showing 45 related works from this author
Amino acid substitutions enhancing thermostability of Bacillus polymyxa beta-glucosidase A
1996
Mutations enhancing the thermostability of β-glucosidase A of Bacillus polymyxa, a family 1 glycosyl hydrolase, have been obtained after hydroxylamine mutagenesis of a plasmid containing the bglA gene, transformation of Escherichia coli with the mutagenized plasmid, and identification of transformant colonies that showed β-glucosidase activity after a thermal treatment that inactivated the wild-type enzyme. Two additive mutations have been characterized that cause replacement of glutamate at position 96 by lysine and of methionine at position 416 by isoleucine respectively. The thermoresistant mutant enzymes showed increased resistance to other denaturing agents, such as pH and urea, while …
The dynamic orientation of membrane-bound peptides: bridging simulations and experiments.
2007
AbstractThe structural organization in a peptide/membrane supramolecular complex is best described by knowledge of the peptide orientation plus its time-dependent and spatial fluctuations. The static orientation, defined by the peptide tilt and a rotation about its molecular axis, is accessible through a number of spectroscopic methods. However, peptide dynamics, although relevant to understand the functionality of these systems, remains largely unexplored. Here, we describe the orientation and dynamics of Trp-flanked and Lys-flanked hydrophobic peptides in a lipid bilayer from molecular dynamics simulations. A novel view is revealed, where collective nontrivial distributions of time-evolvi…
Stability of Asymmetric Lipid Bilayers Assessed by Molecular Dynamics Simulations
2009
The asymmetric insertion of amphiphiles into biological membranes compromises the balance between the inner and outer monolayers. As a result, area expansion of the receiving leaflet and curvature strain may lead to membrane permeation, shape changes, or membrane fusion events. We have conducted both atomistic and coarse-grained molecular dynamics simulations of dipalmitoyl-phosphatidylcholine (DPPC) bilayers to study the effect of an asymmetric distribution of lipids between the two monolayers on membrane stability. Highly asymmetric lipid bilayers were found to be surprisingly stable within the submicrosecond time span of the simulations. Even the limiting case of a monolayer immersed in …
Paramagnetic NMR investigations of Co(II) and Ni(II) amicyanin.
1999
The paramagnetic 1H NMR spectra of the Co(II) and Ni(II) substituted forms of the type 1 blue copper protein (cupredoxin) amicyanin have been assigned. This is the first such analysis of a cupredoxin, which has a distorted tetrahedral active site with the ligands provided by two histidines, a cysteine and a methionine. The isotropic shifts of the resonances in these spectra are compared with those of Co(II) and Ni(II) azurin. A number of interesting similarities and differences are found. The coordination of the metal by the two equatorial histidine ligands is very similar in both proteins. The interaction between the introduced metal and the thiolate sulfur of the equatorial cysteine ligan…
EPR and Magnetic Susceptibility Studies of Cobalt(II)- and Nickel(II)-Substituted Azurins from Pseudomonas aeruginosa. Electronic Structure of the Ac…
1996
The electronic properties of cobalt(II)- and nickel(II)-substituted azurins from Pseudomonas aeruginosa have been investigated. EPR data for the cobalt derivative and paramagnetic susceptibility data for the nickel derivative are reported. The EPR spectrum of Co(II)−azurin shows the typical pattern of a Kramers' doublet (±1/2) associated with an S = 3/2 ground state in a distorted axial symmetry environment. The temperature dependence of the EPR intensities shows that this Kramers' doublet is the excited doublet and, therefore, that the corresponding zero-field splitting parameter D is negative (∼−3.5 cm-1). The mean g value is equal to 2.3. Nickel(II) azurin exhibits an effective magnetic …
Production and characterisation of recombinant forms of human pulmonary surfactant protein C (SP-C):Structure and surface activity
2006
Udgivelsesdato: 2006-Apr Surfactant protein C (SP-C) is an essential component for the surface tension-lowering activity of the pulmonary surfactant system. It contains a valine-rich alpha helix that spans the lipid bilayer, and is one of the most hydrophobic proteins known so far. SP-C is also an essential component of various surfactant preparations of animal origin currently used to treat neonatal respiratory distress syndrome (NRDS) in preterm infants. The limited supply of this material and the risk of transmission of infectious agents and immunological reactions have prompted the development of synthetic SP-C-derived peptides or recombinant humanized SP-C for inclusion in new prepar…
