Hydrophilic Patch Research Articles

Structural basis of intramitochondrial phosphatidic acid transport mediated by Ups1-Mdm35 complex.

Ups1 forms a complex with Mdm35 and is critical for the transport of phosphatidic acid (PA) from the mitochondrial outer membrane to the inner membrane. We report the crystal structure of the Ups1-Mdm35-PA complex and the functional characterization of Ups1-Mdm35 in PA binding and transfer. Ups1 features a barrel-like structure consisting of an antiparallel β-sheet and three α-helices. Mdm35 adopts a three-helical clamp-like structure to wrap around Ups1 to form a stable complex. The β-sheet and α-helices of Ups1 form a long tunnel-like pocket to accommodate the substrate PA, and a short helix α2 acts as a lid to cover the pocket. The hydrophobic residues lining the pocket and helix α2 are critical for PA binding and transfer. In addition, a hydrophilic patch on the surface of Ups1 near the PA phosphate-binding site also plays an important role in the function of Ups1-Mdm35. Our study reveals the molecular basis of the function of Ups1-Mdm35 and sheds new light on the mechanism of intramitochondrial phospholipid transport by the MSF1/PRELI family proteins.

Molecular insight into the dynamical adsorption behavior of nanoscale water droplets on a heterogeneous surface

A heterogeneous surface is constructed by adding one hydrophilic patch at the center of a hydrophobic surface, and the dynamical adsorption process of nanoscale water droplets is investigated adopting molecular dynamics simulations.

Particle Extraction in Plug-based Microfluidics

We describe a novel method to extract magnetic particles from plugs in plug-based microfluidic devices. In general, the extraction of particles from plugs is difficult as the high surface energy of an aqueous phase dispersed in a non-polar carrier liquid usually prevents the penetration of particles through this interface. Key to our approach is to achieve particle extraction by generating channel surface consisting of a hydrophilic patch in a hydrophobic surrounding: The interfaces of plugs contacting this patch, merge with the patch-interface enabling easy extraction. This approach lowers the limit of the concentration of particles, which can be still successfully extracted by more than three orders of magnitude compared to the state of the art.

Colloidosomes formed by nonpolar/polar/nonpolar nanoball amphiphiles.

Fullerene-based amphiphiles are able to form bilayer vesicles in aqueous solution. In this study, the self-assembly behavior of polymer-tethered nanoballs (NBs) with nonpolar/polar/nonpolar (n-p-n') motif in a selective solvent is investigated by dissipative particle dynamics. A model NB bears two hydrophobic polymeric arms (n'-part) tethered on an extremely hydrophobic NB (n-part) with hydrophilic patch (p-part) patterned on its surface. Dependent on the hydrophobicity and length of tethered arms, three types of aggregates are exhibited, including NB vesicle, core-shell micelle, and segmented-worm. NB vesicles are developed for a wide range of hydrophobic arm lengths. The presence of tethered arms perturbs the bilayer structure formed by NBs. The structural properties including the order parameter, membrane thickness, and area density of the inner leaflet decrease with increasing the arm length. These results indicate that for NBs with longer arms, the extent of interdigitation in the membrane rises so that the overcrowded arms in the inner corona are relaxed. The transport and mechanical properties are evaluated as well. As the arm length grows, the permeability increases significantly because the steric bulk of tethered arms loosens the packing of NBs. By contrast, the membrane tension decreases owing to the reduction of NB/solvent contacts by the polymer corona. Although fusion can reduce membrane tension, NB vesicles show strong resistance to fusion. Moreover, the size-dependent behavior observed in small liposomes is not significant for NB vesicles due to isotropic geometry of NB. Our simulation results are consistent with the experimental findings.

