Possible conformation of delta-lysin,a membrane-damaging peptide of Staphylococcus aureus

It is proposed that staphylococcal delta-lysin, a membrane-damaging peptide of 26 residues adopts an α-helical rod-like configuration with separate hydrophilic and hydrophobic faces. Association of six such monomers in a cell membrane may result in the formation of a transmembrane “pore” lined by the hydrophilic faces of the monomers.     

Plasmodium berghei: Architectural analysis by freeze-fracturing of the intraoocyst sporozoite's pellicular system

The technique of freeze-fracturing has been used to study the architecture of the pellicular complex of the intraoocyst sporozoite of Plasmodium berghei. The sporozoite is surrounded by three plasma membranes and a layer of subpellicular microtubules. During freeze-fracturing, each of the three membranes can split along its hydrophobic interior to yield a total of six fracture faces. The most obvious feature of each fracture face is the presence of globular intramembranous particles on the surface. The six fracture faces differ from one another in arrangement, size, and density of these intramembranous particles. Two of the fracture faces exhibit a unique arrangement of particles in well-organized parallel rows along the long axis of the sporozoite. This arrangement has not been reported in either the erythrocytic or the exoerythrocytic forms of Plasmodium spp. Another unique feature in the sporozoite revealed through freeze-fracturing is a single suture line that traverses the long axis of the inner two membranes of the parasite.     

Differentiation of lipid-associating helices by use of three-dimensional molecular hydrophobicity potential calculations.

Several types of lipid-associating helices exist: transmembrane helices such as in receptor proteins, pore-forming helices in ion channel proteins, fusion-inducing peptides in viral proteins, and amphipathic helices such as in plasma apolipoproteins. In order to propose a classification of these helices according to their molecular properties, we introduce the concept of molecular hydrophobicity potential for such helical segments. The calculation of this parameter for alpha-helices enables the visualization of the hydrophobic and hydrophilic envelopes around the peptide and their three-dimensional representation by molecular graphics. We have used this parameter to differentiate between pore-forming helices with a hydrophobic envelope larger than the hydrophilic component, membrane-spanning helices surrounded almost entirely by an hydrophobic envelope, fusiogenic peptides with an hydrophobicity gradient both around the helix and along the axis, and finally, amphipathic helices with a predominantly hydrophilic envelope. The structure of the lipid-protein complexes is determined by a number of different interactions: the hydrophobic interaction of the apolar faces of the helices with lipids, the polar interaction of the hydrophilic sides of different helices with each other, and the interaction of hydrophilic residues with the aqueous solvent. The relative magnitude of the hydrophobic and hydrophilic envelopes accounts for the differences in the structure of the lipid-protein complexes. Purely hydrophobic interactions stabilize transmembrane helical segments, while hydrophobic interactions with the lipid phase and with each other are involved in the stabilization of the pore-forming helices. In contrast, both hydrophobic interactions with the lipids and hydrophilic interactions with the aqueous phase contribute to the arrangement of amphipathic helices around the edges of the discoidal lipid-apoprotein complexes.     

Topology of the hog intrinsic factor receptor in the intestine

The intrinsic factor receptor was isolated from Triton X-100 extract of hog ileal mucosa using affinity chromatography on intrinsic factor bound to cobalamin-Sepharose. We verified that the receptor contains two subunits, alpha and beta. The purified receptor located in the detergent micelle was radioiodinated. The alpha subunit was labeled and dissociated from the receptor. When the receptor was immobilized on intrinsic factor cobalamin-Sepharose, the part of the receptor which binds intrinsic factor evidently faces the gel and the rest faces outward. When such gel-bound pure receptor was iodinated, the beta subunit was labeled. Iodination of the micellar cobalamin-intrinsic factor receptor complex also caused labeling of the beta subunit. This was interpreted as being due to a conformational change in the receptor affected by the binding of the substrate cobalamin-intrinsic factor, exposing groups accessible to iodination. The beta subunit was found to be hydrophobic, but the alpha subunit was soluble in phosphate buffer without detergent. The receptor was liberated from intestinal mucosa by papain treatment. The enzyme seems to solubilize an intrinsic factor-binding part of the receptor, apparently a part of the alpha subunit. The liberated papain-alpha was purified by affinity chromatography. In gel filtration, it seemed to occur in dimeric form, but its true Mr = 45,000, according to findings in sodium dodecyl sulfate-electrophoresis. In the light of the findings, the topology of the receptor is suggested to be as follows: the alpha subunit binding intrinsic factor faces out and the hydrophobic beta subunit faces in.     

