Hydrophobic, hydrophilic, and charged amino acid networks within protein

The native three-dimensional structure of a single protein is determined by the physicochemical nature of its constituent amino acids. The 20 different types of amino acids, depending on their physicochemical properties, can be grouped into three major classes: hydrophobic, hydrophilic, and charged. The anatomy of the weighted and unweighted networks of hydrophobic, hydrophilic, and charged residues separately for a large number of proteins were studied. Results showed that the average degree of the hydrophobic networks has a significantly larger value than that of hydrophilic and charged networks. The average degree of the hydrophilic networks is slightly higher than that of the charged networks. The average strength of the nodes of hydrophobic networks is nearly equal to that of the charged network, whereas that of hydrophilic networks has a smaller value than that of hydrophobic and charged networks. The average strength for each of the three types of networks varies with its degree. The average strength of a node in a charged network increases more sharply than that of the hydrophobic and hydrophilic networks. Each of the three types of networks exhibits the "small-world" property. Our results further indicate that the all-amino-acids networks and hydrophobic networks are of assortative type. Although most of the hydrophilic and charged networks are of the assortative type, few others have the characteristics of disassortative mixing of the nodes. We have further observed that all-amino-acids networks and hydrophobic networks bear the signature of hierarchy, whereas the hydrophilic and charged networks do not have any hierarchical signature.     

不同类型氨基酸网络参量与蛋白质折叠的关系

理论和实验研究表明,蛋白质天然拓扑结构对其折叠过程具有重要的影响．采用复杂网络的方法分析蛋白质天然结构的拓扑特征,并探索蛋白质结构特征与折叠速率之间的内在联系．分别构建了蛋白质氨基酸网络、疏水网、亲水网、亲水-疏水网以及相应的长程网络,研究了这些网络的匹配系数(assortativity coefficient)和聚集系数(clustering coefficient)的统计特性．结果表明,除了亲水-疏水网,上述各网络的匹配系数均为正值,并且氨基酸网和疏水网的匹配系数与折叠速率表现出明显的线性正相关,揭示了疏水残基间相互作用的协同性有助于蛋白质的快速折叠．同时,研究发现疏水网的聚集系数与折叠速率有明显的线性负相关关系,这表明疏水残基间三角结构(triangle construction)的形成不利于蛋白质快速折叠．还进一步构建了相应的长程网络,发现序列上间距较远的残基接触对的形成将使蛋白质折叠进程变慢．     

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.     

Spectroscopic investigations on the conformational changes of lysozyme effected by different sizes of N‐acetyl‐l‐cysteine‐capped CdTe quantum dots

The effect of N‐acetyl‐l ‐cysteine‐capped CdTe quantum dots (NAC‐CdTe QDs) with different sizes on lysozyme was investigated by isothermal titration calorimetry (ITC), enzyme activity assays, and multi‐spectroscopic methods. ITC results proved that NAC‐CdTe QDs can spontaneously bind with lysozyme and hydrophobic force plays a major role in stabilizing QDs–lysozyme complex. Multi‐spectroscopic measurements revealed that NAC‐CdTe QDs caused strong quenching of the lysozyme's fluorescence in a size‐dependent quenching manner. Moreover, the changes of secondary structure and microenvironment in lysozyme caused by the NAC‐CdTe QDs were higher with a bigger size. The results of enzyme activity assays showed that the interaction between lysozyme and NAC‐CdTe QDs inhibited the activity of lysozyme and the inhibiting effect was in a size‐dependent manner. Based on these results, we conclude that NAC‐CdTe QDs with larger particle size had a larger impact on the structure and function of lysozyme.     

Mathematical folding of node chains in a molecular network

Under the framework of network artificial chemistry (NAC), a method to construct a phenotypic machine from genotypic information is proposed. The genotype is expressed as a sequence of nodes (a node chain) with symbol data. According to an implemented algorithm, a chain is agglomerated, tangled, and finally folded into a node cluster that works as a control-flow machine in the network. Two examples of control-flow clusters, splitase and replicase, are presented and their functionality is demonstrated. Several meanings and future research directions are discussed.     