Molecular mechanism of strigolactone perception by DWARF14

The terpenoid, small-compound strigolactones (SLs) are plant hormones that regulate plant shoot branching, which is an important agronomic trait that determines crop yields. An α/β-hydrolase protein, DWARF14 (D14), has been recognized to be an essential component of plant SL signaling. Recently, it has been demonstrated that D14 interacts with a gibberellin (GA)-signaling repressor SLR1 in an SL-dependent manner [1], which suggests that SLR1 mediates crosstalk between the SL and GA signalings in the regulation of plant shoot branching. Although D14 functions in SL perception to promote the interaction with SLR1, its molecular mechanism remains unclear. Here, we report the crystal structure of D14 in the complex with 5-hydroxy-3-methylbutenolide (D-OH), which is a reaction product of SLs. The structure was solved at a 2.10-Å resolution when an SL synthetic analogue, (–)-ent-2'-epi-GR7, was soaked into D14 crystals [1]. In the complex structure, D-OH was located at a site far from the catalytic residues including H297 and appeared to function as a plug for the catalytic cavity to induce an overall hydrophobic surface with a hydrophilic patch between the two α-helices in the cap structure of D14. In the binding site, the indole amine of Trp205 formed a hydrogen bond with the oxygen atom of the C2' hydroxy group, which arose from the catalytic reaction of D14, instead of a water molecule in the structure of apo D14. In addition, the side chain of Phe245 moved 1.3 Å toward D-OH. Mutational analyses of D14 showed that the interaction between D14 and SLR1 required an enzymatic activity of D14 and the residues Trp205 and Phe245 were essential for the SL-dependent SLR1-binding of D14. These results suggest that the D14–D-OH complex mediates the interaction with SLR1 in which the D-OH-induced surface and/or structural change is crucial.

Development of a Novel Transdermal Delivery System of Alendronate, a Nitrogen Containing Bisphosphonate, Using a New Type of Hydrophilic Patch and Dissolving Microneedle Arrays

Alendronate is a nitrogen-containing bisphosphonate that is widely used for the treatment of osteoporosis in postmenopausal women. However, the oral bioavailability (BA) of alendronate is approximately 0.9 to 1.8%. In addition, the oral administration of alendronate has been associated with mucosal damage including gastritis, gastric ulcer, and erosive esophagitis. To prevent these adverse effects of alendronate, patients with osteoporosis should sit up or walk for more than 30 min after oral administration, leading to poor compliance and QOL in these patients. To improve the compliance and QOL of these patients, we developed a new transdermal delivery system for alendronate using a new type of hydrophilic patch and dissolving microneedle arrays. The skin permeation of alendronate observed using our transdermal delivery system was sufficient for the treatment of osteoporosis in rat models. These findings indicate that our transdermal delivery system of alendronate is a promising approach that may result in improved therapeutic effects and better compliance and QOL in patients undergoing treatment for osteoporosis.

Structural characterization of a ligand‐bound form of Bacillus subtilis FadR involved in the regulation of fatty acid degradation

Bacillus subtilis FadR (FadR(Bs)), a member of the TetR family of bacterial transcriptional regulators, represses five fad operons including 15 genes, most of which are involved in β-oxidation of fatty acids. FadR(Bs) binds to the five FadR(Bs) boxes in the promoter regions and the binding is specifically inhibited by long-chain (C14-C20 ) acyl-CoAs, causing derepression of the fad operons. To elucidate the structural mechanism of this regulator, we have determined the crystal structures of FadR(Bs) proteins prepared with and without stearoyl(C18)-CoA. The crystal structure without adding any ligand molecules unexpectedly includes one small molecule, probably dodecyl(C12)-CoA derived from the Escherichia coli host, in its homodimeric structure. Also, we successfully obtained the structure of the ligand-bound form of the FadR(Bs) dimer by co-crystallization, in which two stearoyl-CoA molecules are accommodated, with the binding mode being essentially equivalent to that of dodecyl-CoA. Although the acyl-chain-binding cavity of FadR(Bs) is mainly hydrophobic, a hydrophilic patch encompasses the C1-C10 carbons of the acyl chain. This accounts for the previous report that the DNA binding of FadR(Bs) is specifically inhibited by the long-chain acyl-CoAs but not by the shorter ones. Structural comparison of the ligand-bound and unliganded subunits of FadR(Bs) revealed three regions around residues 21-31, 61-76, and 106-119 that were substantially changed in response to the ligand binding, and particularly with respect to the movements of Leu108 and Arg109. Site-directed mutagenesis of these residues revealed that Arg109, but not Leu108, is a key residue for maintenance of the DNA-binding affinity of FadR(Bs).

