Regulation of fatty acid composition in Escherichia coli: a proposed common mechanism for changes induced by ethanol, chaotropic agents, and a reduction of growth temperature.

Growth of Escherichia coli in the presence of ethanol and chaotropic salts resulted in the synthesis of lipids containing elevated levels of unsaturated fatty acids analogous to the effect of a reduction in growth temperature. Both ethanol and chaotropic agents acted at the level of fatty acid biosynthesis and altered lipid composition by decreasing the proportion of saturated acyl chains available for the synthesis of phospholipids. A reduction in temperature causes similar effects on fatty acid biosynthesis in vivo and in vitro. Ethanol, chaotropic salts, and a decrease in temperature all weaken hydrophobic interactions. Antichaotropic salts antagonized and effects of these treatments on fatty acid synthesis in vitro. These results are consistent with a common mechanism for the effects of chaotropic agents, temperature, and ethanol on fatty acid synthesis. The biosynthesis of saturated and unsaturated acyl chains may be regulated by the strength of hydrophobic interactions. Changes in the strength of hydrophobic interactions could alter enzyme structure, substrate structure, or the equilibrium between the soluble enzymes of fatty acid synthesis and their respective acyl carrier protein substrates.     

Effective interactions between chaotropic agents and proteins

Chaotropic agents are cosolutes that can disrupt the hydrogen bonding network between water molecules and reduce the stability of the native state of proteins by weakening the hydrophobic effect. In this work, we represent the chaotropic agent as a factor that reduces the amount of order in the structures formed by water molecules, both in the bulk and the hydration shells around hydrophobic amino acids. In this framework we show that low chaotrope concentrations lead to a destabilization of the native state of proteins, and that high concentrations induce complete denaturation. We also find that the reduction of the number of bulk ordered states of water molecules can give origin to an effective interaction between chaotropic molecules and proteins.     

Activation of Clostridium perfringens spores under conditions that disrupt hydrophobic interactions of biological macromolecules

The effect of hydrophobic interactions on the activation of C. perfringens NCTC 8679 spores was examined by heating spores under conditions that modify the hydrophobic properties of biological macromolecules. After the activation treatment and a washing procedure, germination was determined by measuring the decrease in optical density of spores suspended in an enriched germination medium. Activation was inhibited for spores that were treated under conditions that strengthen hydrophobic interactions, i.e., a decrease in pH or the presence of structure-stabilizing neutral salts. Activation was enhanced by treatment under conditions that disrupt hydrophobic interactions, i.e., an increase in pH or the presence of urea, dibucaine, or denaturing neutral salts. A deactivation treatment with the antichaotropic salt (NH4)2SO4 reversed activation by the chaotropic salt CaCl2 and to a lesser extent reversed activation by sublethal heat (75 degrees C) or urea. Most treatments that enhanced activation increased spore injury at higher temperatures, which resulted in decreased germination. However, (NH4)2SO4 and a decrease in pH from 5.6 to 3.8, which inhibited activation, also favored injury. The results suggest that activation involves a conformational change of a spore protein(s) through weakening of hydrophobic molecular forces and that activation and injury occur at different spore sites.     

Activation of Clostridium perfringens spores under conditions that disrupt hydrophobic interactions of biological macromolecules.

The effect of hydrophobic interactions on the activation of C. perfringens NCTC 8679 spores was examined by heating spores under conditions that modify the hydrophobic properties of biological macromolecules. After the activation treatment and a washing procedure, germination was determined by measuring the decrease in optical density of spores suspended in an enriched germination medium. Activation was inhibited for spores that were treated under conditions that strengthen hydrophobic interactions, i.e., a decrease in pH or the presence of structure-stabilizing neutral salts. Activation was enhanced by treatment under conditions that disrupt hydrophobic interactions, i.e., an increase in pH or the presence of urea, dibucaine, or denaturing neutral salts. A deactivation treatment with the antichaotropic salt (NH4)2SO4 reversed activation by the chaotropic salt CaCl2 and to a lesser extent reversed activation by sublethal heat (75 degrees C) or urea. Most treatments that enhanced activation increased spore injury at higher temperatures, which resulted in decreased germination. However, (NH4)2SO4 and a decrease in pH from 5.6 to 3.8, which inhibited activation, also favored injury. The results suggest that activation involves a conformational change of a spore protein(s) through weakening of hydrophobic molecular forces and that activation and injury occur at different spore sites.     

Chemical factors influencing adsorption of bacteriophage MS2 to membrane filters.

Antichaotropic salts, such as magnesium sulfate, and metal chelators, such as citrate ions, promoted adsorption of bacteriophage MS2 to membrane filters. In contrast, compounds that disrupt hydrophobic interactions, such as chaotropic salts, urea, Tween 80, and ethanol, did not promote adsorption of MS2 to membrane filters and counteracted the ability of magnesium sulfate to promote such adsorption. These results provide evidence that magnesium sulfate promotes the association of MS2 with membrane filters primarily by strengthening hydrophobic interactions between the virus and the filters.     