Solid State NMR Structure Analysis of the Antimicrobial Peptide Gramicidin S in Lipid Membranes: Concentration-Dependent Re-alignment and Self-Assemb…
2008
Antimicrobial peptides can kill bacteria by permeabilizing their cell membrane, as these amphiphilicmolecules interact favourably with lipid bilayers. This mechanism of action is attributed eitherto the formation of a peptide “carpet” on the membrane surface, or to a transmembranepore. However, the structure of such a pore has not yet been resolved under relevant conditions.Gramicidin S is a symmetrical cyclic β-sheet decapeptide, which has been previouslyshown by solid state NMR to lie flat on the membrane surface at low peptide:lipid ratios (≤ 1:80).Using highly sensitive 19F-NMR, supported by 15N-labelling,we found that gramicidin S can flip into an upright transmembrane alignment at hig…
Pores Formed by Baxα5 Relax to a Smaller Size and Keep at Equilibrium
2010
AbstractPores made by amphipathic cationic peptides (e.g., antimicrobials and fragments of pore-forming proteins) are typically studied by examining the kinetics of vesicle leakage after peptide addition or obtaining structural measurements in reconstituted peptide-lipid systems. In the first case, the pores have been considered transient phenomena that allow the relaxation of the peptide-membrane system. In the second, they correspond to equilibrium structures at minimum free energy. Here we reconcile both approaches by investigating the pore activity of the α5 fragment from the proapoptotic protein Bax (Baxα5) before and after equilibrium of peptide/vesicle complexes. Quenching assays on …
µ-Calpain conversion of antiapoptotic Bfl-1 (BCL2A1) into a prodeath factor reveals two distinct alpha-helices inducing mitochondria-mediated apoptos…
2011
Anti-apoptotic Bfl-1 and pro-apoptotic Bax, two members of the Bcl-2 family sharing a similar structural fold, are classically viewed as antagonist regulators of apoptosis. However, both proteins were reported to be death inducers following cleavage by the cysteine protease µ-calpain. Here we demonstrate that calpain-mediated cleavage of full-length Bfl-1 induces the release of C-terminal membrane active α-helices that are responsible for its conversion into a pro-apoptotic factor. A careful comparison of the different membrane-active regions present in the Bfl-1 truncated fragments with homologous domains of Bax show that helix α5, but not α6, of Bfl-1 induces cell death and cytochrome c r…
A photoswitchable helical peptide with light-controllable interface / transmembrane topology in lipidic membranes
2021
AbstractAccording to the three-step model, the spontaneous insertion and folding of helical transmembrane (TM) polypeptides into lipid bilayers is driven by three sequential equilibria: solution-to-membrane interface (MI) partition, unstructured-to-helical folding, and MI-to-TM helix insertion. However, understanding these three steps with molecular detail has been challenged by the lack of suitable experimental approaches to rapidly and reversibly perturb membrane-bound hydrophobic polypeptides out of equilibrium. Here, we report on a 24-residues-long hydrophobic α-helical polypeptide, covalently coupled to an azobenzene photoswitch (KCALP-azo), which displays a light-controllable TM/MI eq…
Global stability of protein folding from an empirical free energy function
2013
The principles governing protein folding stand as one of the biggest challenges of Biophysics. Modeling the global stability of proteins and predicting their tertiary structure are hard tasks, due in part to the variety and large number of forces involved and the difficulties to describe them with sufficient accuracy. We have developed a fast, physics-based empirical potential, intended to be used in global structure prediction methods. This model considers four main contributions: Two entropic factors, the hydrophobic effect and configurational entropy, and two terms resulting from a decomposition of close-packing interactions, namely the balance of the dispersive interactions of folded an…
Pore Formation by a Bax-Derived Peptide: Effect on the Line Tension of the Membrane Probed by AFM
2007
AbstractBax is a critical regulator of physiological cell death that increases the permeability of the outer mitochondrial membrane and facilitates the release of the so-called apoptotic factors during apoptosis. The molecular mechanism of action is unknown, but it probably involves the formation of partially lipidic pores induced by Bax. To investigate the interaction of Bax with lipid membranes and the physical changes underlying the formation of Bax pores, we used an active peptide derived from helix 5 of this protein (Bax-α5) that is able to induce Bax-like pores in lipid bilayers. We report the decrease of line tension due to peptide binding both at the domain interface in phase-separa…
