Hydrophobicity Gradient Research Articles

Transmembrane Protein Insertion Orientation in Yeast Depends on the Charge Difference across Transmembrane Segments, Their Total Hydrophobicity, and Its Distribution

The determinants of transmembrane protein insertion orientation at the endoplasmic reticulum have been investigated in Saccharomyces cerevisiae using variants of a Type III (naturally exofacial N terminus (Nexo)) transmembrane fusion protein derived from the N terminus of Ste2p, the alpha-factor receptor. Small positive and negative charges adjacent to the transmembrane segment had equal and opposite effects on orientation, and this effect was independent of N- or C-terminal location, consistent with a purely electrostatic interaction with response mechanisms. A 3:1 bias toward Nexo insertion, observed in the absence of a charge difference, was shown to reflect the Nexo bias conferred by longer transmembrane segments. Orientation correlated best with total hydrophobicity rather than length, but it was also strongly affected by the distribution of hydrophobicity within the transmembrane segment. The most hydrophobic terminus was preferentially translocated. Insertion orientation thus depends on integration of responses to at least three parameters: charge difference across a transmembrane segment, its total hydrophobicity, and its hydrophobicity gradient. Relative signal strengths were estimated, and consequences for topology prediction are discussed. Responses to transmembrane sequence may depend on protein-translocon interactions, but responses to charge difference may be mediated by the electrostatic field provided by anionic phospholipids.

Interfacial self-assembly of a Schizophyllum commune hydrophobin into an insoluble amphipathic protein membrane depends on surface hydrophobicity

Hydrophobins are small secreted fungal proteins rich in hydrophobic amino acids with a characteristic hydropathy pattern and conserved location of eight cysteine residues. It was previously shown that purified SC3p hydrophobin of Schizophyllum commune self-assembles at hydrophilic/hydrophobic interfaces into a sodium dodecyl sulphate (SDS)-insoluble amphipathic protein membrane typified at the hydrophobic side by a mosaic of parallel rodlets, similar to those seen at the hydrophobic surface of aerial hyphae of this fungus. Here we show the assembly of SC3p at the interface between water and a continuous hydrophobicity gradient surface obtained by coating glass with dichlorodimethylsilane and displaying contact angles of water ranging from 20° up to 107°. The amount of assembled SC3p (defined as SC3p becoming insoluble in hot SDS) sharply increased in the region of the gradient surface displaying advancing water contact angles between 60° and 90° and then more slowly towards the 107° region, i.e. the hydrophobic end. Here, the adsorbed SC3p decreases the advancing water contact angle from 107° on the bare gradient surface to 60° on the protein coated surface (to 39° before extraction with SDS). The SDS-insoluble SC3p was removed by trifluoroacetic acid, which is known to dissociate assembled hydrophobins, hence restoring the wettability characteristics close to those of the original hydrophobicity gradient surface. Since fungal hyphae firmly attach to natural surfaces which are often hydrophobic, these results suggest a role for hydrophobins in fungal pathogenicity and other fungal host interactions.

New concept of a hydrophobicity motor based on local hydrophobicity transition of functional polymer substrate for micro/nano machines

Abstract Hydrophobic interaction is widely known as one of the most important forces acting between biomolecules. This paper proposes a new concept of driving mechanism for macromolecules or microscale objects, such as hydrogel particles as a biomimetic motor. The idea is based on not only utilizing a tangential gradient of hydrophobicity of the substrate but also that such a gradient should be induced by energy input from the environment through the motor unit. Assuming a simple model for a hydrophobicity motor, numerical analysis revealed the basic dynamical behavior. The movement of the motor was in many cases oscillatory and in certain special conditions, a constant speed motion. The force-velocity curves consisted of three regions: the region simply decreasing with load, the sustaining region for large load with zero speed and the yielding region. This result is very similar to that of muscle contraction. This concept could be the engineering basis for handling biomolecular interactions.

How to Make Water Run Uphill

A surface having a spatial gradient in its surface free energy was capable of causing drops of water placed on it to move uphill. This motion was the result of an imbalance in the forces due to surface tension acting on the liquid-solid contact line on the two opposite sides ("uphill" or "downhill") of the drop. The required gradient in surface free energy was generated on the surface of a polished silicon wafer by exposing it to the diffusing front of a vapor of decyltrichlorosilane, Cl(3)Si(CH(2))(9)CH(3). The resulting surface displayed a gradient of hydrophobicity (with the contact angle of water changing from 97 degrees to 25 degrees ) over a distance of 1 centimeter. When the wafer was tilted from the horizontal plane by 15 degrees , with the hydrophobic end lower than the hydrophilic, and a drop of water (1 to 2 microliters) was placed at the hydrophobic end, the drop moved toward the hydrophilic end with an average velocity of approximately 1 to 2 millimeters per second. In order for the drop to move, the hysteresis in contact angle on the surface had to be low (</=10 degrees ).