Visualization of the nature of protein folding by a study of a distance constraint approach in two-dimensional models

A distance constraint approach is applied to two-dimensional models of proteins in order to visualize the nature of protein folding and to examine the relative roles of different ranges of interaction. Three different native structures (I, II, and III) are considered; they have two different kinds of residues, viz., hydrophobic and hydrophilic, and different sequences of these residues. We examine how the distance constraint approach functions in the prediction of protein folding when we know the sequence of the residues, the (fixed) bond lengths, the mean distances between residues i and i + 2, and i and i + 3, and the mean distances for hydrophobic–hydrophobic, hydrophobic–hydrophilic, and hydrophilic–hydrophilic contacts between residues i and i + j, where j ≥ 4. This approach involves optimization of an object function with respect to 98 variables and is not free of the multiple-minimum problem. The optimization is always terminated if the chain is entangled and/or the segments (residues) are packed too compactly to move. In order to escape from such situations and to take the excluded-volume effect into account, a Monte Carlo method is used after the optimization is trapped in local minima. Success in the prediction of folding is found to depend on the starting conformations and on the native conformations. Fair success is obtained in predicting the helix-like structure in protein I and the overall structure of protein III, but not the β-like structures of proteins I and II. Insofar as the prediction of the structure of protein III is reasonable, it appears that some sequences of residues produce greater constraints on their conformations than others, if one considers only the hydrophobic and hydrophilic nature of the residues. These results imply that, in the folding of real proteins in three dimensions, the competition for hydrophobic (and hydrophilic) residues for inside (outside) positions in the molecule probably constitutes a necessary but not a sufficient condition to form and stabilize the native structure. The failure to predict the structure of protein II, and part of that of protein I, suggests that there are two types of long-range interactions. One (which we considered here) is nonspecific (i.e., is defined only in terms of contacts between residues of the same or different polarity) and acts at any stage of protein folding; the other (which we did not consider here) is a specific interaction between residues in pairs and contributes only when the residues in the specific pair take on the native conformation. Presumably, incorporation of such specific long-range interactions, together with the nonspecific ones, is necessary for successful protein folding, using the distance constraint approach.     

Molecular docking: The role of noncovalent interactions in the formation of protein-nucleotide and protein-peptide complexes

Knowledge of the spatial structure of complexes formed by cellular proteins and membrane receptors with their respective ligands is an important step towards understanding the mechanisms of their functioning. Rational drug design and the search for new therapeutically active compounds also require structural information on the interaction of prototypic drugs with the target protein. The present review briefly describes the main computational methods of molecular docking that are used to predict the conformation of a ligand bound to the active center of a protein. Approaches enabling an increase of the precision and efficiency of the currently used docking algorithms are exemplified by the recent projects of the Laboratory of Biomolecular Modeling of IBCh RAS. Special attention is paid to hydrophilic and hydrophobic interactions, as well as to the stacking phenomena that account for the molecular recognition of specific ligand fragments. These types of contacts are often inadequately described by the algorithms of the estimation of the intermolecular interaction energy of the existing docking programs (scoring functions), this ultimately leading to erroneous predictions of the three-dimensional structure of complexes. Therefore, a thorough consideration of these interactions is one of the most important tasks of molecular modeling.     

Swapping core residues in homologous proteins swaps folding mechanism

Rat intestinal fatty acid binding protein (IFABP) displays an intermediate with little if any secondary structure during unfolding, while the structurally homologous rat ileal lipid binding protein (ILBP) displays an intermediate during unfolding with nativelike secondary structure. Double-jump experiments indicate that these intermediates are on the folding path for each protein. To test the hypothesis that differences in the number of buried hydrophobic atoms in a folding initiating site are responsible for the different types of intermediates observed for these proteins, two mutations (F68C-IFABP and C69F-ILBP) were made that swapped a more hydrophobic residue for a more hydrophilic residue in the respective cores of these two proteins. F68C-IFABP followed an unfolding path identical to that of WT-ILBP with an intermediate that showed nativelike secondary structure, whereas C69F-ILBP followed an unfolding path that was identical to that of WT-IFABP with an intermediate that lacked secondary structure. Further, a hydrophilic residue was introduced at an identical hydrophobic structural position in both proteins (F93S-IFABP and F94S-ILBP). Replacement of phenylalanine with serine at this site led to the appearance of an intermediate during refolding that lacked secondary structure for both proteins that was not detected for either parental protein. Altering the chemical characteristics and/or size of residues within an initiating core of hydrophobic interactions is critical to the types of intermediates that are observed during the folding of these proteins.     

