Yinon Shafrir

Viscoelastic Properties of Living Embryonic Tissues: a Quantitative Study

A number of properties of certain living embryonic tissues can be explained by considering them as liquids. Tissue fragments left in a shaker bath round up to form spherical aggregates, as do liquid drops. When cells comprising two distinct embryonic tissues are mixed, typically a nucleation-like process takes place, and one tissue sorts out from the other. The equilibrium configurations at the end of such sorting out phenomena have been interpreted in terms of tissue surface tensions arising from the adhesive interactions between individual cells. In the present study we go beyond these equilibrium properties and study the viscoelastic behavior of a number of living embryonic tissues. Using a specifically designed apparatus, spherical cell aggregates are mechanically compressed and their viscoelastic response is followed. A generalized Kelvin model of viscoelasticity accurately describes the measured relaxation curves for each of the four tissues studied. Quantitative results are obtained for the characteristic relaxation times and elastic and viscous parameters. Our analysis demonstrates that the cell aggregates studied here, when subjected to mechanical deformations, relax as elastic materials on short time scales and as viscous liquids on long time scales.

Models of membrane-bound Alzheimer's Abeta peptide assemblies†

Although it is clear that amyloid beta (Aβ) peptides play a pivotal role in the development of Alzheimers disease, the precise molecular model of action remains unclear. Aβ peptide forms assemble both in aqueous solution and in lipid membranes. It has been proposed that deleterious effects occur when the peptides interact with membranes, possibly by forming Ca2+ permeant ion channels. In the accompanying manuscript, we propose models in which the C‐terminus third of six Aβ42 peptides forms a six‐stranded β‐barrel in highly toxic soluble oligomers. Here we extend this hypothesis to membrane‐bound assemblies. In these Aβ models, the hydrophobic β‐barrel of a hexamer may either reside on the surface of the bilayer, or span the bilayer. Transmembrane pores are proposed to form between several hexamers. Once the β‐barrels of six hexamers have spanned the bilayer, they may merge to form a more stable 36‐stranded β‐barrel. We favor models in which parallel β‐barrels formed by N‐terminus segments comprise the lining of the pores. These types of models explain why the channels are selective for cations and how metal ions, such as Zn2+, synthetic peptides that contain histidines, and some small organic cations may block channels or inhibit formation of channels. Our models were developed to be consistent with microscopy studies of Aβ assemblies in membranes, one of which is presented here for the first time. Proteins 2010.

Models of Voltage-Dependent Conformational Changes in NaChBac Channels

Models of the transmembrane region of the NaChBac channel were developed in two open/inactivated and several closed conformations. Homology models of NaChBac were developed using crystal structures of Kv1.2 and a Kv1.2/2.1 chimera as templates for open conformations, and MlotiK and KcsA channels as templates for closed conformations. Multiple molecular-dynamic simulations were performed to refine and evaluate these models. A striking difference between the S4 structures of the Kv1.2-like open models and MlotiK-like closed models is the secondary structure. In the open model, the first part of S4 forms an alpha-helix, and the last part forms a 3(10) helix, whereas in the closed model, the first part of S4 forms a 3(10) helix, and the last part forms an alpha-helix. A conformational change that involves this type of transition in secondary structure should be voltage-dependent. However, this transition alone is not sufficient to account for the large gating charge movement reported for NaChBac channels and for experimental results in other voltage-gated channels. To increase the magnitude of the motion of S4, we developed another model of an open/inactivated conformation, in which S4 is displaced farther outward, and a number of closed models in which S4 is displaced farther inward. A helical screw motion for the alpha-helical part of S4 and a simple axial translation for the 3(10) portion were used to develop models of these additional conformations. In our models, four positively charged residues of S4 moved outwardly during activation, across a transition barrier formed by highly conserved hydrophobic residues on S1, S2, and S3. The S4 movement was coupled to an opening of the activation gate formed by S6 through interactions with the segment linking S4 to S5. Consistencies of our models with experimental studies of NaChBac and K(v) channels are discussed.

