Effects of a -Maleimide-derivatized Phosphatidylethanolamine on the Architecture and Properties of Lipid Bilayers.

-maleimide-derivatized phospholipids are often used to facilitate protein anchoring to membranes. In autophagy studies, this is applied to the covalent binding of Atg8, an autophagy protein, to a phosphatidylethanolamine (PE) in the nascent autophagosome. However, the question remains on how closely the -maleimide PE derivative (PE-mal) mimicks the native PE in the bilayer.

Molecular recognition of a membrane-anchored HIV-1 pan-neutralizing epitope.

Antibodies against the carboxy-terminal section of the membrane-proximal external region (C-MPER) of the HIV-1 envelope glycoprotein (Env) are considered as nearly pan-neutralizing. Development of vaccines capable of producing analogous broadly neutralizing antibodies requires deep understanding of the mechanism that underlies C-MPER recognition in membranes. Here, we use the archetypic 10E8 antibody and a variety of biophysical techniques including single-molecule approaches to study the molecular recognition of C-MPER in membrane mimetics.

Functional Delineation of a Protein-Membrane Interaction Hotspot Site on the HIV-1 Neutralizing Antibody 10E8.

Antibody engagement with the membrane-proximal external region (MPER) of the envelope glycoprotein (Env) of HIV-1 constitutes a distinctive molecular recognition phenomenon, the full appreciation of which is crucial for understanding the mechanisms that underlie the broad neutralization of the virus. Recognition of the HIV-1 Env antigen seems to depend on two specific features developed by antibodies with MPER specificity: (i) a large cavity at the antigen-binding site that holds the epitope amphipathic helix; and (ii) a membrane-accommodating Fab surface that engages with viral phospholipids. Thus, besides the main Fab-peptide interaction, molecular recognition of MPER depends on semi-specific (electrostatic and hydrophobic) interactions with membranes and, reportedly, on specific binding to the phospholipid head groups.

Truncation-Driven Lateral Association of α-Synuclein Hinders Amyloid Clearance by the Hsp70-Based Disaggregase.

The aggregation of α-synuclein is the hallmark of a collective of neurodegenerative disorders known as synucleinopathies. The tendency to aggregate of this protein, the toxicity of its aggregation intermediates and the ability of the cellular protein quality control system to clear these intermediates seems to be regulated, among other factors, by post-translational modifications (PTMs). Among these modifications, we consider herein proteolysis at both the N- and C-terminal regions of α-synuclein as a factor that could modulate disassembly of toxic amyloids by the human disaggregase, a combination of the chaperones Hsc70, DnaJB1 and Apg2.

Conformational plasticity underlies membrane fusion induced by an HIV sequence juxtaposed to the lipid envelope.

Envelope glycoproteins from genetically-divergent virus families comprise fusion peptides (FPs) that have been posited to insert and perturb the membranes of target cells upon activation of the virus-cell fusion reaction. Conserved sequences rich in aromatic residues juxtaposed to the external leaflet of the virion-wrapping membranes are also frequently found in viral fusion glycoproteins. These membrane-proximal external regions (MPERs) have been implicated in the promotion of the viral membrane restructuring event required for fusion to proceed, hence, proposed to comprise supplementary FPs.

Exploring polar headgroup interactions between sphingomyelin and ceramide with infrared spectroscopy.

Ceramide is a major actor in the sphingolipid signaling pathway elicited by various kinds of cell stress. Under those conditions ceramide (Cer) is produced in the plasma membrane as a product of sphingomyelin (SM) hydrolysis, and this may lead to apoptosis. Thus, SM and Cer coexist in the membrane for some time, and they are known to separate laterally from the (more abundant) glycerolipids, giving rise to highly rigid domains or platforms.

Conjugative Coupling Proteins and the Role of Their Domains in Conjugation, Secondary Structure and Subcellular Location.

Type IV Coupling Proteins (T4CPs) are essential elements in many type IV secretion systems (T4SSs). The members of this family display sequence, length, and domain architecture heterogeneity, being the conserved Nucleotide-Binding Domain the motif that defines them. In addition, most T4CPs contain a Transmembrane Domain (TMD) in the amino end and an All-Alpha Domain facing the cytoplasm.

Cholesterol Constrains the Antigenic Configuration of the Membrane-Proximal Neutralizing HIV-1 Epitope.

The envelope glycoprotein (Env) enables HIV-1 cell entry through fusion of host-cell and viral membranes induced by the transmembrane subunit gp41. Antibodies targeting the C-terminal sequence of the membrane-proximal external region (C-MPER) block the fusogenic activity of gp41 and achieve neutralization of divergent HIV-1 strains and isolates. Thus, recreating the structure that generates broadly neutralizing C-MPER antibodies during infection is a major goal in HIV vaccine development.

The Binding of Aβ42 Peptide Monomers to Sphingomyelin/Cholesterol/Ganglioside Bilayers Assayed by Density Gradient Ultracentrifugation.

The binding of Aβ42 peptide monomers to sphingomyelin/cholesterol (1:1 mol ratio) bilayers containing 5 mol% gangliosides (either GM1, or GT1b, or a mixture of brain gangliosides) has been assayed by density gradient ultracentrifugation. This procedure provides a direct method for measuring vesicle-bound peptides after non-bound fraction separation. This centrifugation technique has rarely been used in this context previously.

Nucleophosmin interaction with APE1: Insights into DNA repair regulation.

Nucleophosmin (NPM1), an abundant, nucleolar protein with multiple functions affecting cell homeostasis, has also been recently involved in DNA damage repair. The roles of NPM1 in different repair pathways remain however to be elucidated. NPM1 has been described to interact with APE1 (apurinic apyrimidinic endonuclease 1), a key enzyme of the base excision repair (BER) pathway, which could reflect a direct participation of NPM1 in this route.

