Induction of changes in the secondary structure of globular proteins by a hydrophobic surface

Circular dichroism, ellipsometry and radiolabeling techniques were employed to study the induction of changes in the secondary structure of BSA, myoglobin and cytochrome C by a hydrophobic surface. The results showed that adsorbed protein molecules lose their ordered native structure in the initial stage of adsorption and the structure appears to be a random or disordered conformation. Protein molecules adsorbed in later stages adopt a more ordered secondary structure ( helix and structure). The changes of secondary structure of globular proteins induced by a hydrophobic surface can be explained by the steric interaction between adsorbed proteins as well as by hydrophobic interactions during the adsorption process. In addition, there is obviously an intermediate stage in which the protein molecules are mainly in the structure, indicating that for certain proteins, the structure may be a more stable secondary structure than helix on the hydrophobic surface. Correspondence to: S.-F. Sui     

Role of hydrophobic interactions and salt-bridges in β-hairpin folding

β-Hairpins are the simplest form of β-sheets which, due to the presence of long-range interactions, can be considered as tertiary structures. Molecular dynamics simulation is a powerful tool that can unravel whole pathways of protein folding/unfolding at atomic resolution. We have performed several molecular dynamics simulations, to a total of over 250 ns, of a β-hairpin peptide in water using GROMACS. We show that hydrophobic interactions are necessary for initiating the folding of the peptide. Once formed, the peptide is stabilized by hydrogen bonds and disruption of hydrophobic interactions in the folded peptide does not denature the structure. In the absence of hydrophobic interactions, the peptide fails to fold. However, the introduction of a salt-bridge compensates for the loss of hydrophobic interactions to a certain extent. Figure Model of b-hairpin folding: Folding is initiated by hydrophobic interactions (Brown circles). The folded structure, once formed, is stabilized by hydrogen bonds (red lines) and is unaffected by loss of hydrophobic contacts     

The hydrophobicity of bacteria — An important factor in their initial adhesion at the air-water inteface

Bacteria isolated from the surface and the subsurface water at four stations along the Swedish west coast were assessed for their hydrophobicity with hydrophobic interaction chromatography (HIC). The surface bacteria were sampled by the Teflon sheet technique. [³H]-l-leucine metabolically labeled isolates were run on a column packed with Octyl-Sepharose CL-4B gel. The relative hydrophobicity of the bacteria was expressed as the ratio, g/e, between the radioactivity of the gel and the eluate. The results revealed a positive correlation between the degree of enrichment of bacteria at the surface and their hydrophobicity. The subsurface bacteria exhibited a broader spectrum of g/e-values than the surface bacteria. The initial adhesion of bacteria to the surface microlayer depends on several factors of which the hydrophobic interaction may be one of the most important.Abbreviations HIC hydrophobic interaction chromatography - NSS nine salt solution     

疏水相互作用层析分离重组人干扰素α2b

目的采用疏水相互作用层析分离重组人干扰素α2b,去除干扰素样品中的二聚体,得到高纯度的干扰素用于进一步的研究。方法首先采用阳离子交换层析纯化复性重组人干扰素α2b,去除了大部分的杂蛋白,然后采用疏水相互作用层析纯化重组人干扰素α2b,去除复性过程中产生的错误折叠体和二聚体,并考察盐浓度、pH值、流速和洗脱液中尿素对疏水相互作用层析纯化效果的影响。结果硫酸铵初始浓度1.2 mol/L、缓冲液pH值6.0、流速2.5 mL/min、洗脱液中添加尿素浓度为2 mol/L时疏水相互作用层析纯化效果最佳。最终得到的重组人干扰素α2b非还原型SDS-PAGE电泳均呈单一条带。结论确定了疏水层析纯化重组人干扰素α2b的最优条件,成功提取到具有高活性、高纯度的重组人干扰素α2b纯品。     

Determination of molecular associations of some hydrophobic and hydrophilic bile acids by infrared and Raman spectroscopy

