Free energy barrier for melittin reorientation from a membrane-bound state to a transmembrane state

An important step in a phospholipid membrane pore formation by melittin antimicrobial peptide is a reorientation of the peptide from a surface into a transmembrane conformation. In this work we perform umbrella sampling simulations to calculate the potential of mean force (PMF) for the reorientation of melittin from a surface-bound state to a transmembrane state and provide a molecular level insight into understanding peptide and lipid properties that influence the existence of the free energy barrier. The PMFs were calculated for a peptide to lipid (P/L) ratio of 1/128 and 4/128. We observe that the free energy barrier is reduced when the P/L ratio increased. In addition, we study the cooperative effect; specifically we investigate if the barrier is smaller for a second melittin reorientation, given that another neighboring melittin was already in the transmembrane state. We observe that indeed the barrier of the PMF curve is reduced in this case, thus confirming the presence of a cooperative effect

Determining Peptide Partitioning Properties via Computer Simulation

The transfer of polypeptide segments into lipid bilayers to form transmembrane helices represents the crucial first step in cellular membrane protein folding and assembly. This process is driven by complex and poorly understood atomic interactions of peptides with the lipid bilayer environment. The lack of suitable experimental techniques that can resolve these processes both at atomic resolution and nanosecond timescales has spurred the development of computational techniques. In this review, we summarize the significant progress achieved in the last few years in elucidating the partitioning of peptides into lipid bilayer membranes using atomic detail molecular dynamics simulations. Indeed, partitioning simulations can now provide a wealth of structural and dynamic information. Furthermore, we show that peptide-induced bilayer distortions, insertion pathways, transfer free energies, and kinetic insertion barriers are now accurate enough to complement experiments. Further advances in simulation methods and force field parameter accuracy promise to turn molecular dynamics simulations into a powerful tool for investigating a wide range of membrane active peptide phenomena

Small Changes in the Primary Structure of Transportan 10 Alter the Thermodynamics and Kinetics of its Interaction with Phospholipid Vesicles

ABSTRACT: The kinetics and thermodynamics of binding of transportan 10 (tp10) and four of its variants to phospholipid vesicles, and the kinetics of peptide-induced dye efflux, were compared. Tp10 is a 21-residue, amphipathic, cationic, cell-penetrating peptide similar to helical antimicrobial peptides. The tp10 variants examined include amidated and free peptides, and replacements of tyrosine by tryptophan. Carboxy-terminal amidation or substitution of tryptophan for tyrosine enhance binding and activity. The Gibbs energies of peptide binding to membranes determined experimentally and calculated from the interfacial hydrophobicity scale are in good agreement. The Gibbs energy for insertion into the bilayer core was calculated using hydrophobicity scales of residue transfer from water to octanol and to the membrane/ water interface. Peptide-induced efflux becomes faster as the Gibbs energies for binding and insertion of the tp10 variants decrease. If anionic lipids are included, binding and efflux rate increase, as expected because all tp10 variants are cationic and an electrostatic component is added. Whether the most important effect of peptide amidation is the change in charge or an enhancement of helical structure, however, still needs to be established. Nevertheless, it is clear that the changes in efflux rate reflect the differences in the thermodynamics of binding and insertion of the free and amidated peptide groups. We have recently reported a detailed investigation (1) o

Key Amino Acid Residues of Ankyrin-Sensitive Phosphatidylethanolamine/Phosphatidylcholine-Lipid Binding Site of βI-Spectrin

It was shown previously that an ankyrin-sensitive, phosphatidylethanolamine/phosphatidylcholine (PE/PC) binding site maps to the N-terminal part of the ankyrin-binding domain of β-spectrin (ankBDn). Here we have identified the amino acid residues within this domain which are responsible for recognizing monolayers and bilayers composed of PE/PC mixtures. In vitro binding studies revealed that a quadruple mutant with substituted hydrophobic residues W1771, L1775, M1778 and W1779 not only failed to effectively bind PE/PC, but its residual PE/PC-binding activity was insensitive to inhibition with ankyrin. Structure prediction and analysis, supported by in vitro experiments, suggests that “opening” of the coiled-coil structure underlies the mechanism of this interaction. Experiments on red blood cells and HeLa cells supported the conclusions derived from the model and in vitro lipid-protein interaction results, and showed the potential physiological role of this binding. We postulate that direct interactions between spectrin ankBDn and PE-rich domains play an important role in stabilizing the structure of the spectrin-based membrane skeleton

Eukaryote-wide sequence analysis of mitochondrial β-barrel outer membrane proteins