A hydrophilic polymer based microfluidic system with planar patch clamp electrode array for electrophysiological measurement from cells

This paper presents a microfluidic planar patch clamp system based on a hydrophilic polymer poly(ethylene glycol) diacrylate (PEGDA) for whole cell current recording. The whole chip is fabricated by UV-assisted molding method for both microfluidic channel structure and planar electrode partition. This hydrophilic patch clamp chip has demonstrated a relatively high gigaseal success rate of 44% without surface modification compared with PDMS based patch clamp devices. This chip also shows a capability of rapid intracellular and extracellular solution exchange with high stability of gigaseals. The capillary flow kinetic experiments demonstrate that the flow rates of PEGDA microfluidic channels are around two orders of magnitude greater than those for PDMS-glass channels with the same channel dimensions. This hydrophilic polymer based patch clamp chips have significant advantages over current PDMS elastomer based systems such as no need for surface modification, much higher success rate of cell gigaseals and rapid solution exchange with stable cell gigaseals. Our results indicate the potential of these devices to serve as useful tools for pharmaceutical screening and biosensing tasks.

Facile, Solution-Based Synthesis of Soft, Nanoscale Janus Particles with Tunable Janus Balance

We present a novel, versatile, and simple solution-based routine to produce soft, nanosized Janus particles with tunable structural and physical properties at high volume yield. This process is based on the cross-linking of compartments within precisely defined multicompartment micelles (MCMs), which are themselves formed by the self-assembly of ABC triblock terpolymers. Therein, the C blocks form the stabilizing corona emanating from B compartments, which in turn reside on an A core. Cross-linking of the B compartments allows to permanently fixate the phase-separated state and dissolution in a good solvent for all blocks breaks up the MCMs into single Janus particles. They now consist of a core of cross-linked B blocks and two phase-separated hemispheres of A and C. The process gives access to unprecedented structural features such as tunable core diameter and control over the Janus balance ranging from dominant A side to equal hemispheres to dominant C side. We demonstrate that this simple one-pot approach can be extended to a range of triblock terpolymers with different block lengths and block chemistries to furnish a library of tailor-made Janus particles with widely tunable physical properties. Such a diversity and simplicity has remained unreachable with our previously developed approach using the controlled cross-linking of bulk morphologies. We show that this new synthetic route can be upscaled to a high volume yield of 10 wt %, thereby enabling large-scale applications. We further demonstrate the effect of the Janus balance on colloidal self-assembly. Janus particles with a dominant hydrophobic and a small hydrophilic patch aggregate into large clusters in water, but merely di- or trimerize in chloroform.

46.3: Improved Oil Motion Control and Hysteresis‐Free Pixel Switching of Electrowetting Displays

AbstractHysteresis‐free pixel switching and a high control of the fluid flow are beneficial for making optimal use of electrowetting display technology. As described in the paper, the pixel design is shown to play a dominant role to optimize the pixel switching characteristics and to allow improved gray‐scale reproducibility. Use of pixel features such as a hydrophilic patch or a staircase, are shown to be effective. Advantages and drawbacks of these methods are discussed.

