Hydrophobic Stripes Research Articles

Synthesis and characterization of amphiphilic o-phenylene ethynylene oligomers

We have previously reported the synthesis of short o-phenylene ethynylene oligomers with polar triethylene glycol side chains which adopt a helical conformation in solution with three residues per turn. Two new oligomers have been synthesized, a hexamer and a nonamer, incorporating a repeated triad motif of polar–nonpolar–polar sidechains in order to create a hydrophobic stripe in the folded conformation which we report here for the first time. Helical folding in solution was observed and, unlike the previously-reported oligomers, these new oligomers are ordered solids at room temperature. Although these oligomers were designed to assemble into helical bundle-like structures, no evidence for a quaternary-like structure was found. The difference in polarity between alkyl and triethylene glycol side chains is likely not strong enough to induce self-association of folded helices, especially since the molecules are not water soluble where the driving force for association of the nonpolar stripe would be larger. We expect that more polar side chains, granting water solubility, represent an important target for future research.

Role of the Asymmetry of the Homodimeric b2 Stator Stalk in the Interaction with the F1 Sector of Escherichia coli ATP Synthase

The b subunit dimer in the peripheral stator stalk of Escherichia coli ATP synthase is essential for enzyme assembly and the rotational catalytic mechanism. Recent protein chemical evidence revealed the dimerization domain of b to contain a novel two-stranded right-handed coiled coil with offset helices. Here, the existence of this structure in more complete constructs of b containing the C-terminal domain, and therefore capable of binding to the peripheral F1-ATPase, was supported by the more efficient formation of intersubunit disulfide bonds between cysteine residues that are proximal only in the offset arrangement and by the greater thermal stabilities of cross-linked heterodimers trapped in the offset configuration as opposed to homodimers with the helices trapped in-register. F1-ATPase binding analyses revealed the offset heterodimers to bind F1 more tightly than in-register homodimers. Mutations near the C terminus of b were incorporated specifically into either the N-terminally or the C-terminally shifted polypeptide, bN or bC, respectively, to determine the contribution of each position to F1 binding. Deletion of the last four residues of bN substantially weakened F1 binding, whereas the effect of the deletion in bC was modest. Similarly, benzophenone maleimide introduced at the C terminus of bN, but not bC, mediated cross-linking to the delta subunit of F1. These results imply that the polypeptide in the bN position is more important for F1 binding than the one in the bC position and illustrate the significance of the asymmetry of the b dimer in the enzyme.

The Alexander Disease–Causing Glial Fibrillary Acidic Protein Mutant, R416W, Accumulates into Rosenthal Fibers by a Pathway That Involves Filament Aggregation and the Association of αB-Crystallin and HSP27

Here, we describe the early events in the disease pathogenesis of Alexander disease. This is a rare and usually fatal neurodegenerative disorder whose pathological hallmark is the abundance of protein aggregates in astrocytes. These aggregates, termed "Rosenthal fibers," contain the protein chaperones alpha B-crystallin and HSP27 as well as glial fibrillary acidic protein (GFAP), an intermediate filament (IF) protein found almost exclusively in astrocytes. Heterozygous, missense GFAP mutations that usually arise spontaneously during spermatogenesis have recently been found in the majority of patients with Alexander disease. In this study, we show that one of the more frequently observed mutations, R416W, significantly perturbs in vitro filament assembly. The filamentous structures formed resemble assembly intermediates but aggregate more strongly. Consistent with the heterozygosity of the mutation, this effect is dominant over wild-type GFAP in coassembly experiments. Transient transfection studies demonstrate that R416W GFAP induces the formation of GFAP-containing cytoplasmic aggregates in a wide range of different cell types, including astrocytes. The aggregates have several important features in common with Rosenthal fibers, including the association of alpha B-crystallin and HSP27. This association occurs simultaneously with the formation of protein aggregates containing R416W GFAP and is also specific, since HSP70 does not partition with them. Monoclonal antibodies specific for R416W GFAP reveal, for the first time for any IF-based disease, the presence of the mutant protein in the characteristic histopathological feature of the disease, namely Rosenthal fibers. Collectively, these data confirm that the effects of the R416W GFAP are dominant, changing the assembly process in a way that encourages aberrant filament-filament interactions that then lead to protein aggregation and chaperone sequestration as early events in Alexander disease.