<p>Abstract</p> <p>Background</p> <p>The outer membranes of mitochondria are thought to be homologous to the outer membranes of Gram negative bacteria, which contain 100's of distinct families of <it>β</it>-barrel membrane proteins (BOMPs) often forming channels for transport of nutrients or drugs. However, only four families of mitochondrial BOMPs (MBOMPs) have been confirmed to date. Although estimates as high as 100 have been made in the past, the number of yet undiscovered MBOMPs is an open question. Fortunately, the recent discovery of a membrane integration signal (the <it>β</it>-signal) for MBOMPs gave us an opportunity to look for undiscovered MBOMPs.</p> <p>Results</p> <p>We present the results of a comprehensive survey of eukaryotic protein sequences intended to identify new MBOMPs. Our search employs recent results on <it>β</it>-signals as well as structural information and a novel BOMP predictor trained on both bacterial and mitochondrial BOMPs. Our principal finding is circumstantial evidence suggesting that few MBOMPs remain to be discovered, if one assumes that, like known MBOMPs, novel MBOMPs will be monomeric and <it>β</it>-signal dependent. In addition to this, our analysis of MBOMP homologs reveals some exceptions to the current model of the <it>β</it>-signal, but confirms its consistent presence in the C-terminal region of MBOMP proteins. We also report a <it>β</it>-signal independent search for MBOMPs against the yeast and Arabidopsis proteomes. We find no good candidates MBOMPs in yeast but the Arabidopsis results are less conclusive.</p> <p>Conclusions</p> <p>Our results suggest there are no remaining MBOMPs left to discover in yeast; and if one assumes all MBOMPs are <it>β</it>-signal dependent, few MBOMP families remain undiscovered in any sequenced organism.</p

Arginine in Membranes: The Connection Between Molecular Dynamics Simulations and Translocon-Mediated Insertion Experiments

Several laboratories have carried out molecular dynamics (MD) simulations of arginine interactions with lipid bilayers and found that the energetic cost of placing arginine in lipid bilayers is an order of magnitude greater than observed in molecular biology experiments in which Arg-containing transmembrane helices are inserted across the endoplasmic reticulum membrane by the Sec61 translocon. We attempt here to reconcile the results of the two approaches. We first present MD simulations of guanidinium groups alone in lipid bilayers, and then, to mimic the molecular biology experiments, we present simulations of hydrophobic helices containing single Arg residues at different positions along the helix. We discuss the simulation results in the context of molecular biology results and show that the energetic discrepancy is reduced, but not eliminated, by considering free energy differences between Arg at the interface and at the center of the model helices. The reduction occurs because Arg snorkeling to the interface prevents Arg from residing in the bilayer center where the energetic cost of desolvation is highest. We then show that the problem with MD simulations is that they measure water-to-bilayer free energies, whereas the molecular biology experiments measure the energetics of partitioning from translocon to bilayer, which raises the fundamental question of the relationship between water-to-bilayer and water-to-translocon partitioning. We present two thermodynamic scenarios as a foundation for reconciliation of the simulation and molecular biology results. The simplest scenario is that translocon-to-bilayer partitioning is independent of water-to-bilayer partitioning; there is no thermodynamic cycle connecting the two paths

Structural genomics target selection for the New York consortium on membrane protein structure

The New York Consortium on Membrane Protein Structure (NYCOMPS), a part of the Protein Structure Initiative (PSI) in the USA, has as its mission to establish a high-throughput pipeline for determination of novel integral membrane protein structures. Here we describe our current target selection protocol, which applies structural genomics approaches informed by the collective experience of our team of investigators. We first extract all annotated proteins from our reagent genomes, i.e. the 96 fully sequenced prokaryotic genomes from which we clone DNA. We filter this initial pool of sequences and obtain a list of valid targets. NYCOMPS defines valid targets as those that, among other features, have at least two predicted transmembrane helices, no predicted long disordered regions and, except for community nominated targets, no significant sequence similarity in the predicted transmembrane region to any known protein structure. Proteins that feed our experimental pipeline are selected by defining a protein seed and searching the set of all valid targets for proteins that are likely to have a transmembrane region structurally similar to that of the seed. We require sequence similarity aligning at least half of the predicted transmembrane region of seed and target. Seeds are selected according to their feasibility and/or biological interest, and they include both centrally selected targets and community nominated targets. As of December 2008, over 6,000 targets have been selected and are currently being processed by the experimental pipeline. We discuss how our target list may impact structural coverage of the membrane protein space

Outer membrane protein folding from an energy landscape perspective

The cell envelope is essential for the survival of Gram-negative bacteria. This specialised membrane is densely packed with outer membrane proteins (OMPs), which perform a variety of functions. How OMPs fold into this crowded environment remains an open question. Here, we review current knowledge about OFMP folding mechanisms in vitro and discuss how the need to fold to a stable native state has shaped their folding energy landscapes. We also highlight the role of chaperones and the β-barrel assembly machinery (BAM) in assisting OMP folding in vivo and discuss proposed mechanisms by which this fascinating machinery may catalyse OMP folding