Metastable Sessile Nanodroplets on Nanopatterned Surfaces

Small aqueous droplets on homogeneous surfaces surrounded by a reservoir of vapor are inherently unstable. Depending on the humidity, they keep evaporating and ultimately disappear or grow until they fully wet the surface under supersaturation. We are considering a system departing from this common picture. For nanoscale droplets sitting above hydrophilic patches on a heterogeneous surface, there can exist a range of supersaturated pressures at which the droplets maintain a stable volume, determined by the pertinent contact angle and the size of the patches. The region under the droplet perimeter controls the drop’s curvature. Vapor pressure rises along with increased curvature as soon as the drop extends into the hydrophobic area. The drop size may therefore remain stable when its base just covers the hydrophilic patch. The finite range of water–substrate interactions, however, blurs the boundaries between surface regions with different hydrophilicities; hence, the nanodrop contact angle varies with the patch size in a gradual manner. We use molecular simulations to examine this dependence on model surfaces with either chemical or topological heterogeneities. For both types of heterogeneities, our results show the contact angle of a nanodroplet can be predicted by the local Cassie–Baxter mixing relation applied to the area within the interaction range from the drop’s perimeter, which, in turn, enables predictions for drop condensation and saturated vapor pressure above partially wetted nanopatterned hydrophilic/hydrophobic surfaces.

A Simple Approach for Local Contact Angle Determination on a Heterogeneous Surface

We report a simple approach for measuring the local contact angle of liquids on a heterogeneous surface consisting of intersected hydrophobic and hydrophilic patch arrays, specifically by employing confocal microscopy and the addition of a very low concentration of Rhodamine-B (RB) (2 × 10(-7) mol/L). Interestingly, RB at that concentration was found to be aggregated at the air-liquid and solid (hydrophobic patch only)-liquid interfaces, which helps us to distinguish the liquid and solid interfaces as well as hydrophobic and hydrophilic patches by their corresponding fluorescent intensities. From the measured local contact angles, the line tension can be easily derived and the value is found to be (-2.06-1.53) × 10(-6) J/m.

Rational Design of Tryptophan‐Rich Antimicrobial Peptides with Enhanced Antimicrobial Activities and Specificities

Trp-rich antimicrobial peptides play important roles in the host innate defense mechanism of many plants and animals. A series of short Trp-rich peptides derived from the C-terminal region of Bothrops asper myothoxin II, a Lys49 phospholipase A(2) (PLA(2)), were found to reproduce the antimicrobial activities of their parent molecule. Of these peptides, KKWRWWLKALAKK-designated PEM-2-was found to display improved activity against both Gram-positive and Gram-negative bacteria. To improve the antimicrobial activity of PEM-2 for potential clinical applications further, we determined the solution structure of PEM-2 bound to membrane-mimetic dodecylphosphocholine (DPC) micelles by two-dimensional NMR methods. The DPC micelle-bound structure of PEM-2 adopts an α-helical conformation and the positively charged residues are clustered together to form a hydrophilic patch. The surface electrostatic potential map indicates that two of the three tryptophan residues are packed against the peptide backbone and form a hydrophobic face with Leu7, Ala9, and Leu10. A variety of biophysical and biochemical experiments, including circular dichroism, fluorescence spectroscopy, and microcalorimetry, were used to show that PEM-2 interacted with negatively charged phospholipid vesicles and efficiently induced dye release from these vesicles, suggesting that the antimicrobial activity of PEM-2 could be due to interactions with bacterial membranes. Potent analogues of PEM-2 with enhanced antimicrobial and less pronounced hemolytic activities were designed with the aid of these structural studies.

Solution Structure and Model Membrane Interactions of P‐113, a Clinically Active Antimicrobial Peptide Derived from Human Saliva

AbstractP‐113, AKRHHGYKRKFH‐NH2, was derived from human saliva and found to possess clinical activity against fungus infections in HIV patients with oral candidiasis. We have determined the solution structure of P‐113 bound to membrane‐mimetic SDS micelles by two‐dimensional NMR methods. The SDS micelle‐bound structure of P‐113 adopts an α‐helical segment and the positively charged residues are clustered together to form a hydrophilic patch. A variety of biophysical and biochemical experiments, including circular dichroism, fluorescence spectroscopy and microcalorimetry, were used to show that P‐113 interacted with negatively charged phospholipid vesicles and induced dye release from these vesicles. However, its dye leakage efficiency is much less than the results of previously reported antimicrobial peptides. These results suggest that the antimicrobial activity of P‐113, unlike other antimicrobial peptides, may act not only through binding to and destabilization of the microbial membrane but also through a specific protein receptor on the microbial cell surface.