Analysis of single droplet dynamics on striped surface domains using a lattice Boltzmann method

In this paper, the behavior of a micron-scale fluid droplet on a heterogeneous surface is investigated using a two-phase lattice Boltzmann method (LBM). The two-phase LBM permits the simulation of the time dependent three-dimensional motion of a liquid droplet on solid surface patterned with hydrophobic and hydrophilic strips. A nearest-neighbor molecular interaction force is used to model the adhesive forces between the fluid and solid walls. The solid heterogeneous wall is a uniform hydrophilic substrate painted with hydrophobic strips. The model is validated by demonstrating the consistency of the simulation results with an exact solution for capillary rise and through qualitative comparison of computed dynamic contact line behavior with experimentally measured surface properties and observed surface shapes of a droplet on a heterogeneous surface. The dependence of spreading behavior on wettability, the width of hydrophobic strip, initial location of the droplet relative to the strips, and gravity is investigated. A decrease in contact angle of the liquid on a hydrophilic surface may lead to breakup of the droplet for certain substrate patterns. The simulations suggest that the present lattice Boltzmann (LB) model can be used as a reliable way to study fluidic control on heterogeneous surfaces and other wetting related subjects.

Droplet dynamics on patterned substrates

We present a lattice Boltzmann algorithm which can be used to explore the spreading of droplets on chemically and topologically patterned substrates. As an example we use the method to show that the final configuration of a drop on a substrate comprising hydrophobic and hydrophilic stripes can depend sensitively on the dynamical pathway by which the state is reached. We also consider a substrate covered with micron-scale posts and investigate how this can lead to superhydrophobic behaviour. Finally we model how a Namibian desert beetle collects water from the wind.

Solution Structure of the Tctex1 Dimer Reveals a Mechanism for Dynein-Cargo Interactions

Tctex1 is a light chain found in both cytoplasmic and flagellar dyneins and is involved in many fundamental cellular activities, including rhodopsin transport within photoreceptors, and may function in the non-Mendelian transmission of t haplotypes in mice. Here, we present the NMR solution structure for the Tctex1 dimer from Chlamydomonas axonemal inner dynein arm I1. Structural comparisons reveal a strong similarity with the LC8 dynein light chain dimer, including formation of a strand-switched beta sheet interface. Analysis of the Tctex1 structure enables the dynein intermediate chain binding site to be identified and suggests a mechanism by which cargo proteins might be attached to this microtubule motor complex. Comparison with the alternate dynein light chain rp3 reveals how the specificity of dynein-cargo interactions mediated by these dynein components is achieved. In addition, this structure provides insight into the consequences of the mutations found in the t haplotype forms of this protein.

Immobilization and stretching of DNA molecules above a lithographed surface

We present a method to obtain numerous stretched DNA molecules above a patterned surface. Using standard micro-lithography procedures, we made a patterned surface allowing the fixation DNA molecules by one end on functional hydrophobic strips. DNA molecules are stretched by a hydrodynamic flow. Under these conditions, we observe labeled elongated DNA molecules attached by their ends between the hydrophobic strips above the lithographed surface. We anticipate that the simplicity of this technique will lead to the development of numerous applications, such as the study of protein–DNA interactions at the single molecule level.