The aim of this study was to analyse the Raman and infrared spectra of eight common mammalian bile acids in order to identify intermolecular interactions between hydroxyl and carbonyl groups. The results are considered in the light of the new hydrophilic/hydrophobic classification of bile acids. The alcohol OH group of the hydrophobic bile acids forms different intermolecular bonds. The most hydrophobic bile acid, lithocholic acid forms polymers, and this may explain its very low water solubility. The hydrophilic bile acids have some of their alcohol OH groups free of any intermolecular interaction. The strongly hydrophilic murideoxycholic acid also forms dimers, again consistent with a very low water solubility. The proposed structural arrangements are in agreement with published crystallographic studies. Received: 7 November 1996 / Accepted: 8 December 1996     

Localization of ligand binding site in proteins identified in silico

Knowledge-based models for protein folding assume that the early-stage structural form of a polypeptide is determined by the backbone conformation, followed by hydrophobic collapse. Side chain–side chain interactions, mostly of hydrophobic character, lead to the formation of the hydrophobic core, which seems to stabilize the structure of the protein in its natural environment. The fuzzy-oil-drop model is employed to represent the idealized hydrophobicity distribution in the protein molecule. Comparing it with the one empirically observed in the protein molecule reveals that they are not in agreement. It is shown in this study that the irregularity of hydrophobic distributions is aim-oriented. The character and strength of these irregularities in the organization of the hydrophobic core point to the specificity of a particular protein’s structure/function. When the location of these irregularities is determined versus the idealized fuzzy-oil-drop, function-related areas in the protein molecule can be identified. The presented model can also be used to identify ways in which protein–protein complexes can possibly be created. Active sites can be predicted for any protein structure according to the presented model with the free prediction server at . The implication based on the model presented in this work suggests the necessity of active presence of ligand during the protein folding process simulation. Figure Fuzzy-oil-drop model applied to identify the ligation site in lysozyme complexed with N-acetylglucosamine (PDB ID:1LMQ) in form of hydrophobicity deficiency (ΔH) profile and three-dimensional distribution of on protein surface     

Engineering the hydrophobic residues of a GH11 xylanase impacts its adsorption onto lignin and its thermostability

This study aimed to characterise the parameters governing the non-specific adsorption of a xylanase from Thermobacillus xylanilyticus (Tx-Xyn11) onto lignin isolated from maize stems. Such adsorption may be due to hydrophobic interactions between Tx-Xyn11 and lignin. Our strategy was to mutate hydrophobic residues present on the surface of Tx- Xyn11 into non-hydrophobic residues. Three mutants (P1, P2, and P3) with altered hydrophobic regions were produced and characterised. The thermostability of the P1 mutant was largely decreased compared with the thermostable Tx-Xyn11. The rate of adsorbed enzyme onto lignin was reduced to a similar extent for the P1 and P2 mutants, whereas the adsorption of the P3 mutant was less affected compared with that of Tx-Xyn11. When considered separately, the hydrophobic residues did not affect xylanase adsorption onto lignin. The addition of Tween 20 also led to the decreased adsorption of Tx-Xyn11 onto lignin. These results suggest that hydrophobic interactions are a key parameter in the interaction of Tx-Xyn11 with isolated lignin.     

蛋白质表面疏水性的研究

用Ｐｈｅｎｙｌ－ＳｕｐｅｒｏｓｅＨＲ５／５疏水柱在ＦＰＬＣ仪上测定了一些蛋白的表面疏水性。在被测量的蛋白样品中,细胞色素Ｃ的亲水性最强,胰凝乳蛋白酶的疏水性最强。说明蛋白质的疏水性与其表面性质密切相关,而与蛋白质的分子量、疏水残基总数并不直接相关;去辅基细胞色素Ｃ和Ｃ端缩短的金黄色葡萄球菌核酸酶与天然态比较疏水性变化很大。疏水柱层析还用于监测在低浓度胍的作用下蛋白质的构象变化。以Ｎ－乙酰酪氨酸为模型化合物探测盐酸胍对疏水柱结合能力的影响,在０４Ｍ胍存在时,Ｎ－乙酰酪氨酸在疏水柱上的结合能力略有减弱,但核糖核酸酶Ａ的变化较大,表明胍引起的蛋白质的微小构象变化有效地引起其表面性质的变化;在０．１—０．３Ｍ盐酸胍存在时,甘油醛－３－磷酸脱氢酶表面疏水性明显增大,并伴随聚合态的出现。说明在低胍作用下,酶分子发生的构象变化,导致天然态内埋疏水面的暴露,暴露的疏水面间的相互作用是形成聚合的主要原因。     

