Papers by Margarida Bastos
Pure and Applied Chemistry, 2014
The enthalpies of solution of alcohols were determined by calorimetry in HEPES and (HEPES + treha... more The enthalpies of solution of alcohols were determined by calorimetry in HEPES and (HEPES + trehalose) at 298.15 K. The used methodology and experiment’s design allowed us to extract from a single titration experiment the enthalpy of solution (Δsol H m), the limiting solubility of the alcohol in each aqueous media, and an estimation of the enthalpy of solution of water in the alcohol phase. From these values the changes in Gibbs energy (Δsol G m) and in entropy (Δsol S m) of solution were derived. A decrease in solubility for 1-butanol and 1-pentanol in the crowded media (HEPES + trehalose) was observed which is driven by a significant decrease in the favorable enthalpy of solution. The partial molar heat capacity, in each media was determined in our heat capacity drop calorimeter, also at 298.15 K. A significant decrease of the partial molar heat capacity was observed for both alcohols in (HEPES + trehalose), which together with the obtained decrease in favorable Δsol H m, is consi...
FEBS Letters, 2009
More than 100 transthyretin (TTR) variants are associated with hereditary amyloidosis. Approaches... more More than 100 transthyretin (TTR) variants are associated with hereditary amyloidosis. Approaches for TTR amyloidosis that interfere with any step of the cascade of events leading to fibril formation have therapeutic potential. In this study we tested (À)-epigallocatechin-3-gallate (EGCG), the most abundant catechin of green tea, as an inhibitor of TTR amyloid formation. We demonstrate that EGCG binds to TTR ''in vitro" and ''ex vivo" and that EGCG inhibits TTR aggregation ''in vitro" and in a cell culture system. These findings together with the low toxicity of the compound raise the possibility of using EGCG in a therapeutic approach for familial amyloidotic polyneuropathy, the most frequent form of hereditary TTR amyloidosis.
A multi-laboratory benchmark study of isothermal titration calorimetry (ITC) using Ca2+ and Mg2+ binding to EDTA
European Biophysics Journal
A small-scale ITC benchmarking study was performed involving 9 biophysics laboratories/facilities... more A small-scale ITC benchmarking study was performed involving 9 biophysics laboratories/facilities, to evaluate inter-laboratory and intra-laboratory basal levels of uncertainty. Our prime goal was to assess a number of important factors that can influence both the data gathered by this technique and the thermodynamic parameter values derived therefrom. In its first part, the study involved 5 laboratories and 13 different instruments, working with centrally prepared samples and the same experimental protocol. The second part involved 4 additional laboratories and 6 more instruments, where the users prepared their own samples according to provided instructions and did the experiments following the same protocol as in the first part. The study design comprised: (1) selecting a minimal set of laboratories; (2) providing very stable samples; (3) providing samples not requiring preparation or manipulation; and (4) providing a well-defined and detailed experimental protocol. Thus, we were able to assess: (i) the variability due to instrument and data analysis performed by each user on centrally prepared samples; (ii) the comparability of data retrieved when using 4 different software packages to analyze the same data, besides the data analysis carried out by the different users on their own experimental results; and (iii) the variability due to local sample preparation (second part of the study). Individual values, as well as averages and standard deviations for the binding parameters for EDTA-cation interaction, were used as metrics for comparing the equilibrium association constant (logK), enthalpy of interaction (ΔH), and the so-called "stoichiometry" (n), a concentration-correction factor.