Solution NMR Study of the Interaction Between NTF2 and Nucleoporin FxFG Repeats

Interactions with nucleoporins containing FxFG repeat cores are crucial for the nuclear import of RanGDP mediated by nuclear transport factor 2 (NTF2). We describe here a solution NMR-based study that identifies primary and secondary FxFG-binding sites on NTF2 and accounts for a range of observations on the rate of NTF2 nuclear trafficking. We used three complementary NMR methods, namely amide group chemical shift titrations, NOE and cross-saturation measurements, to show that the major FxFG-binding site on the dimeric rat NTF2 (rNTF2) molecule is centred on Trp7 and is formed by residues from both NTF2 chains. A secondary FxFG-binding site is located at the rNTF2 hydrophobic cavity and these two sites, together with a surface hydrophobic cluster centred on Trp112, merge into an elongated hydrophobic stripe on the rNTF2 surface. The primary site centred on Trp7 is lost in the rNTF2-W7A mutant that has been shown to bind FxFG nucleoporins with greatly reduced affinity, whereas the secondary site at the rNTF2 hydrophobic cavity is retained. The interface between NTF2 and FxFG nucleoporins detected by NMR is more extensive than that detected by X-ray crystallography, and the presence of a secondary site at the NTF2 hydrophobic cavity accounts for the unexpectedly rapid nuclear import of rNTF2-W7R recently observed by others. The structure of the binding interfaces on these transport factors provides a rationale for the specificity of their interactions with nucleoporins that, combined with their weak binding constants, facilitates rapid translocation through NPCs during nuclear trafficking.

Impact of Line Tension on the Equilibrium Shape of Liquid Droplets on Patterned Substrates

We studied the morphology of liquid droplets on substrates with a lateral wettability pattern using numerical calculations. We analyzed the influence of the wettability contrast, the sharpness of the transitions between adjacent hydrophilic and hydrophobic stripes, and the line tension of the three-phase contact line on the modulation amplitude of the latter and on the shape of the liquid-vapor interface. In the presence of lateral variations of the line tension, we found that the modulation of the contact angle along the contact line is controlled by the local wettability of the substrate, by the local curvature of the contact line, and by gradients of the line tension. This result confirms a recently published theoretical extension of the modified Young equation.1 Furthermore, the numerical calculations demonstrate that and show how the line tension can be extracted from atomic force microscopy measurements of liquid droplets on patterned substrates.

Water-Soluble Poly(ferrocenylsilanes) for Supramolecular Assemblies by Layer-by-Layer Deposition

We report on the fabrication and characterization of fully organometallic multilayer thin films, composed of poly(ferrocenylsilane) polyanions and polycations. These polyions were deposited electrostatically onto a variety of substrates including quartz, silicon, gold, and hydrophilic/hydrophobically patterned substrates, using layer-by-layer self-assembly. The deposition process was monitored by means of UV/visible absorption spectroscopy, showing a linear increase in absorption with the number of bilayers. Ellipsometry was used to measure the development of film thickness with the number of bilayers, revealing a linear relationship and a thickness contribution of approximately 0.4 nm/bilayer. The multilayer films were further characterized by X-ray photoelectron spectroscopy (XPS) and by cyclic voltammetry. By integration of the voltammetric signals, the surface concentration of the redox-active ferrocene units was obtained as a function of the number of poly(ferrocenylsilane) bilayers. Selective adsorption of the polyions was achieved onto the hydrophobic stripes of a pattern of CH3- and OH-terminated alkanethiol monolayers on gold, forming patterned organometallic multilayer structures. This selective deposition was explained by taking hydrophobic and hydrogen-bonding interactions into account. The hydrophobic backbone of the poly(ferrocenylsilane) polyions has favorable hydrophobic interactions with the methyl-terminated areas of the patterned substrate but not with hydroxyl-terminated domains, which are hydrogen-bonded with the solvent. The dipping sequence does not influence the selectivity of the multilayer deposition.

