Evaluation of surface hydrophobicity of immobilized protein with a surface plasmon resonance sensor

Immobilization is widely used to isolate agglutinative and associative proteins with large hydrophobic surfaces. Surface hydrophobicities of immobilized proteins were quantified by measuring the adsorption amounts of Triton X-100 as a hydrophobic probe with a biosensor that utilizes the phenomena of surface plasmon resonance (SPR). We measured SPR signal changes derived from adsorption of Triton X-100 to five kinds proteins and calculated the monolayer adsorption capacity using the Brunauer-Emmett-Teller equation, partly modified with a term for correcting an influence of the net charge of immobilized protein. SPR signal changes obtained by this method correlated with the values of surface hydrophobicities obtained by conventional assay using a hydrophobic probe. Thus this measuring method using an SPR sensor and Triton X-100 is expected to be a tool for quantifying surface hydrophobicities of immobilized proteins.     

Analysis of Toxoplasma gondii proteins after Triton X-114 solubilization and hydrophobic chromatography

The distribution of the surface proteins of Toxoplasma gondii radiodinated were studied using the phase separation technique and ability of binding in the phenyl-Sepharose column. Eight polypeptides with Mr 22 to 180 distributed exclusively in the detergent rich-phase, while six polypeptides with mol. wt. 15,000 to 76,000 distributed exclusively in the detergent poor-phase. Two polypeptides with 15,000 and 70,000 distributed in both phase. All the polypeptides present in the detergent rich-phase binding in the phenyl-Sepharose column, and can be isolated in two peak according with their relative hydrophobicities. Two polypeptides hydrophobic with Mr 60 and 66 recognized by human serum were isolated by the association of the two technique. Our result showed that the surface proteins of T. gondii present different degrees of hydrophobicity and that the use of hydrophobic interaction chromatography after Triton X-114 extraction may be an important isolation method of membrane proteins.     

Hydrophobic properties of porcine fibronectin and its functional domains

The relations between surface hydrophobicities and binding properties of the functional domains of porcine plasma fibronectin were investigated. Porcine plasma fibronectin as well as human plasma fibronectin was adsorbed on a hydrophobic column with butyl or phenyl ligands in the presence of 0.5 M ammonium sulfate, and recovered in a single peak by decreasing the concentration of ammonium sulfate to 0 M, indicating that both fibronectins have very high surface hydrophobicities. On digestion with thermolysin, porcine plasma fibronectin yielded five fragments (140-150, 43, 25, 17, and 14 kDa) similar to those reported for human fibronectin, although porcine fibronectin was more resistant to the digestion than human fibronectin. The three heparin-binding fragments were found to have a wide range of surface hydrophobicities, the 140-150 kDa fragment having the lowest, the 25 kDa fragment a higher, and the 14 kDa fragment the highest among all the fragments. The 43 kDa collagen-binding and 17 kDa fragments had surface hydrophobicities as high as that of fibronectin. It is noteworthy that the 43 kDa collagen-binding fragment contributes to the high surface hydrophobicity of intact fibronectin in spite of the high content of carbohydrates.     

Physico-chemical and structural properties of the surfaces of Peptostreptococcus micros and Streptococcus mitis as compared to those of mutans streptococci, Streptococcus sanguis and Streptococcus salivarius.

The surface properties of nine Streptococcus mitis and four Peptostreptococcus micros strains from the oral cavity were examined and compared with a large group of oral streptococci. Zeta potential and contact angle measurements were employed to determine physico-chemical cell surface properties. In addition, elemental surface concentration ratios were obtained via X-ray photoelectron spectroscopy, and surface structures were examined with transmission electron microscopy. The S. mitis and P. micros strains were found to have higher isoelectric points, higher hydrophobicities and higher N/C surface concentration ratios than some other oral streptococci. The combined data suggest that both species possess large amounts of surface protein. All the S. mitis strains displayed abundant surface fibrils in negative staining, but the P. micros strains were devoid of surface appendages indicating that surface protein is present in different forms in the two species. The surfaces of S. mitis and P. micros type strains differed significantly from the other strains examined.     