Adsorption of complement proteins on surfaces with a hydrophobicity gradient

Activation of the complement system is recognized as one of the major problems with respect to biocompatibility of biomaterials. The binding of C3 (central component of complement) and B (factor B, an activator of C3) and H (factor H, an inhibitor of C3 activation) plays a crucial role in the activation of the alternative pathway of complement on the surfaces of biomaterials during extracorporeal procedures. Here we report on the adsorption of C3, B or H on to the silica surface with a hydrophobicity gradient. The amount of native 125I-C3 bound to both hydrophilic and hydrophobic surfaces was very similar (0.8 and 0.9 μg/cm 2; 4 × 10 −12 mol/cm 2). Neither factor H nor factor B was able to displace already adsorbed 125I-C3 from either of the surfaces. The extent of binding of factors B and H to preadsorbed C3 was a function of the surface hydrophobicity: more 125I-B or 125I-H was bound to C3 adsorbed at the hydrophilic end than at the hydrophobic end of the gradient surface. The binding of 125I-B or 125I-H to preadsorbed C3 appeared to be influenced by the availability of their binding sites on adsorbed C3 molecules rather than by the amount of surface-bound C3. At the hydrophobic end of the gradient surface the molar binding ratio of B C3 was considerably smaller than the molar binding ratio of H C3 . It can be speculated that the hydrophobicity of the surface determines orientation and/or conformation of adsorbed C3 molecule; when adsorbed at the hydrophobic end of the gradient, C3 molecule predominantly exposes the binding site to which only factor H can bind. A substantial decrease in binding of C3, B or H on to the hydrophobicity gradient surface was observed in the presence of other serum or plasma proteins, particularly at the hydrophobic end of the gradient surface.

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.

Adsorption of ethylene oxide/propylene oxide copolymers on hydrophobicity gradient surfaces

Adsorption of three commerciably available ethylene oxide(EO)/propylene oxide (PO) block copolymers with the general formula EO a PO b EO a , was studied with ellipsometry on a hydrophobicity gradient surface with contact angles ranging from 0 to 90°. Generally, higher adsorption values were obtained on the hydrophobic side than on the hydrophilic. The isotherm shapes were, strictly speaking non-Langmuir. However, within the range 0–10 ppm the isotherms could reasonably well be fitted to a Langmuir model. Generally, semi-plateau values of around 1.5–2.0 mg m −2 were measured on the hydrophobic side and around 0.0–0.5 mg m −2 on the hydrophilic, and a slowly progressing increase of the adsorption was observed at higher concentrations. The most hydrophilic surfactant, EO 148PO 56EO 148, gave slightly lower values on the hydrophobic side compared to the two others (EO 98PO 69EO 98 and EO 37PO 56EO 37). In contrast, comparatively higher affinity and adsorption capacity was found for EO 148PO 56EO 148 on the hydrophilic side of the gradient possibly indicating weak attraction between EO and surface SiOH groups. The adsorption capacity increased only slightly with temperature, but the adsorption affinity increased significantly between 20 and 38°C for EO 98PO 69EO 98, especially on the hydrophilic side. No spontaneous desorption was observed for any of the EO/PO copolymers. On the other hand, when exposed to an albumin solution, significant desorption and displacement occurred, in particular on the the hydrophobic end of the gradient as verified by ESCA analysis.

Characterization of hydrophobicity gradients prepared by means of radio frequency plasma discharge

A hydrophobicity gradient was created by gradually exposing a polydimethylsiloxane film to a radio frequency glow discharge in an oxygen atmosphere. A change in contact angle from 100 to 0° was measured along the gradient surface by means of the Wilhelmy balance technique. The gradient surface was characterized by studying the adsorption from single protein solutions of human albumin, IgG and fibrinogen using total internal reflectance fluorescence (TIRF). Generally, adsorption values similar to monolayer capacities were measured on the hydrophobic side. The adsorption decreased towards the hydrophilic end in correspondence with the change in contact angle. The displacement of proteins with a ethylene oxide-propylene oxide copolymer (EO 70PO 100EO 70) was higher on the hydrophobic side than on the hydrophilic side. Albumin showed an adsorption peak in the wetting transition region, indicating a very strong affinity.