Anomaly of transport of dipicrylamine anions across the central barrier of planar dipalmitoylphosphatidylcholine bilayer membranes at the phase transition

The rate of translocation of the hydrophobic ion dipicrylamine across planar lipid membranes formed from dipalmitoyllecithin in n-decane was determined by voltage jump relaxation experiments. The activation energy of the rate constant shows a change from a positive to a negative value at about 42°C near the main phase transition temperature of this lipid. Below this temperature, the rate constant was found to increase with decreasing temperature. This anomalous behaviour extends over a temperature range of at least 10 K and may be formally interpreted as an enhanced mobility of dipicrylamine in the solid state of the membrane.     

Molecular dynamics simulations of water within models of ion channels.

The transbilayer pores formed by ion channel proteins contain extended columns of water molecules. The dynamic properties of such waters have been suggested to differ from those of water in its bulk state. Molecular dynamics simulations of ion channel models solvated within and at the mouths of their pores are used to investigate the dynamics and structure of intra-pore water. Three classes of channel model are investigated: a) parallel bundles of hydrophobic (Ala20) alpha-helices; b) eight-stranded hydrophobic (Ala10) antiparallel beta-barrels; and c) parallel bundles of amphipathic alpha-helices (namely, delta-toxin, alamethicin, and nicotinic acetylcholine receptor M2 helix). The self-diffusion coefficients of water molecules within the pores are reduced significantly relative to bulk water in all of the models. Water rotational reorientation rates are also reduced within the pores, particularly in those pores formed by alpha-helix bundles. In the narrowest pore (that of the Ala20 pentameric helix bundle) self-diffusion coefficients and reorientation rates of intra-pore waters are reduced by approximately an order of magnitude relative to bulk solvent. In Ala20 helix bundles the water dipoles orient antiparallel to the helix dipoles. Such dipole/dipole interaction between water and pore may explain how water-filled ion channels may be formed by hydrophobic helices. In the bundles of amphipathic helices the orientation of water dipoles is modulated by the presence of charged side chains. No preferential orientation of water dipoles relative to the pore axis is observed in the hydrophobic beta-barrel models.     

Protein-induced bilayer perturbations: Lipid ordering and hydrophobic coupling

The host lipid bilayer is increasingly being recognized as an important non-specific regulator of membrane protein function. Despite considerable progress the interplay between hydrophobic coupling and lipid ordering is still elusive. We use electron spin resonance (ESR) to study the interaction between the model protein gramicidin and lipid bilayers of varying thickness. The free energy of the interaction is up to −6 kJ/mol; thus not strongly favored over lipid-lipid interactions. Incorporation of gramicidin results in increased order parameters with increased protein concentration and hydrophobic mismatch. Our findings also show that at high protein:lipid ratios the lipids are motionally restricted but not completely immobilized. Both exchange on and off rate values for the lipid ↔ gramicidin interaction are lowest at optimal hydrophobic matching. Hydrophobic mismatch of few Å results in up to 10-fold increased exchange rates as compared to the ‘optimal’ match situation pointing to the regulatory role of hydrophobic coupling in lipid-protein interactions.     

蛋白质热变性前新峰形成机制探讨

蛋白质热变性前新峰是蛋白质热变性过程的共性。通过对蛋白质三级结构特征的理论分析及实验验证的方法,研究了蛋白质热变性前新峰的变化规律,从而揭示了该峰产生的机理。采用ＤＳＣ方法对以不同结构水和十二烷基硫酸钠溶液水合溶菌酶样本进行了研究, 结果表明蛋白质的这种热变性前新峰的存在是由于维持其三级结构的疏水相互作用所造成, 新峰出现的峰温及其焓变与水的结构改变及由此而造成的蛋白质中结合水的含量和结构功能的变化有着直接的关系。     

Identification of nonplanar small molecule for G-quadruplex grooves: molecular docking and molecular dynamic study

DNA G-quadruplex is an attractive drug target for anticancer therapy. Most G-quadruplex ligands have little selectivity, due to π-stacking interaction with common G-tetrads surface. Thanks to the varieties of G-quadruplex grooves, the groove-binding ligand is expected to create high selectivity. Therefore, developing novel molecular geometries that target G-quadruplex groove has been paid growing attention. In this work, steroid FG, a special nonplanar and nonaromatic small molecule, interacting with different conformations of G-quadruplexes has been studied by molecular docking and molecular dynamics simulations. The results showed the selectivity of the hydrophobic group of steroid FG for the wide groove of antiparallel G-quadruplex. The methyl groups on the tetracyclic ring of steroid represent the specific binding ability for the small hydrophobic cavity formed by reversed stacking of G-tetrads in antiparallel G-quadruplex groove. This work provides new insight for developing new classes of G-quadruplex groove-binding ligands.     