Comparative studies of the artificial chaperone-assisted refolding of thermally denatured bovine carbonic anhydrase using different capturing ionic detergents and beta-cyclodextrin

Artificial chaperone-assisted refolding has been shown to be an effective approach for improving the refolding yield of some of the denatured proteins. Since identical concentrations of various detergents do not induce similar variations in the protein structures, we arranged to evaluate the artificial chaperoning capabilities of several ionic detergents as a function of charge, structure, and the hydrophobic tail length of the detergent. Our results indicate that carbonic anhydrase can be refolded from its denatured state via artificial chaperone strategy using both anionic and cationic detergents. However, the extent of refolding assistance (kinetic and refolding yield) were different due to protein and detergent net charges, detergent concentrations, and the length of hydrophobic portion of each detergent. These observed differences were attributed to physical properties of CA-detergent complexes and/or to the kinetics of detergent stripping by beta-cyclodextrin from the protein-detergent complexes which is apparently dependent on the detergent-beta-CD association constants and the nature of the partially stripped complexes.     

Simulation of lipid environment of Ca-dependent ATPase from the sarcoplasmic reticulum of skeletal muscles by non-ionic detergents

To determine the conditions under which the Ca-ATPase from rabbit skeletal sarcoplasmic reticulum can be restored to full activity, a systematic study of reactivation of lipid-depleted enzyme by various non-ionic detergents and surfactants has been carried out. All the non-ionic detergents used were able to reactivate the enzyme. The reactivation potencies of the detergents are closely related to a relative size of their polar head group and hydrophobic hydrocarbon chains. The so-called HLB (hydrophyle/lipophyle balance) numbers were used to estimate the relative hydrophobicities of the detergents studied. A striking correlation between the reactivation potency and the HLB number of any detergent was observed; the inverse correlation coefficient for small samples if equal to -0.95 +/- 0.09. The proper orientation of the ATPase protein in two different phases (lipophyle and hydrophyle) seems to be restored during reactivation. In many cases the protein-bound detergent simulates the lipid environment in the membrane sufficiently well to support the continued activity of the Ca-ATPase.     

Membrane association, electrostatic sequestration, and cytotoxicity of Gly-Leu-rich peptide orthologs with differing functions

The skins of closely related frog species produce Gly-Leu-rich peptide orthologs that have very similar sequences, hydrophobicities, and amphipathicities but differ markedly in their net charge and membrane-damaging properties. Cationic Gly-Leu-rich peptides are hemolytic and very potent against microorganisms. Peptides with no net charge have only hemolytic activity. We have used ancestral protein reconstruction and peptide analogue design to examine the roles of electrostatic and hydrophobic interactions in the biological activity and mode of action of functionally divergent Gly-Leu-rich peptides. The structure and interaction of the peptides with anionic and zwitterionic model membranes were investigated by circular dichroism with 2-dimyristoyl-sn-glycero-3-phosphatidylcholine or 1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol vesicles and surface plasmon resonance with immobilized bilayers. The results, combined with antimicrobial assays, the kinetics of bacterial killing, and membrane permeabilization assays, reveal that Gly, Val, Thr, and Ile can all be accommodated in an amphipathic alpha helix when the helix is in a membrane environment. Binding to anionic and zwitterionic membranes fitted to a 2-stage interaction model (adsorption to the membrane followed by membrane insertion). The first step is governed by hydrophobic interactions between the nonpolar surface of the peptide helix and the membranes. The strong binding of Gly-Leu-rich cationic peptides to anionic membranes is due to the second binding step and involves short-range Coulombic interactions that prolong the residence time of the membrane-inserted peptide. The data demonstrate that evolution has positively selected charge-altering nucleotide substitutions to generate an orthologous cationic variant of neutral hemolytic peptides that bind to and permeate bacterial cell membranes.     