Flexible programs for the prediction of average amphipathicity of multiply aligned homologous proteins: application to integral membrane transport proteins

Simple flexible programs (TREEMOMENT and PILEUPMOMENT) are described for depicting the average amphipathicity (hydrophobic moment) along multiply aligned sequences of a family of evolutionarily related proteins. The programs are applicable to any number of aligned sequences and can be set for any desired angle corresponding to a residue repeat unit in a protein secondary structural element such as 100 per residue for an alpha- helix or 180 per residue for a beta-strand. These programs can be used to identify amphipathic regions common to the members of a protein family. The use of these programs is exemplified by showing that some families of integral membrane transport proteins (i.e. permeases of the bacterial phosphotransferase system (PTS) and the anion exchangers of animals) exhibit strikingly amphipathic alpha-helical structures immediately preceding the first hydrophobic transmembrane segment of their membrane-embedded domain(s). Other families, such as the major facilitator superfamily of uniporters, symporters and antiporters, do not exhibit this structural feature. The amphipathic structures in PTS permeases have been implicated in membrane insertion during biogenesis.     

Flexible programs for the prediction of average amphipathicity of multiply aligned homologous proteins: application to integral membrane transport proteins.

Simple flexible programs (TREEMOMENT and PILEUPMOMENT) are described for depicting the average amphipathicity (hydrophobic moment) along multiply aligned sequences of a family of evolutionarily related proteins. The programs are applicable to any number of aligned sequences and can be set for any desired angle corresponding to a residue repeat unit in a protein secondary structural element such as 100 degrees per residue for an alpha-helix or 180 degrees per residue for a beta-strand. These programs can be used to identify amphipathic regions common to the members of a protein family. The use of these programs is exemplified by showing that some families of integral membrane transport proteins (i.e. permeases of the bacterial phosphotransferase system (PTS) and the anion exchangers of animals) exhibit strikingly amphipathic alpha-helical structures immediately preceding the first hydrophobic transmembrane segment of their membrane-embedded domain(s). Other families, such as the major facilitator superfamily of uniporters, symporters and antiporters, do not exhibit this structural feature. The amphipathic structures in PTS permeases have been implicated in membrane insertion during biogenesis.     

Preliminary evidence of abnormal white matter related to the fusiform gyrus in Williams syndrome: a diffusion tensor imaging tractography study

Williams syndrome (WS) is a genetic condition caused by a hemizygous microdeletion on chromosome 7q11.23. WS is characterized by a distinctive social phenotype composed of increased drive toward social engagement and attention toward faces. In addition, individuals with WS exhibit abnormal structure and function of brain regions important for the processing of faces such as the fusiform gyrus. This study was designed to investigate if white matter tracts related to the fusiform gyrus in WS exhibit abnormal structural integrity as compared to typically developing (TD; age matched) and developmentally delayed (DD; intelligence quotient matched) controls. Using diffusion tensor imaging data collected from 40 (20 WS, 10 TD and 10 DD) participants, white matter fibers were reconstructed that project through the fusiform gyrus and two control regions (caudate and the genu of the corpus callosum). Macro-structural integrity was assessed by calculating the total volume of reconstructed fibers and micro-structural integrity was assessed by calculating fractional anisotropy (FA) and fiber density index (FDi) of reconstructed fibers. WS participants, as compared to controls, exhibited an increase in the volume of reconstructed fibers and an increase in FA and FDi for fibers projecting through the fusiform gyrus. No between-group differences were observed in the fibers that project through the control regions. Although preliminary, these results provide further evidence that the brain anatomy important for processing faces is abnormal in WS.     

STRUCTURAL DIFFERENTIATION OF STACKED AND UNSTACKED CHLOROPLAST MEMBRANES : Freeze-Etch Electron Microscopy of Wild-Type and Mutant Strains of Chlamydomonas

Wild-type chloroplast membranes from Chlamydomonas reinhardi exhibit four faces in freeze-etchreplicas: the complementary Bs and Cs faces are found where the membranes are stacked together; the complementary Bu and Cu faces are found in unstacked membranes. The Bs face carries a dense population of regularly spaced particles containing the large, 160 ± 10 A particles that appear to be unique to chloroplast membranes. Under certain growth conditions, membrane stacking does not occur in the ac-5 strain. When isolated, these membranes remain unstacked, exhibit only Bu and Cu faces, and retain the ability to carry out normal photosynthesis. Membrane stacking is also absent in the ac-31 strain, and, when isolated in a low-salt medium, these membranes remain unstacked and exhibit only Bu and Cu faces. When isolated in a high-salt medium, however, they stack normally, and Bs and Cs faces are produced by this in vitro stacking process. We conclude that certain particle distributions in the chloroplast membrane are created as a consequence of the stacking process, and that the ability of membranes to stack can be modified both by gene mutation and by the ionic environment in which the membranes are found.     