Peptides corresponding to helices 5 and 6 of Bax can independently form large lipid pores
2006
Proteins of the B-cell lymphoma protein 2 (Bcl2) family are key regulators of the apoptotic cascade, controlling the release of apoptotic factors from the mitochondrial intermembrane space. A helical hairpin found in the core of water-soluble folds of these proteins has been reported to be the pore- forming domain. Here we show that peptides including any of the two a-helix fragments of the hairpin of Bcl2 associated protein X (Bax) can independently induce release of large labelled dextrans from synthetic lipid vesicles. The permeability promoted by these peptides is influenced by intrinsic monolayer curvature and accompanied by fast transbilayer redis- tribution of lipids, supporting a to…
1H 2D-NMR characterization of Ni(II)-substituted azurin fromPseudomonas aeruginosa
1993
1 H two-dimemional NMR experiments on nickel(II)-substituted azurin have been succesfully applied. Despite the short relaxation time of the hyperfine-shifted resonances, the combined use of NOESY and COSY spectra allowed the alignment of 15 resonances belonging to the metal-coordinated residues Gly-45, His-46, His-117 and Met-121. Even in the case of the two broad and furthest downfield resonances, the NOESY spectra were successful in assigning these signals to the β-CH 2 protons of Cys-112. The protons of the non-coordinated residues Met-13, Phe-15 and Trp-48 were also assigned via NOESY, COSY and TOCSY experiments
A Hooke's law-based approach to protein folding rate
2014
Kinetics is a key aspect of the renowned protein folding problem. Here, we propose a comprehensive approach to folding kinetics where a polypeptide chain is assumed to behave as an elastic material described by the Hooke[U+05F3]s law. A novel parameter called elastic-folding constant results from our model and is suggested to distinguish between protein with two-state and multi-state folding pathways. A contact-free descriptor, named folding degree, is introduced as a suitable structural feature to study protein-folding kinetics. This approach generalizes the observed correlations between varieties of structural descriptors with the folding rate constant. Additionally several comparisons am…
Solid state NMR analysis of peptides in membranes: Influence of dynamics and labeling scheme.
2010
The functional state of a membrane-active peptide is often defined by its conformation, molecular orientation, and its oligomeric state in the lipid bilayer. These "static" structural properties can be routinely studied by solid state NMR using isotope-labeled peptides. In the highly dynamic environment of a liquid crystalline biomembrane, however, the whole-body fluctuations of a peptide are also of paramount importance, although difficult to address and most often ignored. Yet it turns out that disregarding such motional averaging in calculating the molecular alignment from orientational NMR-constraints may give a misleading, if not false picture of the system. Here, we demonstrate that t…
Orientational landscapes of peptides in membranes: prediction of (2)H NMR couplings in a dynamic context.
2009
Unlike soluble proteins, membrane polypeptides face an anisotropic milieu. This imposes restraints on their orientation and provides a reference that makes structure prediction tractable by minimalistic thermodynamic models. Here we use this framework to build orientational distributions of monomeric membrane-bound peptides and to predict their expected solid-state (2)H NMR quadrupolar couplings when labeled at specific side chain positions. Using a complete rigid-body sampling of configurations relative to an implicit lipid membrane, peptide free energy landscapes are calculated. This allows us to obtain probability distributions of the peptide tilt, azimuthal rotation, and depth of membra…
1H-NMR study of a cobalt-substituted blue copper protein: Pseudomonas aeruginosa Co(II)-azurin.
1995
Substitution of copper by cobalt in blue copper proteins gives a paramagnetic metalloderivative suitable for paramagnetic NMR studies. A thorough analysis of the 1H-NMR spectrum of Pseudomonas aeruginosa Co(II)-azurin is presented here. All the observable contact-shifted signals as well as many other paramagnetic signals from protons placed up to about 1.0 nm around the metal center, including some residues belonging to functionally important parts of the protein like the hydrophobic patch and the His35 region, have been assigned. The results obtained permit the detection and study of structural variations like those originated by the His35 ionization, and allow us to draw a feasible pictur…
Bax-derived membrane-active peptides act as potent and direct inducers of apoptosis in cancer cells.