Characterization of the Tween 20-soluble membrane proteins of Acholephasma laidlawii

Methods for the purification of the four major proteins and two of the minor proteins in the Tween 20-soluble extract of A. laidlawii membranes have previously been described. The last step in the purification procedure involved an electrophoresis in a detergent-free buffer, where the concentration of Tween could be reduced by up to 2000 times. The purified proteins were found to remain soluble after removal of the bulk of the detergent. Solutions of the different protein samples contained 3 30 detergent molecules per protein molecule as determined by gas-liquid liquid chromatography. Some of the protein solutions also contained natural membrane lipids. It was probably only a fraction of detergent molecules and lipids, which was bound to the protein. Complete amino acid analysis showed that none of the proteins contained amino sugars and only one of them contained half-cystine. The specific absorbances and the molar absorption coefficients were calculated from the absorption spectra. The hydrophobicities and the partial specific volumes were calculated from the amino acid composition. The hydrophobicity values did not differ significantly from those of non-membrane proteins. Attempts to determine the sedimentation coefficients and the molecular weights were done ultracentrifugation after removal of the bulk of the detergent. The molecular weights, as determined by ultracentrifugation, were in general higher than the molecular weights determined by polyacrylamid-gel electrophoresis in sodium dodecyl sulphate (SDS), indicating that most of the proteins formed aggregates upon reducing the concentration of Tween 20. The size of these aggregates was not influenced by storage of the proteins at 0 C but it seemed to be highly affected by the speed and the time of centrifugation. The electrophoretic mobolities of the proteins were determined by free zone electrophoresis. Crossed immunoelectrophoresis was utilized to demonstrate that the Tween 20-soluble membrane proteins were not undergoing proteolysis during the preparation procedure.     

Characterization and design of chemically selective cationic displacers using a robotic high‐throughput screen

A robotic high‐throughput displacer screen was developed and employed to identify chemically selective displacers for several protein pairs in cation exchange chromatography. This automated screen enabled the evaluation of a wide range of experimental conditions in a relatively short period of time. Displacers were evaluated at multiple concentrations for these protein pairs, and DC‐50 plots were constructed. Selectivity pathway plots were also constructed and different regimes were established for selective and exclusive separations. Importantly, selective displacement was found to be conserved for multiple protein pairs, demonstrating the technique to be applicable for a range of protein systems. Although chemically selective displacers were able to separate protein pairs that had similar retention in ion exchange but different surface hydrophobicities, they were not able to distinguish protein pairs with similar surface hydrophobicities. This corroborates that displacer‐protein hydrophobic interactions play an important role for this class of selective displacers. Important functional group moieties were established and efficient displacers were identified. These results demonstrate that the design of chemically selective displacers requires a delicate balance between the abilities to displace proteins from the resin and to bind to a selected protein. The use of robotic screening of displacers will enable the extension of chemically selective displacement chromatography beyond hydrophobic displacer‐protein interactions to other secondary interactions and more selective displacement systems. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Relationship between Cell Surface Properties and Transport of Bacteria through Soil

A study was conducted to relate the properties of Enterobacter, Pseudomonas, Bacillus, Achromobacter, Flavobacterium, and Arthrobacter strains to their transport with water moving through soil. The bacteria differed markedly in their extent of transport; their hydrophobicity, as measured by adherence to n-octane and by hydrophobic-interaction chromatography; and their net surface electrostatic charge, as determined by electrostatic interaction chromatography and by measurements of the zeta potential. Transport of the 19 strains through Kendaia loam or their retention by this soil was not correlated with hydrophobicities or net surface charges of the cells or the presence of capsules. Among 10 strains tested, the presence of flagella was also not correlated with transport. Retention was statistically related to cell size, with bacteria shorter than 1.0 μm usually showing higher percentages of cells being transported through the soil. We suggest that more than one characteristic of bacterial cells determines whether the organisms are transported through soil with moving water.     

Hydrophobic peptide tags as tools in bioseparation

Hydrophobic interactions are highly selective, and differences in surface hydrophobicities between proteins can be used as an efficient handle to facilitate protein isolation. Aromatic amino acid residues are of particular importance for molecular recognition because they have a key role in several biological functions. The hydrophobicity of a protein can easily be altered with minor genetic modifications, such as site-directed mutagenesis or fusions of hydrophobic peptide tags. An important advantage of hydrophobic peptide tags over traditional affinity tags is the possibility of exploring simple and inexpensive bioseparation materials. Recent results demonstrate the potential of hydrophobic interaction chromatography and aqueous two-phase systems as tools to study relative hydrophobicities of recombinant proteins with only minor alterations. This review focuses on hydrophobic peptide tags as fusion partners, which can be used as important tools in bioseparation.     