Effect of Substrate and Cell Surface Hydrophobicity on Phosphate Utilization in Bacteria

We measured the rates of utilization of hydrophobic and hydrophilic phosphate compounds in gram-negative bacteria with different surface hydrophobicities, isolated from wetland habitats. Three hydrophobic and two hydrophilic bacterial species were selected for study by measuring cell adherence to hydrocarbons. The bacteria were grown under phosphorus-limited conditions with P(infi), hydrophilic (beta)-glycerophosphate, or hydrophobic phosphatidic acid as the phosphate source. Hydrophilic bacteria grew most rapidly on P(infi), followed by (beta)-glycerophosphate. Phosphatidic acid did not support growth or did so at a much later time (40 h) than did the other phosphate treatments. Although all hydrophobic species grew well on these substrates, the rate of growth of two Acinetobacter baumannii isolates on phosphatidic acid exceeded the rate of growth on phosphate or (beta)-glycerophosphate. A membrane phospholipid and lipopolysaccharide were used as a source of phosphorus by hydrophobic species, whereas hydrophilic species could not use the membrane phospholipids and used lipopolysaccharide to a lesser extent. Besides hydrophobic interaction between cells and substrate, phosphatase activity, which was cell bound in hydrophilic species but 30 to 50% unbound in hydrophobic species, affected cell growth. Dialyzed culture supernatant containing phosphatase from hydrophobic species increased the phosphate availability to hydrophilic species. Additionally, cellular extracts from a hydrophilic species, when added to hydrophilic cells, permitted growth on hydrophobic phosphate sources. Naturally occurring amphiphilic humic acids affected the utilization of P(infi) and (beta)-glycerophosphate in bacteria with hydrophilic surfaces but did not affect hydrophobic bacteria. Our results indicate that hydrophobic phosphate sources can be used by bacteria isolated from aquatic environments as the sole phosphorus source for growth. This utilization, in part, appears to be related to cell surface hydrophobicity and extracellular enzyme production.     

Differentiated analysis of the secondary structure of hydrophilic and hydrophobic regions in alpha- and beta-subunits of Na+,K+-ATPase by Raman spectroscopy

Raman spectra of active Na+,K+-ATPase from pig kidney and membrane-bound products of its two-stage trypsinolysis, including alpha-subunit hydrophobic regions as well as the intact beta-subunit and hydrophobic regions of alpha- and beta-subunits, were measured to calculate the secondary structure of hydrophilic and hydrophobic regions of the enzyme. Consequent comparison demonstrated unambiguously that (i) membrane-bound hydrophobic parts of polypeptide chains of Na+,K+-ATPase subunits are in the alpha-helical conformation; (ii) essential contents of the alpha-helix as well as beta-sheet are estimated to form the hydrophilic (mainly cytoplasmic) domain of the Na+,K+-ATPase alpha-subunit; (iii) the exoplasmic hydrophilic domain of the beta-subunit is shown to include several antiparallel beta-pleated sheets and a small amount of the alpha-helix and unordered conformations. The model of the secondary structure organization of hydrophilic domains as well as 8 hydrophobic transmembrane segments of the enzyme molecule was proposed on the basis of experimental results and predictional calculations.     