2011
SUMMARYAlthough many cancer cells are primed for apoptosis, they usually develop resistance to cell death at multiple levels. Permeabilization of the outer mitochondrial membrane, which is mediated by proapoptotic Bcl-2 family members like Bax, is considered as a point-of-no-return for initiating apoptotic cell death. This crucial role has placed Bcl-2 family proteins as recurrent targets for anticancer drug development. Here, we propose and demonstrate a new concept based on using minimal active version of Bax to induce cell death independently of endogenous Bcl-2 proteins. We show that membrane-active segments of Bax can directly induce the release of mitochondria-residing apoptogenic fac…
A lipocentric view of peptide-induced pores
2010
Although lipid membranes serve as effective sealing barriers for the passage of most polar solutes, nonmediated leakage is not completely improbable. A high activation energy normally keeps unassisted bilayer permeation at a very low frequency, but lipids are able to self-organize as pores even in peptide-free and protein-free membranes. The probability of leakage phenomena increases under conditions such as phase coexistence, external stress or perturbation associated to binding of nonlipidic molecules. Here, we argue that pore formation can be viewed as an intrinsic property of lipid bilayers, with strong similarities in the structure and mechanism between pores formed with participation …
Permeabilization of the Outer Mitochondrial Membrane by Bcl-2 Proteins
2010
The proteins of the Bcl-2 family regulate the release of the apoptotic factors from mitochondria during apoptosis, a key event in physiological cell death. Although their molecular mechanisms remain unclear, the Bcl-2 proteins have been proposed to directly control the permeability of the outer mitochondrial membrane by pore formation. Indeed, they share structural features with the pore forming domains of some bacterial toxins and they can give rise to proteolipidic pores in model membranes. The complex level of regulation needed to decide the fate of the cell is achieved by an intricate interaction network between different members of the family. Current models consider multiple parallel …
Influence of Whole-Body Dynamics on 15N PISEMA NMR Spectra of Membrane Proteins: A Theoretical Analysis
2009
AbstractMembrane proteins and peptides exhibit a preferred orientation in the lipid bilayer while fluctuating in an anisotropic manner. Both the orientation and the dynamics have direct functional implications, but motions are usually not accessible, and structural descriptions are generally static. Using simulated data, we analyze systematically the impact of whole-body motions on the peptide orientations calculated from two-dimensional polarization inversion spin exchange at the magic angle (PISEMA) NMR. Fluctuations are found to have a significant effect on the observed spectra. Nevertheless, wheel-like patterns are still preserved, and it is possible to determine the average peptide til…
Hydrophobic mismatch of mobile transmembrane helices: Merging theory and experiments
2012
Abstract Hydrophobic mismatch still represents a puzzle for transmembrane peptides, despite the apparent simplicity of this concept and its demonstrated validity in natural membranes. Using a wealth of available experimental 2 H NMR data, we provide here a comprehensive explanation of the orientation and dynamics of model peptides in lipid bilayers, which shows how they can adapt to membranes of different thickness. The orientational adjustment of transmembrane α-helices can be understood as the result of a competition between the thermodynamically unfavorable lipid repacking associated with peptide tilting and the optimization of peptide/membrane hydrophobic coupling. In the positive misma…
Design of a bivalent peptide with two independent elements of secondary structure able to fold autonomously.
2008
This article describes a strategy to develop, starting from a de novo design, bivalent peptides containing two different (alpha-helix and beta-hairpin) and independent secondary-structure elements. The design was based on the use of conformationally restricted peptide libraries. Structural characterization by NMR revealed that the peptides were stable and did not show any long-range NOE interactions between the N-terminal beta-hairpin and the C-terminal alpha-helix. These results suggest that the two elements of secondary structure are stable and well folded. Copyright (C) 2008 European Peptide Society and John Wiley & Sons. Ltd.
1H NMR and UV-vis spectroscopic characterization of sulfonamide complexes of nickek(II)-carbonic anhydrase. Resonance assignments based on NOE effects
1992
The binding of acetazolamide, p-fluorobenzensulfonamide, p-toluenesulfonamide, and sulfanilamide to nickel(II)-substituted carbonic anhydrase II has been studied by 1H NMR and electronic absorption spectroscopies. These inhibitors bind to the metal ion forming 1:1 complexes and their affinity constants were determined. The 1H NMR spectra of the formed complexes show a number of isotropically shifted signals corresponding to the histidine ligands. The complexes with benzene-sulfonamides gave rise to very similar 1H NMR spectra. The NMR data suggest that these aromatic sulfonamides bind to the metal ion altering its coordination sphere. In addition, from the temperature dependence of 1H NMR s…
Peptides in apoptosis research
2002
Apoptosis is a complex process that plays a central role in physiological and pathological cell death. This fast evolving research area has experienced incredible development in the past few years. Progress in the knowledge of the structure of many of the main molecular actors of the apoptotic signal transduction pathways has driven the design of synthetic peptides that in some cases can function as simplified versions of their parent proteins. These molecules are contributing to a better understanding of the activity and regulation of apoptotic proteins and also are setting the basis for the discovery of effective drugs to combat important diseases related to apoptosis. Most applications o…
(19)F NMR screening of unrelated antimicrobial peptides shows that membrane interactions are largely governed by lipids.