Macromolecular assembly and membrane activity of antimicrobial D,L-α-Cyclic peptides
Colloids and Surfaces B: Biointerfaces, 2021
Antimicrobial peptides are viewed as a promising alternative to conventional antibiotics, as thei... more Antimicrobial peptides are viewed as a promising alternative to conventional antibiotics, as their activity through membrane targeting makes them less prone to resistance development. Among them, antimicrobial D,L-α-cyclic peptides (CPs) have been proposed as an alternative, specially due to their cyclic nature and to the presence of D-α-amino acids that increases their resistance to proteases. In present work, second generation D,L-α-cyclic peptides with proven antimicrobial activity are shown to form complex macromolecular assemblies in the presence of membranes. We addressed the CPs:membrane interactions through a combination of experimental techniques (DSC and ATR-FTIR) with coarse-grained molecular dynamics (CG-MD) simulations, aiming at understanding their interactions, macromolecular assemblies and eventually unveil their mechanism of action. DSC shows that the interaction depends heavily on the negatively charge content of the membrane and on lipid/peptide ratio, suggesting different mechanisms for the different peptides and lipid systems. CG-MD proved that CPs can self-assemble at the lipid surface as nanotubes or micellar aggregates, depending on the peptide, in agreement with ATR-FTIR results. Finally, our results shed light into possible mechanisms of action of the peptides with pending hydrocarbon tail, namely membrane extensive segregation and/or membrane disintegration through the formation of disk-like lipid/peptide aggregates.
Colloids and Surfaces B: Biointerfaces, 2020
The search of new antibiotics, particularly with new mechanisms of action, is nowadays a very imp... more The search of new antibiotics, particularly with new mechanisms of action, is nowadays a very important public health issue, due to the worldwide increase of resistant pathogens. Within this effort, much research has been done on antimicrobial peptides, because having the membrane as a target, they represent a new antibiotic paradigm. Among these, cyclic peptides (CPs) made of sequences of D-and L-amino acids have emerged as a new class of potential antimicrobial peptides, due to their expected higher resistance to protease degradation. These CPs are planar structures that can form Self-assembled Cyclic Peptide Nanotubes (SCPNs), in particular in the presence of lipid membranes. Aiming at understanding their mechanism of action, we used biophysical experimental techniques (DSC and ATR-FTIR) together with Coarse-grained molecular dynamics (CG-MD) simulations, to characterize the interaction of these CPs with model membranes of different electrostatic charges' contents. DSC results revealed that the CPs show a strong interaction with negatively charged membranes, with differences in the strength of interactions depending on peptide and on membrane charge content, at odds with no or mild interactions with zwitterionic membranes. ATR-FTIR suggested that the peptides self-assemble at the membrane surface, adopting mainly a β-structure. The experiments with polarized light showed that in most cases they lie parallel to the membrane surface, but other forms and orientations are also apparent, depending on peptide structure and lipid:peptide ratio. The nanotube formation and orientation, as well as the dependence on membrane charge were also confirmed by the CG-MD simulations. These provide detail on the position and interactions, in agreement with the experimental results. Based on the findings reported here, we could proceed to the design and synthesis of a second-generation CPs, based on CP2 (soluble peptide), with increased activity and reduced toxicity.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2020
Synuclein (αsyn) is a cytosolic intrinsically disordered protein (IDP) known to fold into an α-he... more Synuclein (αsyn) is a cytosolic intrinsically disordered protein (IDP) known to fold into an α-helical structure when binding to membrane lipids, decreasing protein aggregation. Model membrane enable elucidation of factors critically affecting protein folding/aggregation, mostly using either small unilamellar vesicles (SUVs) or nanodiscs surrounded by membrane scaffold proteins (MSPs). Yet SUVs are mechanically strained, while MSP nanodiscs are expensive. To test the impact of lipid particle size on α-syn structuring, while overcoming the limitations associated with the lipid particles used so far, we compared the effects of large unilamellar vesicles (LUVs) and lipid-bilayer nanodiscs encapsulated by diisobutylene/maleic acid copolymer (DIBMA) on αsyn secondary-structure formation, using human-, elephant-and whale-αsyn. Our results confirm that negatively charged lipids induce αsyn folding in h-αsyn and e-αsyn but not in w-αsyn. When a mixture of zwitterionic and negatively charged lipids was used, no increase in the secondary structure was detected at 45°C. Further, our results show that DIBMA/lipid particles (DIBMALPs) are highly suitable nanoscale membrane mimics for studying αsyn secondary-structure formation and aggregation, as folding was essentially independent of the lipid/protein ratio, in contrast with what we observed for LUVs having the same lipid compositions. This study reveals a new and promising application of polymer-encapsulated lipid-bilayer nanodiscs, due to their excellent efficiency in structuring disordered proteins such as αsyn into nontoxic α-helical structures. This will contribute to the unravelling and modelling aspects concerning protein-lipid interactions and α-helix formation by αsyn, paramount to the proposal of new methods to avoid protein aggregation and disease.