Sensation seeking and men's face preferences

Findings from previous studies suggest that only men who are in good physical condition can afford to pursue high-risk activities and that men who engage in high-risk activities are considered particularly attractive by women. Here, we show that men's interest in high-sensation activities, a personality trait that is known to increase the likelihood of those individuals engaging in high-risk behaviors, is positively related to the strength of their preferences for femininity in women's faces (Studies 1–3) but is not related to the strength of their preferences for femininity in men's faces (Study 2). We discuss these findings as evidence for potentially adaptive condition-dependent mate preferences, whereby men who exhibit signals of high quality demonstrate particularly strong preferences for facial cues of reproductive and medical health in potential mates because they are more likely than lower-quality men to succeed in acquiring such partners.     

Local hydrophobicity stabilizes secondary structures in proteins

The probability of occurrence of helix and β-sheet residues in 47 globular proteins was determined as a function of local hydrophobicity, which was defined by the sum of the Nozaki-Tanford transfer free energies at two nearest-neighbors on both sides of the amino acid sequence. In general, hydrophilic amino acids favor neither helix nor β-sheet formations when neighbor residues are also hydrophilic but favor helix formation at higher local hydrophobicity. On the other hand, some hydrophobic amino acids such as Met, Leu, and Ile favor helix formation when neighbor residues are hydrophilic. None of the hydrophobic amino acids favor β-sheet formation with hydrophilic neighbors, but most of them strongly favor β-sheet formation at high local hydrophobicity. When the average of 20 amino acids is taken, both helix and β-sheet residue probabilities are higher at higher local hydrophobicity, although the increase is steeper for β-sheets. Therefore, β-sheet formation is more influenced by local hydrophobicity than helix formation. Generally, helices are nearer the surface and tend to have hydrophilic and hydrophobic faces at opposite sides. The tendency of alternating regions of hydrophilic and hydrophobic residues in a helical sequence was revealed by calculating the correlation of the Nozaki-Tanford values. Such amphipathic helices may be important in protein–protein and protein–lipid interactions and in forming hydrophilic channels in the membrane. The choice of 30 nonhomologous proteins as the data set did not alter the above results.     

Hydrophobicity scale for proteins based on inverse temperature transitions.

In general, proteins fold with hydrophobic residues buried, away from water. Reversible protein folding due to hydrophobic interactions results from inverse temperature transitions where folding occurs on raising the temperature. Because homoiothermic animals constitute an infinite heat reservoir, it is the transition temperature, Tt, not the endothermic heat of the transition, that determines the hydrophobically folded state of polypeptides at body temperature. Reported here is a new hydrophobicity scale based on the values of Tt for each amino acid residue as a guest in a natural repeating peptide sequence, the high polymers of which exhibit reversible inverse temperature transitions. Significantly, a number of ways have been demonstrated for changing Tt such that reversibly lowering Tt from above to below physiological temperature becomes a means of isothermally and reversibly driving hydrophobic folding. Accordingly, controlling Tt becomes a mechanism whereby proteins can be induced to carry out isothermal free energy transduction.     

Open-and-shut cases in coiled-coil assembly: alpha-sheets and alpha-cylinders

The coiled coil is a ubiquitous protein-folding motif. It generally is accepted that coiled coils are characterized by sequence patterns known as heptad repeats. Such patterns direct the formation and assembly of amphipathic alpha-helices, the hydrophobic faces of which interface in a specific manner first proposed by Crick and termed "knobs-into-holes packing". We developed software, SOCKET, to recognize this packing in protein structures. As expected, in a trawl of the protein data bank, we found examples of canonical coiled coils with a single contiguous heptad repeat. In addition, we identified structures with multiple, overlapping heptad repeats. This observation extends Crick's original postulate: Multiple, offset heptad repeats help explain assemblies with more than two helices. Indeed, we have found that the sequence offset of the multiple heptad repeats is related to the coiled-coil oligomer state. Here we focus on one particular sequence motif in which two heptad repeats are offset by two residues. This offset sets up two hydrophobic faces separated by approximately 150 degrees -160 degrees around the alpha-helix. In turn, two different combinations of these faces are possible. Either similar or opposite faces can interface, which leads to open or closed multihelix assemblies. Accordingly, we refer to these two forms as alpha-sheets and alpha-cylinders. We illustrate these structures with our own predictions and by reference to natural variants on these designs that have recently come to light.     