Models of the Structure and Gating Mechanisms of the Pore Domain of the NaChBac Ion Channel

The NaChBac prokaryotic sodium channel appears to be a descendent of an evolutionary link between voltage-gated K(V) and Ca(V) channels. Like K(V) channels, four identical six-transmembrane subunits comprise the NaChBac channel, but its selectivity filter possesses a signature sequence of eukaryotic Ca(V) channels. We developed structural models of the NaChBac channel in closed and open conformations, using K(+)-channel crystal structures as initial templates. Our models were also consistent with numerous experimental results and modeling criteria. This study concerns the pore domain. The major differences between our models and K(+) crystal structures involve the latter portion of the selectivity filter and the bend region in S6 of the open conformation. These NaChBac models may serve as a stepping stone between K(+) channels of known structure and Na(V), Ca(V), and TRP channels of unknown structure.

Beta-barrel models of soluble amyloid beta oligomers and annular protofibrils.

Both soluble and membrane‐bound prefibrillar assemblies of Abeta (Aβ) peptides have been associated with Alzheimers disease (AD). The size and nature of these assemblies vary greatly and are affected by many factors. Here, we present models of soluble hexameric assemblies of Aβ42 and suggest how they can lead to larger assemblies and eventually to fibrils. The common element in most of these assemblies is a six‐stranded β‐barrel formed by the last third of Aβ42, which is composed of hydrophobic residues and glycines. The hydrophobic core β‐barrels of the hexameric models are shielded from water by the N‐terminus and central segments. These more hydrophilic segments were modeled to have either predominantly β or predominantly α secondary structure. Molecular dynamics simulations were performed to analyze stabilities of the models. The hexameric models were used as starting points from which larger soluble assemblies of 12 and 36 subunits were modeled. These models were developed to be consistent with numerous experimental results. Proteins 2010.

STAM: simple Transmembrane Alignment Method

MOTIVATION The database of transmembrane protein (TMP) structures is still very small. At the same time, more and more TMP sequences are being determined. Molecular modeling is an interim answer that may bridge the gap between the two databases. The first step in homology modeling is to achieve a good alignment between the target sequences and the template structure. However, since most algorithms to obtain the alignments were constructed with data derived from globular proteins, they perform poorly when applied to TMPs. In our application, we automate the alignment procedure and design it specifically for TMP. We first identify segments likely to form transmembrane alpha-helices. We then apply different sets of criteria for transmembrane and non-transmembrane segments. For example, the penalty for insertion/deletions in the transmembrane segments is much higher than that of a penalty in the loop region. Different substitution matrices are used since the frequencies of occurrence of the various amino acids differ for transmembrane segments and water-soluble domains. RESULTS This program leads to better models since it does not treat the protein as a single entity with the same properties, but accounts for the different physical properties of the various segments. STAM is the first multisequence alignment program that is directly targeted at transmembrane proteins. AVAILABILITY Source code and installation package are available on request from the authors. Web access is currently implemented.

Polyhistidine Peptide Inhibitor of the Aβ Calcium Channel Potently Blocks the Aβ-Induced Calcium Response in Cells. Theoretical Modeling Suggests a Cooperative Binding Process

On the basis of the consistent demonstrations that the Abeta peptide of Alzheimers disease forms calcium permeant channels in artificial membranes, we have proposed that the intracellular calcium increase observed in cells exposed to Abeta is initiated by calcium fluxes through Abeta channels. We have found that a small four-histidine peptide, NAHis04, potently inhibits the Abeta-induced calcium channel currents in artificial lipid membranes. Here we report that NaHis04 also potently blocks the intracellular calcium increase which is observed in cells exposed to Abeta. PC12 cells loaded with Fura-2AM show a rapid increase in fluorescence and a rapid return to baseline after Abeta is added to the medium. This fluorescence change occurs even when the medium contains nitrendipine, a voltage-gated calcium channel blocker, but fails to occur when application of Abeta is preceded by addition of NAHis04. Steep dose-response curves of the percentage of responding cells and cell viability show that NAHis04 inhibits in the micromolar range in an apparently cooperative manner. We have developed numerous models of Abeta pores in which the first part of the Abeta sequence forms a large beta-barrel ending at His 13. We have modeled how up to four NAHis04 peptides may block these types of pores by binding to side chains of Abeta residues Glu 11, His 13, and His 14.