Contact Angle Measurement and Cell Hydrophobicity of Granular Sludge from Upflow Anaerobic Sludge Bed Reactors

The contact angle, which is generally used to evaluate the hydrophobicities of pure bacterial strains and solid surfaces, was used to study mixed cell cultures of bacteria involved in anaerobic digestion. Previously published data and data from this study showed that most acidogens are hydrophilic (contact angle, <45(deg)) but most of the acetogens and methanogens isolated from granular sludge are hydrophobic (contact angle, >45(deg)). The hydrophobicities of mixtures of hydrophilic and hydrophobic cells were found to be linearly correlated with the cell mixing ratio. The hydrophobicities of cells present in effluents from upflow anaerobic sludge bed reactors which were treating different types of substrates were different depending on the reactor conditions. When the reactor liquid had a high surface tension, cells sloughing off from sludge granules, as well as cells present on the outer surfaces of the granules, were hydrophobic. Short-term batch enrichment cultures revealed that proteins selected for highly hydrophilic cells. Long-term in-reactor enrichment cultures revealed that sugars selected for hydrophilic acidogens on the surfaces of the granules, while fatty acids tended to enrich for hydrophobic methanogens. When linear alkylbenzenesulfonate was added, the cells on the surfaces of granules became more hydrophilic. Control tests performed with pure cultures revealed that there was no change in the surface properties due to linear alkylbenzenesulfonate; hence, the changes in the wash-out observed probably reflect changes in the species composition of the microbial association. A surface layer with moderate hydrophobicity, a middle layer with extremely high hydrophobicity, and a core with high hydrophobicity could be distinguished in the grey granules which we studied.     

Variation of the Detergent-Binding Capacity and Phospholipid Content of Membrane Proteins When Purified in Different Detergents

Purified membrane proteins are ternary complexes consisting of protein, lipid, and detergent. Information about the amounts of detergent and endogenous phospholipid molecules bound to purified membrane proteins is largely lacking. In this systematic study, three model membrane proteins of different oligomeric states were purified in nine different detergents at commonly used concentrations and characterized biochemically and biophysically. Detergent-binding capacities and phospholipid contents of the model proteins were determined and compared. The insights on ternary complexes obtained from the experimental results, when put into a general context, are summarized as follows. 1), The amount of detergent and 2) the amount of endogenous phospholipids bound to purified membrane proteins are dependent on the size of the hydrophobic lipid-accessible protein surface areas and the physicochemical properties of the detergents used. 3), The size of the detergent and lipid belt surrounding the hydrophobic lipid-accessible surface of purified membrane proteins can be tuned by the appropriate choice of detergent. 4), The detergents n-nonyl-β-D-glucopyranoside and Cymal-5 have exceptional delipidating effects on ternary complexes. 5), The types of endogenous phospholipids bound to membrane proteins can vary depending on the detergent used for solubilization and purification. 6), Furthermore, we demonstrate that size-exclusion chromatography can be a suitable method for estimating the molecular mass of ternary complexes. The findings presented suggest a strategy to control and tune the numbers of detergent and endogenous phospholipid molecules bound to membrane proteins. These two parameters are potentially important for the successul crystallization of membrane proteins for structure determination by crystallographic approaches.     

Sequence hydropathy dominates membrane protein response to detergent solubilization

The ability to predict from amino acid sequence how membrane protein structures will respond to detergent solubilization would significantly facilitate experimental characterization of these molecules. Here we have investigated and compared the response to solubilization by the "mild" n-dodecyl-β-d-maltoside (DDM) and "harsh" sodium dodecyl sulfate (SDS) of wild-type and point mutant "hairpin" (helix-loop-helix) membrane proteins derived from the third and fourth TM segments of the human cystic fibrosis transmembrane conductance regulator (CFTR) and the intervening extracellular loop. Circular dichroism spectroscopy, size-exclusion chromatography, and pyrene fluorescence spectroscopy were used to evaluate the secondary structures, hairpin-detergent complex excluded volumes, and hairpin compactness of the detergent-solubilized sequences. Sequence hydrophobicity is found to be the dominant factor dictating membrane protein response to detergent solubilization by DDM and SDS, with hairpin secondary structure exquisitely sensitive to mutation when DDM is used for solubilization. DDM and SDS differ principally in their ability to promote approach of TM segment ends, although hairpin compactness remains sensitive to point mutations. Our overall findings suggest that protein-protein and protein-detergent interactions are determined concomitantly, with the net hydropathy of residues exposed to detergent dominating the observed properties of the solubilized protein.     