Nisin adsorption on hydrophilic and hydrophobic surfaces: evidence of its interactions and antibacterial activity

Study of peptides adsorption on surfaces remains a current challenge in literature. A complementary approach, combining X‐ray photoelectron spectroscopy (XPS) and time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) was used to investigate the antimicrobial peptide nisin adsorption on hydrophilic and hydrophobic surfaces. The native low density polyethylene was used as hydrophobic support and it was grafted with acrylic acid to render it hydrophilic. XPS permitted to confirm nisin adsorption and to determine its amount on the surfaces. ToF‐SIMS permitted to identify the adsorbed bacteriocin type and to observe its distribution and orientation behavior on both types of surfaces. Nisin was more oriented by its hydrophobic side to the hydrophobic substrate and by its hydrophilic side to the outer layers of the adsorbed peptide, in contrast to what was observed on the hydrophilic substrate. A correlation was found between XPS and ToF‐SIMS results, the types of interactions on both surfaces and the observed antibacterial activity. Such interfacial studies are crucial for better understanding the peptides interactions and adsorption on surfaces and must be considered when setting up antimicrobial surfaces. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Hyaluronic acid by atomic force microscopy.

Hyaluronic acid (HA) of different molecular weights has been examined by atomic force microscopy (AFM) in air. This technique allows 3-D surface images of soft samples without any pretreatment, such as shadowing or staining. In the present study we examined the supermolecular organization of HA chains when deposited on mica and graphite, to better understand the interchain and intrachain interactions of HA molecules in solution. The concentration of the solution deposited varied from 0.001 to 1 mg/ml. On both substrates, and independent of the concentration, high-molecular-mass HA formed networks in which molecules ran parallel for hundreds of nanometers, giving rise to flat sheets and tubular structures that separate and rejoin into similar neighboring aggregates. Accurate measurements of the thickness of the thinnest sheets were consistent with a monolayer of HA molecules, 0.3 nm thick, strongly indicating lateral aggregation forces between chains as well as rather strong hydrophilic interactions between mica and HA. The results agree with an existing model of HA tertiary structure in solution in which the network is stabilized by both hydrophilic and hydrophobic interactions. Our images support this model and indicate that hydrophobic interactions between chains may exert a pivotal role in aqueous solution.     

Amino acid sequence pattern in the regulatory peptides.

The essential properties of the primary structure of regulatory peptides, i.e. amino acid residues and their combinations, which are characteristic of the whole population of regulatory peptides, have been revealed using statistical methodology. These properties are as follows: increased content of certain residues (Gly, Pro, Phe, Arg, Tyr, Met and Trp) as well as an increased rate of occurrence of certain pairs of residue as compared with proteins, a random sequence of residues and "nonregulatory" peptides. By representing regulatory peptides as a sequence of hydrophobic (2 types) and hydrophilic (3 types) segments, the pattern for alternation of these segments in regulatory peptides has been determined. The segments were classified into 5 types according to the peculiarities of mutual localization of hydrophobic and hydrophilic residues within the primary structure of regulatory peptides. As compared with proteins, "nonregulatory" peptides and a random sequence of segments, regulatory peptides were characterized by an increased frequency of 4 particular pairs of segments among 12 theoretically possible pairs. These 4 pairs are fragments of the periodic segment sequence with periods of 4 segments. The revealed pattern indicates that there exists a general principle of the regulatory peptide primary structure organization and possibly a common type of the regulatory peptides flexible peptide chain folding at the ligand-receptor complex formation.     

Analysis of accessible surface of residues in proteins

We analyzed the total, hydrophobic, and hydrophilic accessible surfaces (ASAs) of residues from a nonredundant bank of 587 3D structure proteins. In an extended fold, residues are classified into three families with respect to their hydrophobicity balance. As expected, residues lose part of their solvent-accessible surface with folding but the three groups remain. The decrease of accessibility is more pronounced for hydrophobic than hydrophilic residues. Amazingly, Lysine is the residue with the largest hydrophobic accessible surface in folded structures. Our analysis points out a clear difference between the mean (other studies) and median (this study) ASA values of hydrophobic residues, which should be taken into consideration for future investigations on a protein-accessible surface, in order to improve predictions requiring ASA values. The different secondary structures correspond to different accessibility of residues. Random coils, turns, and beta-structures (outside beta-sheets) are the most accessible folds, with an average of 30% accessibility. The helical residues are about 20% accessible, and the difference between the hydrophobic and the hydrophilic residues illustrates the amphipathy of many helices. Residues from beta-sheets are the most inaccessible to solvent (10% accessible). Hence, beta-sheets are the most appropriate structures to shield the hydrophobic parts of residues from water. We also show that there is an equal balance between the hydrophobic and the hydrophilic accessible surfaces of the 3D protein surfaces irrespective of the protein size. This results in a patchwork surface of hydrophobic and hydrophilic areas, which could be important for protein interactions and/or activity.     