Multivariate Analysis of Conserved Sequence–Structure Relationships in Kinesins: Coupling of the Active Site and a Tubulin-binding Sub-domain

An extensive computational analysis of available sequence and crystal structure data was used to identify functionally important residue interactions within the motor domain of the kinesin molecular motor. Principal component analysis revealed that all current kinesin crystal structures reside in one of two main conformations, which differ at the active site, and in the position of a microtubule-binding sub-domain relative to a rigid central core. This sub-domain consists of secondary structure elements α4-loop12-α5-loop13 and contains a conserved hydrophilic surface patch that may be involved in strong binding to microtubules. A hinge point for the sub-domain motion lies near a conserved glycine at position 292. Statistical coupling analysis revealed a network of co-evolving positions that link this region to the nucleotide-binding site, via a highly conserved histidine in the switch I loop. The data are consistent with a model in which the nucleotide status of the active site shifts kinesin between weak and strong binding conformations via reconfiguration of the identified sub-domain. Our data provide a statistically supported framework for further examination of this and other structure–function relationships in the kinesin family.

Solution structure of a novel D‐naphthylalanine substituted peptide with potential antibacterial and antifungal activities

A new type of Trp-rich peptide, Ac-KWRRWVRWI-NH2, designated as Pac-525, was found to possess improved activity against both gram-positive and negative bacteria. We have synthesized two Pac-525 analogues, D-Pac-525 containing all D-amino acids and D-Nal-Pac-525, the D-Pac-525 analogue with tryptophan replaced by D-beta-naphthylalanine. We have determined the solution structure of D-Nal-Pac-525 bound to membrane-mimetic DPC micelles by two-dimensional NMR methods. The DPC micelle-bound structure of D-Nal-Pac-525 adopts a left-hand alpha-helical segment and the positively charged residues are clustered together to form a hydrophilic patch. The surface electrostatic potential map indicates the three D-beta-naphthylalanines are packed against the peptide backbone and form an amphipathic structure. A variety of biophysical and biochemical experiments, including circular dichroism, fluorescence spectroscopy, and microcalorimetry, were used to show that D-Nal-Pac-525 interacted strongly with negatively charged phospholipid vesicles and induced efficient dye release from these vesicles, suggesting that the strong antimicrobial activity of D-Nal-Pac-525 may be due to interactions with bacterial and fungus membranes.

Solution Structure of Mouse Cripto CFC Domain and Its Inactive Variant Trp107Ala

We report here for the first time the solution structures at pH 3 and pH 6 of the synthetic CFC domain of mouse Cripto and of the point mutated variant W107A that is unable to bind to the Alk4 Cripto receptor. NMR data confirm that the CFC domain has a C1-C4, C2-C6, C3-C5 disulfide pattern and show that structures are rather flexible and globally extended, with three noncanonical antiparallel strands. His104 and Trp107 side chains protrude from a protein edge and are strongly exposed to solvent, supporting previous evidence of direct involvement in receptor binding. On the opposite molecule side, several nonpolar residues are gathered, forming a large hydrophobic patch that supposedly acts as interface with the cell membrane or the adjacent EGF-like domain. A second hydrophilic patch surrounding His104 and Trp107 is present only in the wild type variant, suggesting a possible involvement in modulating Alk4 recognition.