Exploring the role of hydrophilic amino acids in unfolding of protein in aqueous ethanol solution

Hydrophobic association is the key contributor behind the formation of well packed core of a protein which is often believed to be an important step for folding from an unfolded chain to its compact functional form. While most of the protein folding/unfolding studies have evaluated the changes in the hydrophobic interactions during chemical denaturation, the role of hydrophilic amino acids in such processes are not discussed in detail. Here we report the role of the hydrophilic amino acids behind ethanol induced unfolding of protein. Using free energy simulations, we show that chicken villin head piece (HP‐36) protein unfolds gradually in presence of water‐ethanol binary mixture with increasing composition of ethanol. However, upon mutation of hydrophilic amino acids by glycine while keeping the hydrophobic amino acids intact, the compact state of the protein is found to be stable at all compositions with gradual flattening of the free energy landscape upon increasing compositions. The local environment around the protein in terms of ethanol/water number significantly differs in wild type protein compared to the mutated protein. The calculated Wyman‐Tanford preferential binding coefficient of ethanol for wild type protein reveals that a greater number of cosolutes (here ethanol) bind to the unfolded state compared to its folded state. However, no significant increase in binding coefficient of ethanol at the unfolded state is found for mutated protein. Local‐bulk partition coefficient calculation also suggests similar scenarios. Our results reveal that the weakening of hydrophobic interactions in aqueous ethanol solution along with larger preferential binding of ethanol to the unfolded state mediated by hydrophilic amino acids combinedly helps unfolding of protein in aqueous ethanol solution.     

Structural changes and interactions involved in the Ca(2+)-triggered stabilization of the cell-bound cell envelope proteinase in Lactococcus lactis subsp. cremoris SK11

The cell-bound cell envelope proteinase (CEP) of the mesophilic cheese-starter organism Lactococcus lactis subsp. cremoris SK11 is protected from rapid thermal inactivation at 25 degrees C by calcium bound to weak binding sites. The interactions with calcium are believed to trigger reversible structural rearrangements which are coupled with changes in specific activity (F. A. Exterkate and A. C. Alting, Appl. Env. Microbiol. 65:1390-1396, 1999). In order to determine the significance of the rearrangements for CEP stability and the nature of the interactions involved, the effects of the net charge present on the enzyme and of different neutral salts were studied with the stable Ca-loaded CEP, the unstable so-called "Ca-free" CEP and with the Ca-free CEP which was stabilized nonspecifically and essentially in its native conformation by the nonionic additive sucrose. The results suggest that strengthening of hydrophobic interactions is conducive to stabilization of the Ca-free CEP. On the other hand, a hydrophobic effect contributes significantly to the stability of the Ca-loaded CEP; a phased salting-in effect by a chaotropic salt suggests a complex inactivation process of this enzyme due to weakening of hydrophobic interactions and involving an intermediate enzyme species. Moreover, a Ca-triggered increase of a relatively significant hydrophobic effect in the sucrose-stabilized Ca-free CEP occurs. It is suggested that in the Ca-free CEP the absence of both local calcium-mediated backbone rigidification and neutralization of negative electrostatic potentials in the weak Ca-binding sites, and in addition the lack of significant hydrophobic stabilization, increase the relative effectiveness of electrostatic repulsive forces on the protein to an extent that causes the observed instability. The conditions in cheese seem to confer stability upon the cell-bound enzyme; its possible involvement in proteolysis throughout the ripening period is discussed.     

Structural Changes and Interactions Involved in the Ca2+-Triggered Stabilization of the Cell-Bound Cell Envelope Proteinase in Lactococcus lactis subsp. cremoris SK11

The cell-bound cell envelope proteinase (CEP) of the mesophilic cheese-starter organism Lactococcus lactis subsp. cremoris SK11 is protected from rapid thermal inactivation at 25°C by calcium bound to weak binding sites. The interactions with calcium are believed to trigger reversible structural rearrangements which are coupled with changes in specific activity (F. A. Exterkate and A. C. Alting, Appl. Env. Microbiol. 65:1390–1396, 1999). In order to determine the significance of the rearrangements for CEP stability and the nature of the interactions involved, the effects of the net charge present on the enzyme and of different neutral salts were studied with the stable Ca-loaded CEP, the unstable so-called “Ca-free” CEP and with the Ca-free CEP which was stabilized nonspecifically and essentially in its native conformation by the nonionic additive sucrose. The results suggest that strengthening of hydrophobic interactions is conducive to stabilization of the Ca-free CEP. On the other hand, a hydrophobic effect contributes significantly to the stability of the Ca-loaded CEP; a phased salting-in effect by a chaotropic salt suggests a complex inactivation process of this enzyme due to weakening of hydrophobic interactions and involving an intermediate enzyme species. Moreover, a Ca-triggered increase of a relatively significant hydrophobic effect in the sucrose-stabilized Ca-free CEP occurs. It is suggested that in the Ca-free CEP the absence of both local calcium-mediated backbone rigidification and neutralization of negative electrostatic potentials in the weak Ca-binding sites, and in addition the lack of significant hydrophobic stabilization, increase the relative effectiveness of electrostatic repulsive forces on the protein to an extent that causes the observed instability. The conditions in cheese seem to confer stability upon the cell-bound enzyme; its possible involvement in proteolysis throughout the ripening period is discussed.     