A method for three-dimensional protein characterization and its application to a complex plant (corn) extract

Knowledge of host protein properties is critical for developing purification methods for recombinant proteins from a specific host, or for choosing suitable hosts and targeted expression tissues for a specific recombinant protein. A method to obtain a three-dimensional (3D) map (surface hydrophobicity (SH), isoelectric point (pI), and molecular weight (MW)), of a host's aqueous soluble protein properties was developed. The method consists of hydrophobic partitioning in a PEG 3350 (15.7%)-Na(2)SO(4) (8.9%)-NaCl (3%) aqueous two-phase (ATP) system followed by quantitative, 2D-electrophoretic characterization of the proteins of each equilibrium phase and the original extract. The pI and MW of host proteins were obtained directly through 2D electrophoresis. The partition coefficients of individual proteins were obtained by quantitative matching of protein spots in the top and bottom phase gels and calculating the protein partition coefficients from this information. Correlation of the partition coefficient to a SH scale was established by partitioning several model proteins with known surface hydrophobicities in the same ATP system. The inclusion of the extract gel provided for a spot selection criterion based on satisfactory mass balance closure. The method is illustrated by application to a mixture of model proteins and to complex mixtures, that is, corn germ proteins extracted at pH 7 and pH 4.     

Surface proteins of thymus-derived lymphocytes and bone-marrow-derived lymphocytes. Selective isolation of immunoglobulin and the θ-antigen by non-ionic detergents

Accessible surface proteins of thymus-derived lymphocytes (T-cells) of normal CBA mice and bone-marrow-derived lymphocytes (B-cells) of congenitally athymic nu/nu mice were analysed. The surfaces of lymphocytes were radioiodinated by using the enzyme lactoperoxidase (EC 1.11.1.7), then solubilized either in acid-urea or in the non-ionic detergent Nonidet P-40. These lysates were then precipitated with antisera specific to either immunoglobulin or the theta-alloantigen in order to assess the presence of these surface markers. Comparable amounts of radioactivity in proteins specifically precipitable as immunoglobulin were obtained from T-lymphocytes and B-lymphocytes when the cells were disrupted by acid-urea. This immunoglobulin had mol. wt. approx. 180000 and was composed of light chains and mu-type heavy chains. When radioiodinated lymphocytes were solubilized with Nonidet P-40, 3-4% of radioiodinated high-molecular-weight protein of B-cells consisted of immunoglobulin, a result similar to that found with acid-urea extraction. However, with the detergent extraction, only 0.1% of T-cell surface protein was precipitable by anti-globulin reagents. The theta-alloantigen was isolated from CBA T-cells both by acid-urea and by detergent lysis. This protein possessed a mobility on polyacrylamide-gel electrophoresis in sodium dodecyl sulphate which was consistent with a mol. wt. of 60000. An identical component was isolated from the theta-positive thymoma WEHI 105. The theta-antigen was not isolated from B-cells by either of the extraction procedures used. These results provide further evidence that the surface membranes of normal T-cells and B-cells differ in physicochemical properties. In particular, various surface components possess differential solubilities in non-ionic or organic solvents. This observation provides an explanation for discrepant results that have appeared in the literature concerning the isolation of immunoglobulin from T-lymphocytes.     

Purification and disposition of a surface protein associated with virulence of Aeromonas salmonicida.

Virulent strains of Aeromonas salmonicida observed by electron microscopy were characterized by an outer layer exhibiting a tetragonal repeat pattern. Attenuated strains had a 2.5 X 10(3)- to 5 X 10(3)-fold reduction in virulence and lost the outer layer, autoaggregating properties, and a 49-kilodalton protein (A protein) simultaneously. The A protein is the major protein component of outer membrane fractions of virulent strains. A variety of radiolabeling studies showed that this protein was surface localized and that it provided an effective barrier against iodination of other outer membrane proteins with either lactoperoxidase or diazoiodosulfanilic acid; A protein was not labeled with lactoperoxidase but was specifically labeled with diazoidosulfanilic acid. The A protein was purified by selective extraction with detergent and guanidine hydrochloride, and its amino acid composition was determined. The properties of A protein are compared with those of other bacterial surface layer proteins.     