2014
AbstractMany amphiphilic antimicrobial peptides permeabilize bacterial membranes via successive steps of binding, re-alignment and/or oligomerization. Here, we have systematically compared the lipid interactions of two structurally unrelated peptides: the cyclic β-pleated gramicidin S (GS), and the α-helical PGLa. 19F NMR was used to screen their molecular alignment in various model membranes over a wide range of temperatures. Both peptides were found to respond to the phase state and composition of these different samples in a similar way. In phosphatidylcholines, both peptides first bind to the bilayer surface. Above a certain threshold concentration they can re-align and immerse more dee…
Switchable Bactericidal Effects from Novel Silica-Coated Silver Nanoparticles Mediated by Light Irradiation
2011
Here we report on the triggering of antibacterial activity by a new type of silver nanoparticle coated with porous silica, Ag@silica, irradiated at their surface plasmon resonant frequency. The nanoparticles are able to bind readily to the surface of bacterial cells, although this does not affect bacterial growth since the silica shell largely attenuates the intrinsic toxicity of silver. However, upon simultaneous exposure to light corresponding to the absorption band of the nanoparticles, bacterial death is enhanced selectively on the irradiated zone. Because of the low power density used for the treatments, we discard thermal effects as the cause of cell killing. Instead, we propose that …
Overlap and diversity in antimicrobial peptide databases: Compiling a non-redundant set of sequences
2015
Abstract Motivation: The large variety of antimicrobial peptide (AMP) databases developed to date are characterized by a substantial overlap of data and similarity of sequences. Our goals are to analyze the levels of redundancy for all available AMP databases and use this information to build a new non-redundant sequence database. For this purpose, a new software tool is introduced. Results: A comparative study of 25 AMP databases reveals the overlap and diversity among them and the internal diversity within each database. The overlap analysis shows that only one database (Peptaibol) contains exclusive data, not present in any other, whereas all sequences in the LAMP_Patent database are inc…
Role of Membrane Lipids for the Activity of Pore Forming Peptides and Proteins
2010
Bilayer lipids, far from being passive elements, have multiple roles in polypeptide-dependent pore formation. Lipids participate at all stages of the formation of pores by providing the binding site for proteins and peptides, conditioning their active structure and modulating the molecular reorganization of the membrane complex. Such general functions of lipids superimpose to other particular roles, from electrostatic and curvature effects to more specific actions in cases like cholesterol, sphingolipids or cardiolipin.
Double-spanning Plant Viral Movement Protein Integration into the Endoplasmic Reticulum Membrane Is Signal Recognition Particle-dependent, Translocon…
2005
The current model for cell-to-cell movement of plant viruses holds that transport requires virus-encoded movement proteins that intimately associate with endoplasmic reticulum membranes. We have examined the early stages of the integration into endoplasmic reticulum membranes of a double-spanning viral movement protein using photocross-linking. We have discovered that this process is cotranslational and proceeds in a signal recognition particle-dependent manner. In addition, nascent chain photocross-linking to Sec61alpha and translocating chain-associated membrane protein reveal that viral membrane protein insertion takes place via the translocon, as with most eukaryotic membrane proteins, …
Two-dimensional 1 H NMR spectra of ferricytochrome c 551 from Pseudomonas aeruginosa
1993
AbstractThe full assignment of 1H NMR signals of heme proton resonances of ferricytochrome c551 from Pseudomonas aeruginosa has been performed by means of 2D NMR experiments. This technique allows the complete and unequivocal assignment of all heme resonances, including methylene resonances of the propionic groups, directly implicated in the pH dependence of the redox properties of cytochrome c551.