High-recovery one-step purification of the DNA-binding protein Fur by mild guanidinium chloride treatment

Engineering of DNA-binding domains of regulatory proteins aimed to control gene expression requires a deep knowledge of protein–DNA interactions acquired from structural data on purified species. Most DNA-binding proteins work as dimers establishing multiple protein–protein contacts mainly driven by hydrophobic interactions, being its cleansing a difficult task because of solubility problems. One-step purification of soluble, functional recombinant FurA from the cyanobacterium Anabaena sp. PCC 7120 has been achieved using mild chaotropic conditions. FurA was isolated using a Zn-iminodiacetate chromatography of the crude extract obtained after sonication of Escherichia coli in the presence of 2 M guanidium chloride. CD and ¹D NMR spectroscopies demonstrate that FurA conserves the native tertiary structure. Functional analysis reveals FurA ability to recognise and bind target DNAs. We propose that the use of chaotropic agents under mild denaturating conditions might have general application in the purification of DNA-binding proteins and other proteins prone to aggregation.     

Studies on the quaternary structure of class I major histocompatibility complex antigens. Effect of different agents on the interaction between subunits

The effect of temperature, urea, guanidine HCl, ionic and nonionic detergents, organic solvents, chaotropic salts, pH, and divalent cations has been investigated on purified human histocompatibility antigens solubilized by papain (HLApap) or solubilized by sodium cholate (HLAchol). HLApap and HLAchol are fairly stable proteins to agents acting predominantly on hydrogen bonds (temperature, urea) or hydrophobic forces (ionic and nonionic detergents). However, agents which affect ionic interactions (pH, salts, divalent cations) dissociate the molecules into subunits. A single binding site for beta 2-microglobulin with an affinity constant of 1.0 X 10(7) M-1 was found for the alpha chain of HLAchol. The dissociated subunits can be separated by affinity chromatography on Sepharose-rabbit IgG anti-human beta 2-microglobulin and reassociate in vitro when incubated under the appropriate conditions. The results point toward an important role of ionic interactions between subunits in the stabilization of the quaternary structure of HLA.     

Lysis of halophilic Vibrio alginolyticus and Vibrio costicolus induced by chaotropic anions.

High concentration (1.0 M) of KSCN, but not of NaSCN, induced lysis of slightly halophilic Vibrio alginolyticus and moderately halophilic Vibrio costicolus, and the decrease in absorbance of the cell suspension was complete after 30 min at 25 degrees C. Replacement of K+ with Na+ effectively prevented the lysis by SCN-.K+ salts of NO3-, Br- and I-, however, induced no significant lysis. In electron micrographs, a prolonged exposure of the cells of V. alginolyticus to 1.0 M KSCN displaced the nucleoplasm to maintain close contact with the cell membranes. After 40 min of interaction, 50% of the cellular protein, 96% of RNA and 94% of DNA were recovered in the lysed cells. In contrast to lysis in hypotonic conditions, the lysis induced by KSCN is due mainly to a partial release of protein from the cells. V. costicolus was more susceptible to SCN- than V. alginolyticus, whereas nonhalophilic Escherichia coli was resistant to 1.0 M KSCN. Thus, lysis by SCN- is characteristic of halophilic bacteria and cell membranes of more halophilic bacteria are more susceptible to chaotropic anions. The protective effect of Na+ observed here was considered to be manifested by specific interactions of Na+ with components of cell membranes, thereby rendering their structures resistant to the action of chaotropic anions.     

Inhibition of the cooperative adhesion of Streptococcus sanguis to hydroxylapatite

The adhesion of Streptococcus sanguis to hydroxylapatite is a process involving several adhesins and receptors. Binding isotherms and Scatchard plots of the adhesion suggest that cooperative interactions occur at low cell densities. It was found that sulfolane, a hydrophobic-bond diluent, was capable of inhibiting the cooperative adhesion of S. sanguis to saliva-coated hydroxylapatite beads. Sodium thiocyanate, a chaotropic agent, inhibited not only cooperative adhesion, but also the adhesion thought to result from noncooperative interactions. It is suggested that strong chaotropic agents may not only inhibit adhesin-receptor complexes, but also may influence the secondary/tertiary structures of interacting species.     