Surface Pattern Recognition by a Colloidal Particle

This paper's focus is on understanding how surface heterogeneity alters surface forces. An atomic force microscope was used to measure interactions between a 20 μm hydrophobic (−CH3) sphere and a patterned self-assembled monolayer surface of alkanethiols that consists of hydrophobic (−CH3) and hydrophilic (−COOH) stripes of controlled width from 0.27 to 2.9 μm. Of significance to this study is the ratio of the particle radius to the stripe size. This ratio varied from 3.5 to 37, as a heterogeneous patterning on a length scale much larger than the sphere cannot affect the force. The results demonstrate that hydrophobic and hydrophilic interactions are not additive and the net interaction between these kind of surfaces depends on the relative size of hydrophobic and hydrophilic sites on the surface. Independent of whether the hydrophobic sphere is positioned on top of a hydrophobic or hydrophilic area, we measure repulsions, with the same strength and decay length of the electrostatic interaction. The magnitude of the heterogeneous energy is greatest when the patch size is smallest. Our results indicate that interactions, jump into contact distances and adhesion data, between a 20 μm methyl terminated sphere and a heterogeneous surface that has equal methyl and carboxylic acid areas will average. In all other cases, when the surface has unequal amounts of hydrophobic and hydrophilic areas, the hydrophobic sphere will clearly differentiate between the different patches and will accurately map high and low adhesion areas on the surface.

Complementary packing of α-helices in proteins

The packing of α-helices in proteins is restricted by both the principle of close packing and the chemical nature of side chains. As a result, (1) α-helical surfaces forming the interface should be complementary to each other, (2) hydrophobic stripes of the α-helices should fit together like pieces of a jigsaw puzzle, and (3) buried polar side chains (if there are any) should be arranged in a complementary fashion.

Selectivities among capillary bonds in mesoscale self-assembly

This letter describes the capillarity-driven self-assembly of mm-sized plates of poly(dimethylsiloxane) at the perfluorodecalin/H2O interface into complex arrays. The shape of the interacting menisci could be tailored by three strategies: (1) changing the shape of uniformly hydrophobic edges; (2) patterning the distribution of vertical hydrophobic strips on the edges and, in some cases, varying the density of the blocks; and (3) patterning the hydrophobic areas on a face to be chiral.

Mapping the active site of CD59.

CD59 is a widely distributed membrane-bound inhibitor of the cytolytic membrane attack complex (MAC) of complement. This small (77 amino acid) glycoprotein is a member of the Ly6 superfamily of proteins and is important in protecting host cells from the lytic and proinflammatory activity of the MAC. CD59 functions by binding to C8 and/or C9 in the nascent MAC and interfering with C9 membrane insertion and polymerization. We present data obtained from a combination of molecular modeling and mutagenesis techniques, which together indicate that the active site of CD59 is located in the vicinity of a hydrophobic groove on the face of the molecule opposite to a "hydrophobic strip" suggested earlier. In addition, removal of the single N-linked glycosylation site at Asn18 of CD59 resulted in an enhancement of complement inhibitory activity.

1H NMR studies of prototypical helical designer peptides. A comparative study of the amide chemical shift dependency on temperature and polypeptide sequence.