Self-Assembling of Peptide/Membrane Complexes by Atomistic Molecular Dynamics Simulations
2007
Abstract Model biological membranes consisting of peptide/lipid-bilayer complexes can nowadays be studied by classical molecular dynamics (MD) simulations at atomic detail. In most cases, the simulation starts with an assumed state of a peptide in a preformed bilayer, from which equilibrium configurations are difficult to obtain due to a relatively slow molecular diffusion. As an alternative, we propose an extension of reported work on the self-organization of unordered lipids into bilayers, consisting of including a peptide molecule in the initial random configuration to obtain a membrane-bound peptide simultaneous to the formation of the lipid bilayer. This strategy takes advantage of the…
Canonical azimuthal rotations and flanking residues constrain the orientation of transmembrane helices.
2013
AbstractIn biological membranes the alignment of embedded proteins provides crucial structural information. The transmembrane (TM) parts have well-defined secondary structures, in most cases α-helices and their orientation is given by a tilt angle and an azimuthal rotation angle around the main axis. The tilt angle is readily visualized and has been found to be functionally relevant. However, there exist no general concepts on the corresponding azimuthal rotation. Here, we show that TM helices prefer discrete rotation angles. They arise from a combination of intrinsic properties of the helix geometry plus the influence of the position and type of flanking residues at both ends of the hydrop…
Active Fragments from Pro- and Antiapoptotic BCL-2 Proteins Have Distinct Membrane Behavior Reflecting Their Functional Divergence
2010
International audience; BACKGROUND:The BCL-2 family of proteins includes pro- and antiapoptotic members acting by controlling the permeabilization of mitochondria. Although the association of these proteins with the outer mitochondrial membrane is crucial for their function, little is known about the characteristics of this interaction.METHODOLOGY/PRINCIPAL FINDINGS:Here, we followed a reductionist approach to clarify to what extent membrane-active regions of homologous BCL-2 family proteins contribute to their functional divergence. Using isolated mitochondria as well as model lipid Langmuir monolayers coupled with Brewster Angle Microscopy, we explored systematically and comparatively the…
Paramagnetic Cobalt and Nickel Derivatives of Alcaligenes denitrificans Azurin and Its M121Q Mutant. A 1H NMR Study
1996
Using cobalt or nickel to replace copper in native azurin allows one to fingerprint the metal coordination site of the protein. The metal sites of wild type Alcaligenes denitrificans azurin and its M121Q mutant are clearly distinguishable through the paramagnetic 1H NMR spectra of the Ni(II) and Co(II) derivatives. In the wild type azurin, Gly45 coordinates to nickel or cobalt, while Met121 appears as a weak metal ligand. On the contrary, in the M121Q azurin mutant, the metal exhibits a clear preference for the Gln121, which coordinates through the side chain carbonyl oxygen, and Gly45 is not a ligand. Changes in the isotropic shifts and relaxation properties of signals from the Cys112, His…
Interaction of sulphate and chloride with cobalt(II)-carbonic anhydrase
1990
The interaction between Cobalt(II)-Bovine Carbonic Anhydrase II and the inhibitors sulphate and chloride have been investigated through 1H NMR and electronic absorption spectroscopies. Both inhibitors bind to the metal ion forming a 1:1 adduct and the corresponding affinity constants have been determined. These inhibitors interact weakly with CoBCA II and this interaction only occurs at low pH values. The T1 values of the meta-like protons of the coordinated histidines have been measured. The coordination number of the metal ion in the adducts is discussed on the basis of temperature dependence of the isotropic shifts, T1, and molar absorbance values.