Langmuir : the ACS journal of surfaces and colloids, Feb 5, 2018
The understanding of the mechanism of action of antimicrobial peptides is fundamental for the dev... more The understanding of the mechanism of action of antimicrobial peptides is fundamental for the development of new and more active antibiotics. In present work, we use a wide range of techniques (SANS, SAXD, DSC, ITC, CD, confocal and electron microscopy) in order to fully characterize the interaction of a cecropin A-melittin hybrid antimicrobial peptide, CA(1-7)M(2-9), of known antimicrobial activity, with a bacterial model membrane of POPE/POPG, in an effort to unravel its mechanism of action. We found that CA(1-7)M(2-9) disrupts the vesicles inducing membrane condensation, forming an 'onion-like' structure of multilamellar stacks, held together by the intercalated peptides. SANS and SAXD revealed changes induced by the peptide in the lipid bilayer thickness and the bilayer stiffening, in a tightly packed liquid-crystalline lamellar phase. The analysis of the observed abrupt changes in the repeat distance upon the phase transition to the gel state suggests the formation of a...
Physica B: Condensed Matter, 2006
Neutron time-of-flight spectra were measured for an H 2 O-hydrated and a nominally dry sample of ... more Neutron time-of-flight spectra were measured for an H 2 O-hydrated and a nominally dry sample of a 15-residue antibacterial oligopeptide from 99 to 271 K. Proton mobilities, quasielastic broadenings, and changes in low-frequency inelastic intensities characterise the evolution of the peptide energy landscape as a function of momentum transfer and temperature.
The Journal of Physical Chemistry B, 2005
The interaction of two hybrid peptides of cecropin A and melittin [CA(1-8)M(1-18) and CA(1-7)M(2-... more The interaction of two hybrid peptides of cecropin A and melittin [CA(1-8)M(1-18) and CA(1-7)M(2-9)] with liposomes was studied by differential scanning calorimetry (DSC), circular dichroism (CD), and quasi-elastic light scattering (QELS). The study was carried out with large unilamellar vesicles (LUVs) of three different lipid compositions: 1,2-dimyristoil-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoylsn-glycero-3-phospho-rac-(1-glycerol) (DMPG) and a binary mixture of DMPC/DMPG, in a wide range of peptide-to-lipid (P:L) molar ratios (0 to 1:7). DSC results indicate that, for both peptides, the interaction depends on membrane composition, with very different behavior for zwitterionic and anionic membranes. CD data show that, although the two peptides have different secondary structures in buffer (random coil for CA(1-7)M(2-9) and predominantly-sheet for CA(1-8)M(1-18)), they both adopt an R-helical structure in the presence of the membranes. Overall, results are compatible with a model involving a strong electrostatic surface interaction between the peptides and the negatively charged liposomes, which gives place to aggregation in the gel phase and precipitation after a threshold peptide concentration. In the case of zwitterionic membranes, a progressive surface coverage with peptide molecules destabilizes the membrane, eventually leading to membrane disruption. Moreover, delicate modulations in behavior were observed depending on the peptide.