Wetting and plasma-protein adsorption studies using surfaces with a hydrophobicity gradient

Wetting gradients on silica were prepared according to the technique developed by Elwing et al. (J. Colloid Interface Sci., 119 (1987) 1). The contact angle, determined by the Wilhelmy-plate method, showed an S-shaped dependence on the position along the gradient. The advancing water contact angle was 0° on the hydrophilic end and 97±3° on the hydrophobic end. The receding water contact angles were ∼40° smaller in the wetting transition region and ∼20° smaller on the hydrophobic side of the gradient. Total internal reflection fluorescence (TIRF) was used to study the adsorption of three fluorescein-labelled proteins: human serum albumin, IgG and fibrinogen. The gradient surface exposed to a single protein solution resulted in an amount of adsorbed protein on the hydrophobic end that approximately corresponded to a monolayer. As a rule, lower adsorption was found on the hydrophilic end. When the proteins were adsorbed from a mixture, the surface concentration of albumin was generally not affected, the fibrinogen adsorption decreased by 40–50% and the IgG adsorption decreased to 10% of the adsorption measured from a single protein solution. The so-called Vroman effect (for example, transient IgG adsorption maximum in adsorption kinetics) was found on the hydrophobic side of the gradient. The desorption experiments were carried out with a nonionic polymeric surfactant, an ethylene oxide-propylene oxide copolymer: EO100-PO70-EO100. The adsorption affinity of albumin to the wetting transition region, as measured by surfactant induced desorption, was extraordinarily high.

Hydrophobicity gradient on silica surfaces: A study using total internal reflection fluorescence spectroscopy

A silica surface with hydrophobicity gradient was characterized using total internal reflection fluorescence spectroscopy and ANS, 1-anilinonaphthalene-8-sulfonate, as a surface probe. The probe responded to the hydrophobicity gradient of the surface. Strong enhancement of ANS fluorescence was found at the hydrophobic side of the gradient and no fluorescence enhancement at the hydrophilic side. Similar results were obtained with ANS binding to bovine serum albumin adsorbed on the gradient surface.

Separation of proteins by polymeric adsorbents containing azobenzene moiety as a ligand

AbstractPolymeric adsorbents containing an azobenzene moiety as a ligand were prepared, and the photoinduced adsorption behavior of proteins to the adsorbent was studied. This method regulates by light the adsorption/desorption of proteins in hydrophobic chromatography. In the dark, the amount of protein adsorbed onto the adsorbent increased with increasing hydrophobicity of either adsorbent or protein. On irradiation with UV light, the amount of protein adsorbed decreased. Such a photoinduced change of the adsorption of protein was considered to be caused by the change of the hydrophobic interaction between the adsorbent and the protein due to the photoisomerization of the azobenzene moiety accompanying the polarity change of the adsorbent. It is also found that the desorption of protein was dependent on the balance of the hydrophobicity between the adsorbents and proteins. When column chromatography was carried out, the proteins were adsorbed in the dark and could be eluted after photoirradiation, with water as the single solvent. Furthermore, mixture of proteins could be separated by using a hydrophobic gradient column which was constituted by two polymeric adsorbents having different hydrophobicity.

Non-ionic adsorption chromatography of proteins

Evidence is presented indicating that protein separation by adsorption chromatography, based on differential non-ionic interaction with immobilized hydrophobic ligands, potentially is as generally applicable as ion-exchange chromatography. A procedure for the normalization of binding capacities of amine-substituted agaroses has been presented. Attempts have been made at the separation of proteins in normal human blood serum by means of hydrophobicity gradients consisting of series of interconnected columns of n-alkylamino-agaroses of increasing hydrophobicities and equilibrated with 3 M NaCl. The γ-globulin fraction, or components thereof, can be bound hydrophobically as well as through another type of salt(NaCl)-stable but non-hydrophobic binding.

Fractionation of protein mixtures through differential adsorption on a gradient of substituted agaroses of increasing hydrophobicity.

Data are presented which indicate the feasibility of protein fractionation at high salt concentrations (greater than or equal to 3 M NaLl) through differential hydrophobic (non-ionic) adsorption on a series of columns of agaroses substituted with ligands of increasing hydrophobicity. By means of such a hydrophobicity gradient of connected columns, separation of mixtures of gamma-globulin and serum albumin, as well as group separation of proteins in dialyzed human blood serum, has been achieved.