior of zwitterionic and negatively-charged lipid vesicles, with the largest effects seen for the latter. Not only the charge but also the lipid headgroup structure seems to be relevant for the energetics of the interaction. ESR showed that both peptides were capable of increasing lipid packing and head group ordering only in the presence of negatively charged lipids, which was further corroborated by NMR on aligned lipids deposited into nanoporous anodic aluminum oxide substrates. The observed effects are well correlated to those caused by well-known membrane fusion promoters and are in contrast to those promoted by membrane fusion inhibitors. The combined effect of an increased chain-packing energy, which induces bending moment in the bilayer, and membrane surface ordering, which is related to lipid dehydration, might promote negative curvature for negatively charged lipid-containing membranes, thus, helping to overcome the high kinetic barrier of the membrane fusion.

The Conformational Flexibility of an Internal Fusion Peptide from Sars-Cov Spike Glycoprotein is Modulated by Lipid Membrane Composition

The S2 domain of the spike glycoprotein from SARS-CoV is responsible for driving viral and host cell membrane fusion. An internal fusion peptide (SARSIFP), located near the S2 domain N-terminus, plays a crucial role in the fusion process. Although much information has been obtained in recent years on membrane fusion, many aspects of the molecular mechanisms behind virus-host cell membrane fusion are not totally understood yet. Questions regarding structure, dynamics, and topology of fusion peptides in lipid membranes still remain and are addressed in this work by means of a variety of experimental and computational techniques. The minimum-energy conformation of the peptide was determined by GSA/MD simulations in different conditions. Cluster analysis and free-energy surfaces revealed a conformational promiscuity for SARSIFP, including α-helices, β-strands, and random coil conformations. The distance distribution between the N- and C-termini was also experimentally determined from four-pulse DEER experiments of a doubly labeled derivative. Different membrane mimetics modulated the average peptide end-to-end distance. Moreover, SARSIFP spontaneously adsorbed at the membrane-solvent interface in a random conformation and then forms a helix. Free-energy values and secondary structure content were modulated by the presence of negative membranes, in agreement with our CD and fluorescence data. MD and electron spin resonance (ESR) results indicated a water-exposed C-terminal and a membrane-inserted N-terminal. Spin-labeled lipids and ESR showed that SARSIFP increased lipid packing and head group ordering only in the presence of negatively charged lipids. To our knowledge, this is the first report that provides dynamics, structural and topological information of SARSIFP in model membranes at atomic resolution, which may shed light on the mechanism by which the peptide folds and inserts into membranes.ACKNOLEDGEMENTS: FAPESP, CAPES, CNPq.

Interaction of biologically-relevant peptides with membrane model systems

In this work, we monitor the alterations caused in membrane model systems upon the addition of biologically-relevant peptides. Our first study reports on the interaction with model membranes of an internal fusion peptide (SARSIFP) from the S2 subunit of the SARS coronavirus spike glycoprotein. It is believed that SARSIFP might be fundamental for the later steps of the fusion between the viral and host cellular membranes. Non-linear least-squares fits of stearic acid spin labels ESR spectra showed that the rotational dynamics of the HPS headgroup region and of the whole carbon chain of SDS surfactants was perturbed by the peptide. Additionally, Tyr fluorescence quenching promoted by spin labels locates this residue in the aqueous interface of HPS and in the hydrophobic core of SDS micelles. The second investigation deals with the conformational changes induced by interactions with model membranes of three TOAC-labeled peptide analogues derived from a new antimicrobial peptide extracted from the skin secretion of the frog Hypsiboas albopunctatus. Our results shed light on how the peptides labeled at positions 0, 2, and 13 interact with DPPC/DPPA/X (X = DPPE, SM, and CL) liposomes and LPC micelles. The findings allowed the description of the peptide topology into the membrane, where the N-terminal region is solvent-exposed, position 2 is at the interface, and position 13 is fully inserted. Financial Support: FAPESP, CNPq, CAPES.