Synthetic Ligands Able to Interact with the P53 Tetramerization Domain. Towards Understanding a Protein Surface Recognition Event

The applied interaction of synthetic molecules with defined regions of protein surfaces is an emerging strategy for the modulation of protein activity and/or stability. In spite of recent advances, the design of these molecules is not trivial. Among the most challenging aspects in designing these compounds is that they must compete with water molecules for interaction with polar patches of protein surfaces. Herein is reported the preparation of an arginine-rich peptide that interacts in aqueous solution with a very hydrophilic patch at the surface of the tetramerization domain of the tumor suppressor protein p53. The interaction has been studied by several complementary techniques. By using this peptide as a template, a library of peptides has been prepared and evaluated in order to examine the different factors that contribute to the recognition event. The conclusions extracted from this work could be useful for the design of ligands directed at highly hydrophilic protein surface patches.

Solution Structure of a Novel Tryptophan-Rich Peptide with Bidirectional Antimicrobial Activity

Trp-rich antimicrobial peptides play important roles in the host innate defense mechanisms of many plants, insects, and mammals. A new type of Trp-rich peptide, Ac-KWRRWVRWI-NH(2), designated Pac-525, was found to possess improved activity against both gram-positive and -negative bacteria. We have determined that the solution structures of Pac-525 bound to membrane-mimetic sodium dodecyl sulfate (SDS) micelles. The SDS micelle-bound structure of Pac-525 adopts an alpha-helical segment at residues Trp2, Arg3, and Arg4. The positively charged residues are clustered together to form a hydrophilic patch. The three hydrophobic residues Trp2, Val6, and Ile9 form a hydrophobic core. The surface electrostatic potential map indicates the three tryptophan indole rings are packed against the peptide backbone and form an amphipathic structure. Moreover, the reverse sequence of Pac-525, Ac-IWRVWRRWK-NH(2), designated Pac-525(rev), also demonstrates similar antimicrobial activity and structure in membrane-mimetic micelles and vesicles. A variety of biophysical and biochemical methods, including circular dichroism, fluorescence spectroscopy, and microcalorimetry, were used to show that Pac-525 interacted strongly with negatively charged phospholipid vesicles and induced efficient dye release from these vesicles, suggesting that the antimicrobial activity of Pac-525 may be due to interactions with bacterial membranes.

Structural Insights into Endosomal Sorting Complex Required for Transport (ESCRT-I) Recognition of Ubiquitinated Proteins

The endosomal sorting complex required for transport (ESCRT-I) is a 350-kDa complex of three proteins, Vps23, Vps28, and Vps37. The N-terminal ubiquitin-conjugating enzyme E2 variant (UEV) domain of Vps23 is required for sorting ubiquitinated proteins into the internal vesicles of multivesicular bodies. UEVs are homologous to E2 ubiquitin ligases but lack the conserved cysteine residue required for catalytic activity. The crystal structure of the yeast Vps23 UEV in a complex with ubiquitin (Ub) shows the detailed interactions made with the bound Ub. Compared with the solution structure of the Tsg101 UEV (the human homologue of Vps23) in the absence of Ub, two loops that are conserved among the ESCRT-I UEVs move toward each other to grip the Ub in a pincer-like grasp. The contacts with the UEV encompass two adjacent patches on the surface of the Ub, one containing several hydrophobic residues, including Ile-8(Ub), Ile-44(Ub), and Val-70(Ub), and the second containing a hydrophilic patch including residues Asn-60(Ub), Gln-62(Ub), Glu-64(Ub). The hydrophobic Ub patch interacting with the Vps23 UEV overlaps the surface of Ub interacting with the Vps27 ubiquitin-interacting motif, suggesting a sequential model for ubiquitinated cargo binding by these proteins. In contrast, the hydrophilic patch encompasses residues uniquely interacting with the ESCRT-I UEV. The structure provides a detailed framework for design of mutants that can specifically affect ESCRT-I-dependent sorting of ubiquitinated cargo without affecting Vps27-mediated delivery of cargo to endosomes.