The Journal of Physical Chemistry B, 2005
Physical Chemistry Chemical Physics, 2013
In the present work we bridge neutron scattering and calorimetry in the study of a low-hydration ... more In the present work we bridge neutron scattering and calorimetry in the study of a low-hydration sample of a 15-residue hybrid peptide from cecropin and mellitin CA(1-7)M(2-9) of proven antimicrobial activity. Quasielastic and low-frequency inelastic neutron spectra were measured at defined hydration levelsa nominally 'dry' sample (specific residual hydration h = 0.060 g/g), a H 2 O-hydrated (h = 0.49) and a D 2 Ohydrated one (h = 0.51). Averaged mean square proton mobilities were derived over a large temperature range (50-300 K) and the vibrational density of states (VDOS) were evaluated for the hydrated samples. The heat capacity of the H 2 O-hydrated CA(1-7)M(2-9) peptide was measured by adiabatic calorimetry in the temperature range 5-300 K, for different hydration levels. The glass transition and water crystallization temperatures were derived in each case. The existence of different types of water was inferred and their amounts calculated. The heat capacities as obtained from direct calorimetric measurements were compared to the values derived from the neutron spectroscopy by way of integrating appropriately normalized VDOS functions. While there is remarkable agreement with respect to both temperature dependence and glass transition temperatures, the results also show that the VDOS derived part represents only a fraction of the total heat capacity obtained from calorimetry. Finally our results indicate that both hydration water and the peptide are involved in the experimentally observed transitions.
Journal of Pharmacy and Bioallied Sciences, 2011
Lactoferrin is a glycoprotein with two globular lobes, each having two domains. Since the discove... more Lactoferrin is a glycoprotein with two globular lobes, each having two domains. Since the discovery of its antimicrobial properties, efforts have been made to find peptides derived from this protein showing antimicrobial properties. Most peptides initially studied were derived from Lactoferricin B, obtained from the protein by digestion with pepsin. More recently, a new family of antimicrobial peptides (AMPs) derived from Lactoferrin was discovered by Bolcher et al, and named Lactoferrampin (LFampin). The original sequence of LFampin contained residues 268-284 from the N1 domain of Lactoferrin. From this peptide, the Bolscher's group synthesized a collection of peptides obtained by extension and / or truncation at the C or N-terminal sides, in order to unravel the main structural features responsible for antimicrobial action. Here, we present results for three of these peptides, namely LFampin 265-284, LFampin 265-280, and LFampin 270-284. The peptides were tested against bacteria (E. coli and S. sanguinis), fungi (C. albicans), and model membranes of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPG), and their mixtures at a ratio of 3 : 1 (DMPC : DMPG (3 : 1)). The ability to adopt a helical conformation was followed by a circular dichroism (CD), and the perturbation of the gel to the liquid-crystalline phase transition of the membrane was characterized by differential scanning calorimetry (DSC). Distinct behavior was observed in the three peptides, both from the microbiology and model membrane studies, with the biophysical results showing excellent correlation with the microbiology activity studies. LFampin 265-284 was the most active peptide toward the tested microorganisms, and in the biophysical studies it showed the highest ability to form an α-helix and the strongest interaction with model membranes, followed by LFampin 265-280. LFampin 270-284 was inactive, showing marginal secondary structure and no interaction with the pathogen model membranes.
Journal of Peptide Science, 2008
Lycotoxin I and Lycotoxin II are natural anti-microbial peptides that were identified in the veno... more Lycotoxin I and Lycotoxin II are natural anti-microbial peptides that were identified in the venom of the Wolf Spider Lycosa carolinensis. These peptides were found to be potent growth inhibitors for bacteria (Escherichia coli) and yeast (Candida glabrata) at micromolar concentrations. Recently, shortened analogues of LycoI and LycoII have been reported to have decreased haemolytic effects. A shorter Lyco-I analogue studied, LycoI 1-15 (H-IWLTALKFLGKHAAK-NH 2), was active only above 10 µM, but was also the least haemolytic. On the basis of these findings, we became interested in obtaining a deeper insight into the membrane activity of LycoI 1-15, as this peptide may represent the first major step for the future development of selective, i.e. non-haemolytic, Lycotoxin-based antibiotics. The interaction of this peptide with liposomes of different composition was studied by microcalorimetry [differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC)] and CD. The results obtained from the calorimetric and spectroscopic techniques were jointly discussed in an attempt to further understand the interaction of this peptide with model membranes.