Effects of surface-bound water and surface stereochemistry on cell adhesion to crystal surfaces

Crystals of calcium-(R,S)-tartrate trihydrate were used as adhesion substrates (for A6 epithelial cells), to study specific stages in cell adhesion. Events such as surface recognition, cell attachment, spreading, motility, cell-cell aggregation, and cell penetration into the crystal bulk are all shown to depend on the molecular structure of the various crystal faces. These crystals exhibit three chemically equivalent, yet structurally distinct, faces. On the {100}, a layered surface exposing bound water, the cells attach, are motile, and tend to form multicellular aggregates, but do not spread and do not form focal contacts. Following prolonged incubation, single cells attached to the {100} surface undergo apoptosis, while those interacting with other cells are rescued. Macroscopic spiral dislocations emerging on the {100} face of the crystal are highly adhesive for cells. Cells attached to these sites develop long protrusions that penetrate into the crystal. The {011} faces expose mainly hydroxyls attached to the chiral carbons. The cells interact extensively with these faces, are immobilized, do not spread, do not form focal contacts, and subsequently die. The faces belonging to the {0kl}? family are characterized by molecular and topographical steps. The cells attach to these faces, spread, and form focal contacts and stress fibers. Thus the molecular character of the crystal surfaces, including the presence of bound water, the exposure of determinants that promote rapid surface recognition, and the effective association with extracellular adhesive proteins, affect the patterns of cell adhesive behavior and fate.     

Ranking the factors that contribute to protein beta-sheet folding

The formation of beta-sheet domains in proteins involves five energetically important factors: the formation of networks of hydrogen bonds and hydrophobic faces, and the residue propensities, or preferences, to be found at the edges of the beta-sheet, to adopt the extended conformation, and to make contact with other residues. These relative energy contributions define a potential energy function. Here, we show how optimizing this potential energy function reveals the formation of hydrophobic faces as the utmost factor. The potential energy function was optimized to minimize the Z-scores of the native topologies among the exhaustive sets of over 400 different beta-sheets. These results corroborate with experimental data that showed the environment of a protein is an important modulator of beta-sheet folding. The contact propensities were found to be the least important, which could explain the poor predictive power of beta-strand alignment methods based on pair-wise contact matrices.     

Binding preferences, surface attachment, diffusivity, and orientation of a family 1 carbohydrate-binding module on cellulose

Cellulase enzymes often contain carbohydrate-binding modules (CBMs) for binding to cellulose. The mechanisms by which CBMs recognize specific surfaces of cellulose and aid in deconstruction are essential to understand cellulase action. The Family 1 CBM from the Trichoderma reesei Family 7 cellobiohydrolase, Cel7A, is known to selectively bind to hydrophobic surfaces of native cellulose. It is most commonly suggested that three aromatic residues identify the planar binding face of this CBM, but several recent studies have challenged this hypothesis. Here, we use molecular simulation to study the CBM binding orientation and affinity on hydrophilic and hydrophobic cellulose surfaces. Roughly 43 μs of molecular dynamics simulations were conducted, which enables statistically significant observations. We quantify the fractions of the CBMs that detach from crystal surfaces or diffuse to other surfaces, the diffusivity along the hydrophobic surface, and the overall orientation of the CBM on both hydrophobic and hydrophilic faces. The simulations demonstrate that there is a thermodynamic driving force for the Cel7A CBM to bind preferentially to the hydrophobic surface of cellulose relative to hydrophilic surfaces. In addition, the simulations demonstrate that the CBM can diffuse from hydrophilic surfaces to the hydrophobic surface, whereas the reverse transition is not observed. Lastly, our simulations suggest that the flat faces of Family 1 CBMs are the preferred binding surfaces. These results enhance our understanding of how Family 1 CBMs interact with and recognize specific cellulose surfaces and provide insights into the initial events of cellulase adsorption and diffusion on cellulose.     