Adsorption of synthetic homo- and hetero-oligodeoxynucleotides onto highly oriented pyrolytic graphite: atomic force microscopy characterization

DNA adsorption on electrode surfaces is of fundamental interest for the development of DNA-based biosensors. The free adsorption of 10-mer synthetic oligodeoxynucleotides (ODNs) onto highly oriented pyrolytic graphite (HOPG) surfaces was studied using Magnetic AC mode atomic force microscopy (MAC Mode AFM). The mechanism of interaction of nucleic acids with carbon electrode surfaces was elucidated, using 10-mer synthetic homo- and hetero-ODNs sequences of known base sequences, because they allow clear interpretation of the experimental data. AFM images in air revealed different adsorption patterns and degree of HOPG surface coverage for the ODNs, and correlation with the individual structure and base sequence of each ODN molecule will be presented. The results demonstrated that the hydrophobic interactions with the HOPG hydrophobic surface explain the main adsorption mechanism, although other effects such as electrostatic and Van der Waals interactions may contribute to the free adsorption process. The ODNs interacted differently with the HOPG surface, according to the ODN sequence hydrophobic characteristics, being directly depending on the molecular mass, the hydrophobic character of the individual bases and on the secondary structure of the molecule. The importance of the type of base existent at the ODN chain extremities on the adsorption process was investigated and different adsorption patterns were obtained with ODN sequences composed by the same group of bases aligned in a different order.     

Evaluation of the protein interfaces that form an intermolecular four-helix bundle as studied by computer simulation

Rational design of protein surface is important for creating higher order protein structures, but it is still challenging. In this study, we designed in silico the several binding interfaces on protein surfaces that allow a de novo protein–protein interaction to be formed. We used a computer simulation technique to find appropriate amino acid arrangements for the binding interface. The protein–protein interaction can be made by forming an intermolecular four-helix bundle structure, which is often found in naturally occurring protein subunit interfaces. As a model protein, we used a helical protein, YciF. Molecular dynamics simulation showed that a new protein–protein interaction is formed depending on the number of hydrophobic and charged amino acid residues present in the binding surfaces. However, too many hydrophobic amino acid residues present in the interface negatively affected on the binding. Finally, we found an appropriate arrangement of hydrophobic and charged amino acid residues that induces a protein–protein interaction through an intermolecular four-helix bundle formation.     

The molten globule state of a chimera of human alpha-lactalbumin and equine lysozyme.

The molten globule state of equine lysozyme is more stable than that of alpha-lactalbumin and is stabilized by non-specific hydrophobic interactions and native-like hydrophobic interactions. We constructed a chimeric protein which is produced by replacing the flexible loop (residues 105-110) in human alpha-lactalbumin with the helix D (residues 109-114) in equine lysozyme to investigate the possible role of the helix D for the high stability and native-like packing interaction in the molten globule state of equine lysozyme. The stability of the molten globule state formed by the chimeric protein to guanidine hydrochloride-induced unfolding is the same as that of equine lysozyme and is substantially greater than that of human alpha-lactalbumin, although only six residues come from equine lysozyme. Our results also suggest that the non-native interaction in the molten globule state of alpha-lactalbumin changes to the native-like packing interaction due to helix substitution. The solvent-accessibility of the Trp residues in the molten globule state of the chimeric protein is similar to that in the molten globule state of equine lysozyme in which packing interaction around the Trp residues in the native state is partially preserved. Therefore, the helix D in equine lysozyme is one of the contributing factors to the high stability and native-like packing interaction in the molten globule state of equine lysozyme. Our results indicate that the native-like packing interaction can stabilize the rudimentary intermediate which is stabilized by the non-specific hydrophobic interactions.     