The effect of mutations in the synthesis of lipopolysaccharides and calcofluor-binding polysaccharides on biofilm formation by Azospirillum brasilense

The thickness and antigenic properties of biofilms produced by Azospirillum brasilense Sp245 and its mutants deficient in the synthesis of lipopolysaccharides (Lps) and calcofluor-binding polysaccharides (CBPS) at the interface between water and hydrophilic or hydrophobic solid surfaces were compared. The mutants deficient in acidic LpsI synthesis produce thicker biofilms on hydrophilic surfaces. Biofilms produced on hydrophobic surfaces by bacteria that are unable to synthesize CBPS are less pronounced. Defects in CBPS production in Azospirillum mutants with impaired flagellar motility can cause adverse effects on the cell ability to attach to hydrophobic and hydrophilic surfaces. The loss of the neutral LpsII antigen by the mutants capable of producing CBPS does not affect their behavior on hydrophobic surfaces, which is probably due to the compensatory increase in the total polysaccharide production. The fundamental change in the Lps structure correlates with the activation of biofilm formation by the relevant mutants on hydrophilic and hydrophobic surfaces.     

Amino acid network and its scoring application in protein-protein docking

Protein-protein complex, composed of hydrophobic and hydrophilic residues, can be divided into hydrophobic and hydrophilic amino acid network structures respectively. In this paper, we are interested in analyzing these two different types of networks and find that these networks are of small-world properties. Due to the characteristic complementarity of the complex interfaces, protein-protein docking can be viewed as a particular network rewiring. These networks of correct docked complex conformations have much more increase of the degree values and decay of the clustering coefficients than those of the incorrect ones. Therefore, two scoring terms based on the network parameters are proposed, in which the geometric complementarity, hydrophobic-hydrophobic and polar-polar interactions are taken into account. Compared with a two-term energy function, a simple scoring function HPNet which includes the two network-based scoring terms shows advantages in two aspects, not relying on energy considerations and better discrimination. Furthermore, combing the network-based scoring terms with some other energy terms, a new multi-term scoring function HPNet-combine can also make some improvements to the scoring function of RosettaDock.     

Charged residues are involved in membrane fusion mediated by a hydrophilic peptide located in vesicular stomatitis virus G protein

Membrane fusion is an essential step of the internalization process of the enveloped animal viruses. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion at the acidic environment of the endosomal compartment. In a previous work, we identified a specific sequence in VSV G protein, comprising the residues 145 to 164, directly involved in membrane interaction and fusion. Unlike fusion peptides from other viruses, this sequence is very hydrophilic, containing six charged residues, but it was as efficient as the virus in catalyzing membrane fusion at pH 6.0. Using a carboxyl-modifying agent, dicyclohexylcarbodiimide (DCCD), and several synthetic mutant peptides, we demonstrated that the negative charges of peptide acidic residues, especially Asp153 and Glu158, participate in the formation of a hydrophobic domain at pH 6.0, which is necessary to the peptide-induced membrane fusion. The formation of the hydrophobic region and the membrane fusion itself were dependent on peptide concentration in a higher than linear fashion, suggesting the involvement of peptide oligomerization. His148 was also necessary to hydrophobicity and fusion, suggesting that peptide oligomerization occurs through intermolecular electrostatic interactions between the positively-charged His and a negatively-charged acidic residue of two peptide molecules. Oligomerization of hydrophilic peptides creates a hydrophobic region that is essential for the interaction with the membrane that results in fusion.     