Fluorous effect in proteins: de novo design and characterization of a four-alpha-helix bundle protein containing hexafluoroleucine

Several studies have demonstrated that proteins incorporating fluorinated analogues of hydrophobic amino acids such as leucine and valine into their hydrophobic cores exhibit increased stability toward thermal denaturation and unfolding by guanidinium chloride. However, estimates for the increase in the thermodynamic stability of a protein (DeltaDeltaG(unfold)) afforded by the substitution of a hydrophobic amino acid with its fluorinated analogue vary quite significantly. To address this, we have designed a peptide that adopts an antiparallel four-helix bundle structure in which the hydrophobic core is packed with leucine, and investigated the effects of substituting the central two layers of the core with L-5,5,5,5',5',5'-hexafluoroleucine (hFLeu). We find that DeltaDeltaG(unfold) is increased by 0.3 kcal/mol per hFLeu residue. This is in good agreement with the predicted increase in DeltaDeltaG(unfold) of 0.4 kcal/mol per residue arising from the increased hydrophobicity of the hFLeu side chain, which we determined experimentally from partitioning measurements on hFLeu and leucine. The increased stability of this fluorinated protein may therefore be ascribed to simple hydrophobic effects, rather than specific "fluorous" interactions between the hFLeu residues.     

The fluorinated anesthetic halothane as a potential NMR biologic probe

Fluorinated anesthetics such as halothane preferentially partition into hydrophobic environments such as cell membranes. The 19F-NMR spectrum of halothane in a rat adenocarcinoma (with known altered lipid metabolism and membrane composition) shows an altered chemical shift pattern compared to the anesthetic in normal tissue. In eight tumor samples examined, the 19F-NMR spectra exhibit two distinct resonances, compared to a single resonance observed in normal tissues. This is explained by an enhanced or altered hydrophobic component in the tumor tissue giving rise to two discrete halothane environments. Another fluorinated anesthetic, isoflurane, shows similar behavior in distinguishing normal from diseased tissue. Given the large chemical shift range of fluorine and the inherent sensitivity of this nucleus, 19F-NMR spectra of fluorinated anesthetics can also be used to follow anesthetic degradation by the liver. The ability of fluorinated anesthetics to discriminate tissues and to monitor metabolic processes is potentially useful for in vivo 19F-NMR surface coil and imaging studies.     

Hydrogen bond connectivity patterns and hydrophobic interactions in crystal structures of small, acyclic peptides.

Crystal structures of all available unblocked linear peptides with two to five residues were retrieved from the Cambridge Structural Database and their intermolecular contacts and packing modes studied using molecular graphics. This survey reveals that interactions between hydrophobic portions of the molecules are critically important in determining the overall features of their crystal packing patterns. Distinct hydrophobic columns or layers are observed in almost all crystal structures. Analyses of the relationships between these interactions and crystal growth properties of small peptides are given. It is suggested that needle growth is promoted by hydrophobic packing, usually along a short crystallographic axis (4.6-6.0 angstroms). Also contributing to these morphologic characteristics are entropic factors associated with hydrophobic aggregation as well as tightly bound water molecules on hydrophobic faces. The paper also provides a comprehensive overview of hydrogen bond patterns in acyclic peptide crystals. It is demonstrated that one of their primary roles is to provide a scaffolding within which hydrophobic groups can aggregate. Even though there is a high density of hydrogen bonds in the crystals, often with complex patterns and networks, certain motifs are found to recur in a number of structures indicating specific hydrogen bond preferences. Water, for example, is an integral part of the hydrogen bond networks in these crystals, usually acting as the primary donor for main-chain carboxylate groups in peptide hydrates.     

NMR observable-based structure refinement of DAP12-NKG2C activating immunoreceptor complex in explicit membranes

NMR observables, such as NOE-based distance measurements, are increasingly being used to characterize membrane protein structures. However, challenges in membrane protein NMR studies often yield a relatively small number of such restraints that can create ambiguities in defining critical side chain-side chain interactions. In the recent solution NMR structure of the DAP12-NKG2C immunoreceptor transmembrane helix complex, five functionally required interfacial residues (two Asps and two Thrs in the DAP12 dimer and one Lys in NKG2C) display a surprising arrangement in which one Asp side chain faces the membrane hydrophobic core. To explore whether these side-chain interactions are energetically optimal, we used the published distance restraints for molecular dynamics simulations in explicit micelles and bilayers. The structures refined by this protocol are globally similar to the published structure, but the side chains of those five residues form a stable network of salt bridges and hydrogen bonds, leaving the Asp side chain shielded from the hydrophobic core, which is also consistent with available experimental observations. Moreover, the simulations show similar short-range interactions between the transmembrane complex and lipid/detergent molecules in micelles and bilayers, respectively. This study illustrates the efficacy of NMR membrane protein structure refinements in explicit membrane systems.