2D IR Correlation Spectroscopy in the Determination of Aggregation and Stability of KH Domain GXXG Loop Peptide in the Presence and Absence of Trifluoroacetate.

Trifluoroacetate (TFA) is a strong anion byproduct of solid-phase peptide synthesis. Fourier transform infrared (FT-IR) spectroscopy can be used to ascertain the presence of this excipient in peptide samples for quality assessment. TFA absorbs as a strong sharp peak (1675 cm) within the amide I' band of the spectral region.

Solid lipid nanoparticles for delivery of Calendula officinalis extract.

Solid lipid nanoparticles (SLN) composed of long-chain fatty acids (palmitic acid, stearic acid or arachidic acid), Epikuron 200 (purified phosphatidylcholine), and bile salts (cholate, taurocholate or taurodeoxycholate) have been prepared by dilution of a microemulsion. A total of five different systems were prepared, and characterized by photon correlation spectroscopy, transmission electron microscopy, differential scanning calorimetry, and infrared spectroscopy. The SLN formulation showing optimal properties (lowest size and polydispersity index and highest zeta potential) was obtained with stearic acid and taurodeoxycholate as cosurfactant.

Structure and immunogenicity of a peptide vaccine, including the complete HIV-1 gp41 2F5 epitope: implications for antibody recognition mechanism and immunogen design.

The membrane-proximal external region (MPER) of gp41 harbors the epitope recognized by the broadly neutralizing anti-HIV 2F5 antibody, a research focus in HIV-1 vaccine development. In this work, we analyze the structure and immunogenic properties of MPERp, a peptide vaccine that includes the following: (i) the complete sequence protected from proteolysis by the 2F5 paratope; (ii) downstream residues postulated to establish weak contacts with the CDR-H3 loop of the antibody, which are believed to be crucial for neutralization; and (iii) an aromatic rich anchor to the membrane interface. MPERp structures solved in dodecylphosphocholine micelles and 25% 1,1,1,3,3,3-hexafluoro-2-propanol (v/v) confirmed folding of the complete 2F5 epitope within continuous kinked helices.

Amyloid fibril formation from human and bovine serum albumin followed by quasi-simultaneous Fourier-transform infrared (FT-IR) spectroscopy and static light scattering (SLS).

Human and bovine serum albumins are widely known proteins that can form amyloid fibrils under destabilizing conditions. Use of well-known proteins with easily-controlled aggregation process, and comparison of these processes for similar proteins from different species, could help elucidate the nature of the aggregation process implicated in many degenerative diseases, for example Alzheimer's, Parkinson's, or type II diabetes. In this work both amyloidogenic mechanisms have been studied by use of infrared spectroscopy in combination with static light scattering, enabling analysis of intra and intermolecular processes and measurement of prefibril and fibril growing quasi-simultaneously.

Deletion of a single helix from the transmembrane domain causes large changes in membrane insertion properties and secondary structure of the bacterial conjugation protein TrwB.

TrwB is an essential protein in the conjugative transfer of plasmid R388. The protein consists of a bulky cytosolic domain containing the catalytic site, and a small transmembrane domain (TMD). Our previous studies support the idea that the TMD plays an essential role in the activity, structure and stability of the protein.

A conventional an 2DCOS infrared approach to the kinetics of protein misfolding.

Cell viability depends on the correct folding of the proteins involved in metabolism. Proteins are synthesized on the endoplasmic reticulum and must follow a pathway to a correct, metastable, tridimensional structure. Changes in structure or in environmental conditions can drive an instability of the folding conditions and produce non-active aggregates that in principle are proteolysed by the cellular mechanisms.

Membrane insertion stabilizes the structure of TrwB, the R388 conjugative plasmid coupling protein.

TrwB is an integral membrane protein that plays a crucial role in the conjugative process of plasmid R388. We have recently shown [Vecino et al., Biochim.

Influence of aggregation propensity and stability on amyloid fibril formation as studied by Fourier transform infrared spectroscopy and two-dimensional COS analysis.

Understanding the process of amyloidogenesis is important for the future treatment of misfolding-based diseases, such as Alzheimer's, spongiform encephalopathies, and other important disorders affecting humans. In this work, we have used one of the best-characterized models for folding and misfolding, the activation domain of human procarboxypeptidase A2 (ADA2h). The wild type (WT) and three mutants affecting the kinetics of aggregation have been studied by IR from the folded state at acidic pD to fibril formation, showing the disappearance of structured features prior to a dramatic increase in the magnitude of the amyloid-characteristic band upon temperature induction.

Structural constraints imposed by the conserved fusion peptide on the HIV-1 gp41 epitope recognized by the broadly neutralizing antibody 2F5.

The HIV-1 gp41 epitope recognized by the broadly neutralizing 2F5 antibody has focused much attention as a suitable target in the design of peptide immunogens. Peptides mimicking the linear 2F5 epitope (2F5ep) are however intrinsically disordered, while the structural constraints existing in the cognate gp41 native structure recognized by the antibody are presently unknown. In recent reports, we have shown that core residues of the amino-terminal fusion peptide (FP) increase MAb2F5 affinity.

Structural analysis and assembly of the HIV-1 Gp41 amino-terminal fusion peptide and the pretransmembrane amphipathic-at-interface sequence.

The amino-terminal region within the HIV-1 gp41 aromatic-rich pretransmembrane domain is an amphipathic-at-interface sequence (AIS). AIS is highly conserved between different viral strains and isolates and recognized by the broadly neutralizing 2F5 antibody. The atomic structure of the native Fab2F5-bound AIS appears to involve a nonhelical extended region and a beta-turn structure.