Some morphologic changes induced by Nd:YAG laser on the noncoated enamel surface: A scanning electron microscopy study
1992
The enamel surface layer of some human teeth was treated with the low-energy Nd:YAG laser at 8 mJ pulse energy. These samples were previously etched with 0.05 M orthophosphoric acid to reduce the surface reflection. The treated samples, as well as the control samples, were widely studied by scanning electron microscopy, and, in the lased group, significant morphologic changes affecting the enamel surface were observed. Those changes reveal principally the loss of the typical surface structure of the acid-etched enamel. The hydroxyapatite prisms were not discernible, and there was a decrease in the roughness of the lased surface enamel. These laser-induced structural changes may be related t…
Peptides Derived from Apoptotic Bax and Bid Reproduce the Poration Activity of the Parent Full-Length Proteins
2005
Bax and Bid are proapoptotic proteins of the Bcl-2 family that regulate the release of apoptogenic factors from mitochondria. Although they localize constitutively in the cytoplasm, their apoptotic function is exerted at the mitochondrial outer membrane, and is related to their ability to form transbilayer pores. Here we report the poration activity of fragments from these two proteins, containing the first alpha-helix of a colicinlike hydrophobic hairpin (alpha-helix 5 of Bax and alpha-helix 6 of Bid). Both peptides readily bind to synthetic lipid vesicles, where they adopt predominantly alpha-helical structures and induce the release of entrapped calcein. In planar lipid membranes they fo…
The Crystal Structure of Nickel(II)-Azurin
1995
The nickel(II)-azurin metalloderivative has been crystallized and its structure solved at 0.205-nm resolution by X-ray diffraction. The overall structure is not modified by the metal exchange and the only differences with regard to the native copper(II)-azurin occur in the metal site region. These variations affect principally the axial ligands. Nickel co-ordinates more strongly to the carbonyl oxygen of Gly45 while its distance to the Met121 S4 enlarges up to 0.330 nm. The resulting metal center structure is intermediate between those of the Cu(II) and Zn(II) azurins, and can be described as distorted tetrahedral. However, the existence of contact interaction between Met121 and the nickel …
Direct Observation of Nanometer-Scale Pores of Melittin in Supported Lipid Monolayers
2015
Melittin is the most studied membrane-active peptide and archetype within a large and diverse group of pore formers. However, the molecular characteristics of melittin pores remain largely unknown. Herein, we show by atomic force microscopy (AFM) that lipid monolayers in the presence of melittin are decorated with numerous regularly shaped circular pores that can be distinguished from nonspecific monolayer defects. The specificity of these pores is reinforced through a statistical evaluation of depressions found in Langmuir-Blodgett monolayers in the presence and absence of melittin, which eventually allows characterization of the melittin-induced pores at a quantitative low-resolution leve…
Orientation and Dynamics of Peptides in Membranes Calculated from 2H-NMR Data
2009
Solid-state (2)H-NMR is routinely used to determine the alignment of membrane-bound peptides. Here we demonstrate that it can also provide a quantitative measure of the fluctuations around the distinct molecular axes. Using several dynamic models with increasing complexity, we reanalyzed published (2)H-NMR data on two representative alpha-helical peptides: 1), the amphiphilic antimicrobial peptide PGLa, which permeabilizes membranes by going from a monomeric surface-bound to a dimeric tilted state and finally inserting as an oligomeric pore; and 2), the hydrophobic WALP23, which is a typical transmembrane segment, although previous analysis had yielded helix tilt angles much smaller than ex…
The dynamic properties of the M121H azurin metal site as studied by NMR of the paramagnetic Cu(II) and Co(II) metalloderivatives
1998
The M121H azurin mutant in solution presents various species in equilibrium that can be detected and studied by 1H NMR of the Cu(II) and Co(II) paramagnetic metalloderivatives. In both cases up to three species are observed in slow exchange, the proportions of which are different for the two metalloderivatives. Above pH 5 the major species displays a tetrahedral coordination in which the His121 can be observed as a coordinated residue. Its metal site corresponds to a new type of site that is defined as a type 1.5 site. The second and third species resemble the wild type (type 1) azurin and, above pH 4.5, they are present only at a low concentration. At low pH a protonation process increases…
Influence of proline residues in transmembrane helix packing
2003
Integral membrane proteins often contain proline residues in their alpha-helical transmembrane (TM) fragments, which may strongly influence their folding and association. Pro-scanning mutagenesis of the helical domain of glycophorin A (GpA) showed that replacement of the residues located at the center abrogates helix packing while substitution of the residues forming the ending helical turns allows dimer formation. Synthetic TM peptides revealed that a point mutation of one of the residues of the dimerization motif (L75P) located at the N-terminal helical turn of the GpA TM fragment, adopts a secondary structure and oligomeric state similar to the wild-type sequence in detergents. In additi…
Membrane-insertion fragments of Bcl-xL, Bax, and Bid.
2004
Apoptosis regulators of the Bcl-2 family associate with intracellular membranes from mitochondria and the endoplasmic reticulum, where they perform their function. The activity of these proteins is related to the release of apoptogenic factors, sequestered in the mitochondria, to the cytoplasm, probably through the formation of ion and/or protein transport channels. Most of these proteins contain a C-terminal putative transmembrane (TM) fragment and a pair of hydrophobic alpha helices (alpha5-alpha6) similar to the membrane insertion fragments of the ion-channel domain of diphtheria toxin and colicins. Here, we report on the membrane-insertion properties of different segments from antiapopt…