Comparison of contact angles and adhesion to hexadecane of urogenital, dairy, and poultry lactobacilli: effect of serial culture passages.

The aim of this study was to examine the hydrophobicities of 23 urogenital, dairy, poultry, and American Type Culture Collection isolates of lactobacilli and to determine the effect on hydrophobicity of serially passaging the strains in liquid medium. To this end, strains were grown after isolation and identification and then serially passaged up to 20 times. Hydrophobicity was assessed through contact angle measurements on lawns of cells by using water, formamide, methylene iodide, 1-bromonaphthalene, and hexadecane as wetting agents and through measurement of their partitioning in a hexadecane-water system. The hydrophobicities of these strains varied widely, with Lactobacillus casei strains being predominantly hydrophilic and L. acidophilus strains being mostly hydrophobic. For some isolates, serial passaging was accompanied by a clear loss of hydrophobic surface properties, whereas for other strains, cultures became heterogeneous in that some cells had already lost their hydrophobic surface properties while others were still hydrophobic. Adhesion of this collection of lactobacilli to hexadecane droplets in microbial adhesion to hexadecane (MATH) tests was driven by their aversion to water rather than by their affinity for hexadecane, as concluded from the fact that hexadecane contact angles were zero for all strains. Furthermore, adhesion of the lactobacilli to hexadecane in MATH tests occurred only when the water contact angle on the cells was above 60 degrees.     

Comparison of contact angles and adhesion to hexadecane of urogenital, dairy, and poultry lactobacilli: effect of serial culture passages.

The aim of this study was to examine the hydrophobicities of 23 urogenital, dairy, poultry, and American Type Culture Collection isolates of lactobacilli and to determine the effect on hydrophobicity of serially passaging the strains in liquid medium. To this end, strains were grown after isolation and identification and then serially passaged up to 20 times. Hydrophobicity was assessed through contact angle measurements on lawns of cells by using water, formamide, methylene iodide, 1-bromonaphthalene, and hexadecane as wetting agents and through measurement of their partitioning in a hexadecane-water system. The hydrophobicities of these strains varied widely, with Lactobacillus casei strains being predominantly hydrophilic and L. acidophilus strains being mostly hydrophobic. For some isolates, serial passaging was accompanied by a clear loss of hydrophobic surface properties, whereas for other strains, cultures became heterogeneous in that some cells had already lost their hydrophobic surface properties while others were still hydrophobic. Adhesion of this collection of lactobacilli to hexadecane droplets in microbial adhesion to hexadecane (MATH) tests was driven by their aversion to water rather than by their affinity for hexadecane, as concluded from the fact that hexadecane contact angles were zero for all strains. Furthermore, adhesion of the lactobacilli to hexadecane in MATH tests occurred only when the water contact angle on the cells was above 60 degrees.     

Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons.

We demonstrate in this work that the surface tension, water-organic solvent, transfer-free energies and the thermodynamics of melting of linear alkanes provide fundamental insights into the nonpolar driving forces for protein folding and protein binding reactions. We first develop a model for the curvature dependence of the hydrophobic effect and find that the macroscopic concept of interfacial free energy is applicable at the molecular level. Application of a well-known relationship involving surface tension and adhesion energies reveals that dispersion forces play little or no net role in hydrophobic interactions; rather, the standard model of disruption of water structure (entropically driven at 25 degrees C) is correct. The hydrophobic interaction is found, in agreement with the classical picture, to provide a major driving force for protein folding. Analysis of the melting behavior of hydrocarbons reveals that close packing of the protein interior makes only a small free energy contribution to folding because the enthalpic gain resulting from increased dispersion interactions (relative to the liquid) is countered by the freezing of side chain motion. The identical effect should occur in association reactions, which may provide an enormous simplification in the evaluation of binding energies. Protein binding reactions, even between nearly planar or concave/convex interfaces, are found to have effective hydrophobicities considerably smaller than the prediction based on macroscopic surface tension. This is due to the formation of a concave collar region that usually accompanies complex formation. This effect may preclude the formation of complexes between convex surfaces.