Large scale simulations of two-species annihilation, A+B→0, with drift

Abstract We present results of computer simulations of the diffusion-limited reaction process A + B →0, on the line, under extreme drift conditions, for lattices of up to 2 27 sites, and where the process proceeds to completion (no particles left). These enormous simulations are made possible by the renormalized reaction-cell method (RRC). Our results allow us to resolve an existing controversy about the rate of growth of domain sizes, and about corrections to scaling of the concentration decay.

Mapping discontinuous epitopes for MRK-16, UIC2 and 4E3 antibodies to extracellular loops 1 and 4 of human P-glycoprotein

P-glycoprotein (P-gp), an ATP-dependent efflux pump, is associated with the development of multidrug resistance in cancer cells. Antibody-mediated blockade of human P-gp activity has been shown to overcome drug resistance by re-sensitizing resistant cancer cells to anticancer drugs. Despite the potential clinical application of this finding, the epitopes of the three human P-gp-specific monoclonal antibodies MRK-16, UIC2 and 4E3, which bind to the extracellular loops (ECLs) have not yet been mapped. By generating human-mouse P-gp chimeras, we mapped the epitopes of these antibodies to ECLs 1 and 4. We then identified key amino acids in these regions by replacing mouse residues with homologous human P-gp residues to recover binding of antibodies to the mouse P-gp. We found that changing a total of ten residues, five each in ECL1 and ECL4, was sufficient to recover binding of both MRK-16 and 4E3 antibodies, suggesting a common epitope. However, recovery of the conformation-sensitive UIC2 epitope required replacement of thirteen residues in ECL1 and the same five residues replaced in the ECL4 for MRK-16 and 4E3 binding. These results demonstrate that discontinuous epitopes for MRK-16, UIC2 and 4E3 are located in the same regions of ECL1 and 4 of the multidrug transporter.

Structural models of NaChBac: Does the secondary structure of S4 change during gating?

NaChBac is a prokaryotic 6TM tetrameric voltage-gated sodium channel with a locus point in homology space connecting channels from all major voltage gated channels superfamilies. The voltage-sensing domain of NaChBac exhibits the familiar RxxRxxR motif of S4 and conserved negative residues on S2 and S3. Thus, the voltage sensing mechanism of NaChBac is probably shared with other voltage gated channels. We have used the crystal structure of the Kv1.2/2.1 chimera to model NaChBacs open conformation and that of the MlotiK channel to model its closed conformation. In the closed MlotiK structure the first part of S4 forms a 310 helix and the last part forms an α helix, whereas in the open Kv1.2/2.1 structure the first part is an α helix while the rest is a 310 helix. This elastic type of transition between secondary structures during gating can explain some apparent discrepancies regarding the magnitude of S4 motion reported for several potassium channels. However, this type of transition alone is not sufficient to explain the large gating charge movement reported for NaChBac and other channels. To account for this, we have incorporated the α-310 transition into the “helical screw model” in which the α-helix part of S4 moves in a screw-like fashion while the 310 part of S4 moves in a simple axial translation. In our models four positively charged residues of S4 moves outwardly during activation across a transition barrier formed by highly conserved hydrophobic residues on S1, S2, and S3. S4 movement is coupled to opening of the activation gate formed by S6 through interactions with the segment linking S4 to S5. Consistencies of our models with experimental studies of the NaChBac and Kv channels will be discussed.