Journal of Biological Chemistry, 2004
General Physiology and Biophysics, 2009
The structure and temperature behaviour of the DNA+dipalmitoylphosphatidylcholine (DPPC) bilayer ... more The structure and temperature behaviour of the DNA+dipalmitoylphosphatidylcholine (DPPC) bilayer as a function of ZnCl 2 concentration were examined using differential scanning calorimetry (DSC), small-angle neutron scattering (SANS) and small-angle X-ray diffraction (SAXD). Experiments revealed the coexistence of two lamellar phases in the mixture: the L PC phase, formed due to Zn 2+ binding to the DPPC bilayers, and the condensed lamellar phase L DNA+PC with DNA strands packed between the DPPC bilayers. With increasing concentration of zinc, the temperature of the gel-liquid-crystal phase transition of DPPC increases in both phases, and the volume fraction of L DNA+PC phase decreases. In the gel state (at 20°C), the repeat distance of L DNA+PC phase is constant, d DNA+PC ~ 8.3 nm, up to 20 mmol/l of ZnCl 2 , and increases for higher concentrations of the salt. The periodicity of the L PC lamellar phase decreases substantially with the increasing concentration of the salt in the mixture. In the liquid-crystalline state, concentrations above 20 mmol/l ZnCl 2 promote the dissolution of the L DNA+PC phase into DPPC + Zn 2+ unilamellar vesicles and DNA is neutralized by Zn 2+ ions. The screening of Zn 2+ charge and the formation of a diffuse double layer due to increasing ionic strength of solution are responsible for the observed changes.
Carbohydrate Polymers, 2011
Food-grade galacto-oligosaccharides (GOS) are commercially available as transparent syrups or dri... more Food-grade galacto-oligosaccharides (GOS) are commercially available as transparent syrups or dried powders. Food powders can be found in an amorphous metastable state which is very sensitive to changes in temperature and moisture content. In this work the impact of water content on thermal behavior and relative humidity on water sorption behavior of amorphous GOS powders were studied. Results from differential scanning calorimetry (DSC) and sorption isotherms suggest that GOS mixture studied, with high content of oligosaccharides, has low ability to crystallize. A dramatic decrease in the stability of GOS powders occurred above critical water content (12-14 g/100 g) and corresponding critical water activity (0.55-0.62). Above these conditions GOS powder lost its amorphous character, collapsed and shrank, as the powder became a transparent "solution-like" material. The knowledge about the physicochemical changes, acquired during the present study, should be used to a proper control of processing and storage conditions to achieve and maintain optimum powder quality with desired properties.
Biophysical Journal, 2010
The structure and function of the innate defence regulatory peptide 1018 was investigated. This p... more The structure and function of the innate defence regulatory peptide 1018 was investigated. This peptide, whose sequence is distantly related to that of the 12 residue linear antimicrobial peptide Bac2A, a synthetic peptide derivative of the bovine cathelicidin Bactenecin, has both innate immune regulatory and direct antimicrobial activities. We present the solution state NMR structure of 1018 in DPC micelles, as well as its secondary structure in SDS and POPC/ PG (1:1 molar ratio) from CD measurements. These structures reveal that 1018 can adopt a variety of folds, tailored to its different functions. The structural data is discussed in light of the ability of 1018 to induce cytokine and chemokine responses, to reduce the LPS-induced TNF-a response, and finally, to directly kill both Gram positive and Gram negative bacteria.