A series of designer alpha-helical peptides with hydrophobic residues located at different positions along the sequence (PH-1.0 = LYQELQKLTQTLK, PH-1.19 = LYQELQKLTQTFK, PH-1.12 = LYQELQKLLQTLK, PH-1.13 = LYQELQKLTLTLK, PH-1.4 = LYQELQKLTQTTK) were analyzed using one- and two-dimensional NMR methods (TOCSY and NOESY). The central feature of these designer peptides is the incorporation of a maximal hydrophobic strip which may play a role in antigen processing and the nucleation of alpha-helices in proteins (J. Immunol. 145, 899, 1990). Using the 2D-NMR, sequence specific assignments and NOE connectivities were determined in all peptides when dissolved in H2O/TFE mixtures. NOE connectivities indicated that all these peptides are helical in this medium. An unusually large number of NOEs was found for all these designer peptides. This is in accord with ultracentrifugation studies that showed that PH-1.0 forms a trimer in 50% H2O/TFE mixtures. Other peptides in the series behave in similar manner as PH-1.0. The structural differences among these peptides was addressed using the backbone amide chemical shift temperature coefficients, [symbol: see text], and the differences between the observed and random coil values, delta delta HN. The delta delta HN patterns along the peptide sequence are consistent with those expected for amphiphilic alpha-helices, where most delta delta HN values are below zero. However, no significant differences among the peptides in this series can be detected on the delta delta HN patterns, with the exception of PH-1.12. The [symbol: see text] values reveal differences among the peptides of the series. The patterns of [symbol: see text] along the peptide sequences are similar to that found for delta delta HN for PH-1.0, PH-1.19 and PH-1.4. The other peptides in the series, PH-1.12 and PH-1.13, showed different patterns for [symbol: see text]. The latter parameter was used to evaluate the helicity of this series of peptides. According to this parameter the relative helicity of this series is as follows: PH-1.12 > PH-1.0 > PH-1.4 > PH-1.19 > PH-1.13 The NMR data shown here correlated well with the helical propensities predicted for polypeptide sequences using statistical arguments (Proc. Natl. Acad. Sci. USA, 90, 9100, 1993).

Wetting characteristics of liquid drops at heterogeneous surfaces

Well-defined heterogeneous surfaces consisting of hydrophobic and hydrophilic regions were prepared on gold (a 2000 Å gold film supported on an Si/SiO 2/Ti substrate) by patterning self-assembled monolayers (SAMs), using an elastomer stamp. One surface was composed of alternating and parallel hydrophobic (2.5 μm) and hydrophilic (3 μm) strips, and the second surface consisted of alternating hydrophilic squares (3 μm × 3 μm) separated by hydrophobic strips (2.5 μm). The wetting characteristics of these well-defined heterogeneous solid surfaces were examined by contact angle measurements. The contact angles for water drops, which varied in pH from 5.8 to 10.0, were measured with the strips both tangential to and normal to the three-phase contact line. The experimental contact angles are in good agreement with theory as calculated from the Cassie equation when the three-phase contact line is non-contorted (i.e. the three-phase contact line is situated along the hydrophobic strip). On the other hand, when the strips are normal to the drop edge, corrugation of the three-contact line affects the contact angle significantly. Contact angles, measured with the strips normal to the drop edge, were lower by 7–16° than those calculated from the Cassie equation. Analysis of these measurements, together with contact angle/drop size measurements for fully hydrophobic and hydrophilic surfaces, demonstrate the validity of a modified Cassie equation that includes a term describing the line tension contribution.

Prediction of α-Helices in Proteins with the Hydrophobic Strip-of-Helix Template and Distributions of Other Amino Acids around the Hydrophobic Strip

Upon addition of requirements for highly characteristic distributions of helix-stabilizing residues to an α-helix predictor based upon identification of a longitudinal, hydrophobic strip-of-helix pattern, maximal sensitivity and efficiency scores approached 50% levels at a high degree of stringency. The hydrophobic strip-of-helix template (□○○○□○○□○○○□○○□○○○, joined in a circle) was applied to 247 helices to maximize the strip-of-helix hydrophobicity index (SOHHI; the mean hydrophobicity of residues in □ positions). Statistically significant increases or decreases in the frequencies of certain residues are observed in some □ and ○ positions: □ in the helix (including N- and C-terminal □ in the helix): Leu, Ile, Val, Phe, and Met; first □ positions in extensions of the template to surrounding segments: not increased Leu, Ile, Val, Phe, or Met; N-terminal ○: Asp, Glu; C-terminal ○ and the first □ after the helix: His, Lys, Arg; N-terminal ○: Asn; C-terminal ○: Gln; smallest residue in longitudinal strip-of-helix: Ala or Val at crossing points between helices. An algorithm was then derived to indicate α-helices by merging predictions with templates ○○○□○○○□○○□○○ and ○○○□○○□○○○□○○, where the SOHHI ≥ 3.0 in the Kyte-Doolittle scale. Relative to the baseline prediction using only the template, more elaborate rules using combinations of other structural patterns produced more efficient (49 versus 42%, respectively) but less sensitive (32 versus 42%, respectively) predictions when the prediction was required to overlap substantially with the known helix. These maximal sensitivities and efficiencies imply that some helices may be formed in folding intermediates but are lost upon coalescence of the final form. Better predictions of protein structure from the primary sequence may require modeling of competing interactions of local structures in folding intermediates.