Influence of hydrophobic and hydrophilic spacer-containing enzyme conjugates on functional parameters of steroid immunoassay

Introduction of spacers in coating steroid antigen or enzyme conjugates or immunogen is known to exert an influence on the sensitivity of steroid enzyme immunoassays. We have introduced hydrophobic and hydrophilic spacers between enzyme and steroid moieties and studied their effects on functional parameters of enzyme immunoassays, using cortisol as a model steroid. Cortisol-3-O-carboxymethyloxime-bovine serum albumin (F-3-O-CMO-BSA) was used as immunogen to raise the antiserum in New Zealand white rabbits. Three enzyme conjugates were prepared using cortisol-21-hemisuccinate (F-21-HS) as carboxylic derivative of cortisol and horseradish peroxidase (HRP) as an enzyme label. These were F-21-HS-HRP (without spacer), F-21-HS-adipic acid dihydrazide-HRP (adipic acid dihydrazide as hydrophobic spacer), and F-21-HS-urea-HRP (urea as hydrophilic spacer). The influence of hydrophobic and hydrophilic spacers on the functional parameters of assays such as lower detection limit, ED50, and specificity was studied with reference to enzyme conjugate without spacer. The results of the present investigation revealed that the presence of a hydrophilic spacer in the enzyme conjugate decreases the lower detection limit, decreases the ED50, and marginally improves the specificity of assays. These improvements in functional parameters of assays may be due to the decreased magnitude of the overall hydrophobic interactions existing between the spacer in enzyme conjugate and the antigen binding site of the antibody.     

Protein chemistry of the Neurospora crassa plasma membrane H+-ATPase

A highly effective procedure for fragmenting the Neurospora crassa plasma membrane H+-ATPase and purifying the resulting peptides is described. The enzyme is cleaved with trypsin to form a limit digest containing both hydrophobic and hydrophilic peptides, and the hydrophobic and hydrophilic peptides are then separated by extraction with an aqueous ammonium bicarbonate solution. The hydrophilic peptides are fractionated by Sephadex G-25 column chromatography into three pools, and the individual peptides in each pool are purified by high-performance liquid chromatography. The hydrophobic peptides are dissolved in neat trifluoroacetic acid (TFA), diluted with chloroform-methanol (1:1), and the hydrophobic peptide solution thus obtained is then fractionated by Sephadex LH-60 column chromatography in chloroform-methanol (1:1) containing 0.1% TFA. The recoveries in all of the above procedures are greater than 90%. The N-terminal amino acid sequences of three of the hydrophobic H+-ATPase peptides purified by this methodology have been determined, which establishes the position of these peptides in the 100,000 Da polypeptide chain by reference to the published gene sequence, and documents the sequencability of the hydrophobic peptides purified in this way. This methodology should facilitate the identification of a variety of amino acid residues important for the structure and function of the H+-ATPase molecule. Moreover, the overall strategy for working with the protein chemistry of the H+-ATPase should be applicable to other amphiphilic integral membrane proteins as well.     

Influence of far-ultraviolet radiation on the permeability of the outer membrane of Escherichia coli

Far-ultraviolet radiation (254 nm) at a dose of 10, 20, and 30 J/m2 was found to disrupt the outer membrane permeability barrier of Escherichia coli to various antibiotics, dyes, and detergents. The degree of sensitization to these agents was proportional to the radiation dose. The irradiated cells showed a significant increase in the sensitivity of hydrophilic antibiotics (ampicillin, carbenicillin, penicillin), whereas much less sensitization was found towards hydrophobic probes (kanamycin, erythromycin, rifamycin SV, crystal violet, phenol, novobiocin) and detergents (dodecyl sulfate, bile salt, Triton X-100). The biochemical data and ultrastructural analysis of the outer membrane by freeze-etching have shown that the increase in phospholipid:protein ratio after irradiation had changed the architecture of the outer membrane from a highly asymmetric bilayer structure with densely packed lipopolysaccharide--protein particles on the outer half, to one predominantly exhibiting smooth phospholipid bilayer characteristics. The structure, composition, and barrier function of the outer membrane were restored to normal within 3 h of postirradiation incubation in nonproliferative medium. During this period, the acquisition of resistance towards a hydrophilic antibiotic (ampicillin) was faster than that for a hydrophobic agent (phenol).