Influence of sodium chloride on hydrophobic adsorption of PEGylated lysozyme

Interaction of macromolecules in aqueous salt‐containing solution with a hydrophobic adsorbent is studied by adsorption equilibrium measurements and by independent isothermal titration calorimetry. The macromolecules are native as well as mono‐, di‐, and tri‐PEGylated lysozyme and four pure PEGs. The hydrophobic adsorbent is Toyopearl PPG‐600M. The salt is sodium chloride. The sodium chloride concentration in the aqueous 25 mM sodium phosphate buffer is varied from 2000 to 4500 mM at pH 7.0 and 25°C. PEGylation of the lysozyme is carried using 5 and 10 kDa PEG chains. The molar enthalpy of adsorption is calculated from the adsorption equilibrium and the calorimetric data. The results show that the adsorption of the PEGylated lysozyme is caused by both the interaction of the lysozyme and the interaction of the PEG chains with the adsorbent, respectively, but the interaction of the lysozyme is stronger than that of PEG. The comparison of the results of the present study on the influence of sodium chloride with a corresponding study on the influence of ammonium sulfate shows that the adsorption mechanism changes upon the variation of the salt. The knowledge of the adsorption mechanisms supports the systematic development of chromatographic purification steps.     

The binding of anionic and nonionic surfactants to collagen through the hydrophobic effect

The adsorption of nonionic surfactants on hide powder previously treated with anionic surfactants has been studied. The adsorption of nonionic surfactants takes place through hydrophobic interactions. A mechanism has been proposed for this interaction, assuming that the nonionic surfactant has been fixed by means of secondary adsorption (hydrophobic interaction) after the primary adsorption of the anionic surfactant (ionic and hydrophobic interaction) which makes it possible.     

Surface Energetics to Assess Microbial Adhesion onto Fluidized Chromatography Adsorbents

Cell‐to‐support interaction and cell‐to‐cell aggregation phenomena have been studied in a model system composed of intact yeast cells and agarose‐based chromatography adsorbent surfaces. Biomass components and beaded adsorbents were characterized by contact angle determinations with three diagnostic liquids and, complementarily, by zeta potential measurements. Such experimental characterization of the interacting surfaces has allowed the calculation of interfacial free energy of interaction in aqueous media vs. distance profiles. The extent of biomass adhesion was inferred from calculations performed assuming standard chromatographic conditions, but different adsorption modes. Several stationary support/mobile phase systems were considered, i.e., ion exchange, hydrophobic interaction, and pseudo‐affinity. The calculated interaction energy minima revealed marginal attraction between cells and cation exchangers or agarose‐matrix beads (U ≤ |10–20| kT) but strong attraction with anion exchangers (U ≥ |200–1000| kT). Other systems including hydrophobic interaction and chelating beads showed intermediate energy minimum values (U <$>\approx<$> |40–100| kT) for interaction with biological particles. However, the calculations also showed that working conditions in the presence of salt can promote cell aggregation apart from cell‐to‐support interaction. Predictions based on the application of the XDLVO approach were confirmed by independent experimental methods, e.g., biomass deposition experiments and laser diffraction spectroscopy. The understanding of biomass attachment onto chromatographic supports can help in alleviating process limitations normally encountered during direct (primary) sequestration of bioproducts.     

Phosphoinositide-dependent kinase-1 orthologues from five eukaryotes are activated by the hydrophobic motif in AGC kinases

Phosphoinositide-dependent kinase-1 (PDK1) mediates activation of many AGC kinases by docking onto a phosphorylated hydrophobic motif located C-terminal of the catalytic domain in the AGC kinase. The interaction shifts PDK1 into a conformation with increased catalytic activity and leads to autophosphorylation of PDK1. We demonstrate here that addition of a hydrophobic motif peptide increases the catalytic activity of PDK1 orthologues from Homo sapiens, Aplysia californica, Arabidopsis thaliana, Schizosaccharomyces pombe (ksg1), and Saccharomyces cerevisiae (Pkh1 and Pkh2) 2- to 12-fold. Furthermore, the hydrophobic motif peptide increases autophosphorylation of PDK1 from Homo sapiens, S. pombe, and S. cerevisiae (Phk2). Our results suggest that PDK1 interaction and activation by the hydrophobic motif of AGC kinases is a central mechanism in PDK1 function, which is conserved during eukaryotic evolution.