A repeating 11‐mer amino acid motif and plant desiccation

Among the proteins that accumulate as plant seeds desiccate are several protein families that are composed principally of a tandemly repeated 11-mer amino acid motif. Proteins containing the same motif accumulate in the desiccating leaves of a desiccation-tolerant plant species. This motif is characterized by apolar residues in positions 1, 2, 5 and 9, and charged or amide residues in positions 3, 6, 7, 8 and 11. An alpha helical arrangement of the 11-mer repeating unit gives an amphiphilic helix whose hydrophobic stripe twists in a right-handed fashion around the helix. Should these proteins dimerize via binding of their hydrophobic faces, a right-handed coiled coil would be formed. Such a structure has not previously been observed. A conceivable function for these proteins in ion sequestration in the desiccated state is proposed.

Adsorption and helical coiling of amphipathic peptides on lipid vesicles leads to negligible protection from cathepsin B or cathepsin D.

The processing of antigenic peptides for presentation by MHC molecules to T cells, may depend upon the function of a second, consensus sequence in or near the T cell-presented epitope. One such processing-regulating sequence appears to be composed of amino acids Leu, Ile, Val, Phe, and Met recurring in a fashion to form a longitudinal, hydrophobic strip when the excised peptide is coiled as an alpha-helix. Such a hydrophobic strip-of-helix may: (a) scavenge peptides from lumens onto lipid membranes of digestion vesicles, (b) stabilize peptides there as protease-resistant helices, (c) specify recognition by the antigenic peptide-binding sites of chaperonin proteins, transmembranal transporters, or MHC molecules. By circular dichroism and electron paramagnetic resonance, we demonstrated that peptides with recurrent hydrophobic residues potentially forming longitudinal strips adsorbed to, and partially coiled as helices on, di-O-hexadecyl, D-L-alpha-phosphatidylcholine (DHPC) vesicles. Cathepsin B or cathepsin D cleavages of three such peptides were identified. With either enzyme, it made no significant difference whether a peptide substrate was in solution or bound to vesicles in terms of efficiency and specificity of peptide bond cleavages. We conclude that protease resistance, per se, of membrane-adsorbed, helically coiled peptides is not a major factor in the selection for T cell presentation of epitopes in peptides which have a motif with a longitudinal hydrophobic strip.

Critical functional role of the COOH-terminal ends of longitudinal hydrophobic strips in alpha-helices of T4 lysozyme.

The sensitivity of bacteriophage T4 lysozyme function to amino acid substitutions at defined positions in and around the longitudinal, hydrophobic strips of 9 alpha-helices was assessed after systematic replacement of each residue in the protein with a series of 13 amino acids. The hydrophobic strips were defined by identifying the longitudinal sectors in the helices with the highest mean residue hydrophobicities. Sensitivity to mutation (the percentage of replacements leading to loss of function) was calculated for each residue in the following positions: whole protein, helices, hydrophobic strips, other positions within the helices, and various positions within the hydrophobic strips as well as their extensions beyond the helices. Substitutions at positions in the hydrophobic strips led more frequently to loss of function than substitutions in the protein as a whole. One subset, the COOH-terminal hydrophobic strip residues, is apparently critical; substitutions of these residues (but not of their NH2-terminal counterparts) led at least as frequently to loss of function as substitutions of solvent-inaccessible residues, and nearly as frequently as substitutions of the most highly conserved residues.