Biophysical Journal, 2011
The observation of a micellar cubic phase is reported for a mixture of an antimicrobial peptide f... more The observation of a micellar cubic phase is reported for a mixture of an antimicrobial peptide from the Lactoferrin family, LFampin 265-284, and a model membrane system of dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (3:1), as derived from small-angle x-ray diffraction (SAXD) measurements. The system shows remarkable thermotropic polymorphism: the peptide disrupts the lipid bilayer, forming a cubic phase of the space group Pm3n (t < 28 C), and as the temperature increases it shows a complex phase behavior (not fully clarified by SAXD). The onset, volume fraction of each phase, and phase parameters are seen to vary with peptide/lipid ratio and temperature. The obtained SAXD data represent the first experimental evidence, to our knowledge, of a micellar cubic phase in the context of antimicrobial peptide/membrane interaction. We propose that the micellization of the membrane according to the carpet model, for long proposed as a possible mechanism of action, can go through the formation of a cubic micellar phase.
Biophysical Journal, 2004
Isothermal titration calorimetry was used to characterize and quantify the partition of indometha... more Isothermal titration calorimetry was used to characterize and quantify the partition of indomethacin and acemetacin between the bulk aqueous phase and the membrane of egg phosphatidylcholine vesicles. Significant electrostatic effects were observed due to binding of the charged drugs to the membrane, which implied the use of the Gouy-Chapman theory to calculate the interfacial concentrations. The binding/partition phenomenon was quantified in terms of the partition coefficient (K p), and/or the equilibrium constant (K b). Mathematical expressions were developed, either to encompass the electrostatic effects in the partition model, or to numerically relate partition coefficients and binding constants. Calorimetric titrations conducted under a lipid/drug ratio [100:1 lead to a constant heat release and were used to directly calculate the enthalpy of the process, DH, and indirectly, DG and DS. As the lipid/drug ratio decreased, the constancy of reaction enthalpy was tested in the fitting process. Under low lipid/drug ratio conditions simple partition was no longer valid and the interaction phenomenon was interpreted in terms of binding isotherms. A mathematical expression was deduced for quantification of the binding constants and the number of lipid molecules associated with one drug molecule. The broad range of concentrations used stressed the biphasic nature of the interaction under study. As the lipid/drug ratio was varied, the results showed that the interaction of both drugs does not present a unique behavior in all studied regimes: the extent of the interaction, as well as the binding stoichiometry, is affected by the lipid/drug ratio. The change in these parameters reflects the biphasic behavior of the interaction-possibly the consequence of a modification of the membrane's physical properties as it becomes saturated with the drug.
Biophysical Journal, 2008
The energetics and partition of two hybrid peptides of cecropin A and melittin (CA(1-8)M(1-18) an... more The energetics and partition of two hybrid peptides of cecropin A and melittin (CA(1-8)M(1-18) and CA(1-7)M(2-9)) with liposomes of different composition were studied by time-resolved fluorescence spectroscopy, isothermal titration calorimetry, and surface plasmon resonance. The study was carried out with large unilamellar vesicles of three different lipid compositions: 1,2-dimyristoil-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DMPG), and a 3:1 binary mixture of DMPC/DMPG in a wide range of peptide/lipid ratios. The results are compatible with a model involving a strong electrostatic surface interaction between the peptides and the negatively charged liposomes, giving rise to aggregation and precipitation. A correlation is observed in the calorimetric experiments between the observed events and charge neutralization for negatively charged and mixed membranes. In the case of zwitterionic membranes, a very interesting case study was obtained with the smaller peptide, CA(1-7)M(2-9). The calorimetric results obtained for this peptide in a large range of peptide/lipid ratios can be interpreted on the basis of an initial and progressive surface coverage until a threshold concentration, where the orientation changes from parallel to perpendicular to the membrane, followed by pore formation and eventually membrane disruption. The importance of negatively charged lipids on the discrimination between bacterial and eukaryotic membranes is emphasized.
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Papers by Margarida Bastos