Chemical cross-linking of mitochondrial NADH dehydrogenase from bovine heart.

The structure of bovine heart mitochondrial NADH dehydrogenase was investigated by using two cleavable cross-linking agents, disuccinimidyl tartrate and (ethylene glycol)yl bis-(succinimidyl succinate). Cross-linking was analysed primarily by immunoblotting to detect products containing subunits of the iron-protein fraction from chaotropic resolution of the enzyme, namely those of 75, 49, 30 and 13 kDa. By using both the isolated iron-protein fraction and the intact dehydrogenase, cross-links were identified between these four subunits, from these subunits to the largest subunit of the flavoprotein fraction, which contains the active site for NADH, and from these subunits to polypeptides in the hydrophobic shell, which surrounds the hydrophilic iron-protein and flavoprotein fractions.     

A method for isolation of RNA from Pneumocystis carinii

Total RNA from Pneumocystis carinii obtained directly from the rat lung and from short term culture on A549 cells was evaluated for size and purity. An isolation procedure using guanidine isothiocyanate and lithium chloride was preferable to a hot phenol method. Host cells were eliminated by hypotonic lysis and a series of microfiltrations. Pneumocystis carinii were pretreated with Zymolyase for increased susceptibility to chaotropic agents. The major ribosomal species of P. carinii RNA migrated similarly to Saccharomyces cerevisiae rRNA. The 28s-like species migrated well ahead of rat and A549 cell rRNA and well behind the prokaryotic large rRNA species.     

A Method for Isolation of RNA from Pneumocystis carinii

Toial RNA from Pneumocystis carinii obtained directly from the rat lung and from short term culture on A549 cells was evaluated for size and purity. An isolation procedure using guanidine isothiocyanate and lithium chloride was preferable to a hot phenol method. Host cells were eliminated by hypotonic lysis and a series of microfiltrations. Pneumocystis carinii were pretreated with Zymolyase for increased susceptibility to chaotropic agents. The major ribosomal species of P. carinii RNA migrated similarly to Saccharomyces cerevisiae rRNA. The 28s-like species migrated well ahead of rat and A549 cell rRNA and weli behind the prokaryotic large rRNA species.     

-Haemolysin from E. coli purification and self-aggregation properties

An improved, straightforward purification procedure for E. coli -haemolysin has been developed. The protein exists in the form of large aggregates, held together mainly by hydrophobic forces. In the presence of urea or other chaotropic agents, the size of the aggregates decreases, while the specific activity is increased.     

Method for the isolation of high-quality RNA from grape berry tissues without contaminating tannins or carbohydrates

Grape berries contain compounds that aggregate with and precipitate RNA in the presence of chaotropic agents or phenol. The procedure described here extracts RNA from finely ground tissues using mild denaturants, and selectively precipitates the aggregate-forming material with 30% ethanol. The resulting RNA is suitable for northern blot analysis and translationin vitro.     

Protein disulfide isomerase appears necessary to maintain the catalytically active structure of the microsomal triglyceride transfer protein

Protein disulfide isomerase (PDI) is a component of the microsomal triglyceride transfer protein (MTP) complex. This study was initiated to help elucidate the role of PDI in MTP. The 88-kDa polypeptide of MTP (88K) was dissociated from PDI by using chaotropic agents (NaClO4 and KSCN), low concentrations of a denaturant (guanidine hydrochloride) or a nondenaturing detergent (octyl glucoside). As assessed by fluorescence and circular dichroism spectroscopy, these three different approaches appeared to dissociate the components of MTP under mild, nondenaturing conditions. The dissociating agents were diluted or removed by dialysis, and the free PDI and 88K were further characterized. In all cases, the dissociation coincided with the loss of triglyceride transfer activity. The free 88-kDa polypeptide readily aggregated, suggesting that it is a hydrophobic peptide. Even in the presence of chaotropic agents, when 88K was not aggregated, transfer activity was not expressed. These results suggest that the association of PDI with 88K is necessary to maintain the catalytically active form of the triglyceride transfer protein and prevent the aggregation of 88K.     

Transmembrane diffusion of hydrophobic antimicrobial agents and cell surface hydrophobicity in Bacteroides fragilis

The transmembrane diffusion of hydrophobic antimicrobial agents, e.g. lincomycin and clindamycin, was examined in Bacteroides fragilis which is sensitive to these agents. The results showed that these agents penetrate efficiently through the outer membrane. Cell surface hydrophobicity measured by the partition assay between water and p-xylene revealed that the cell surface of B. fragilis is more hydrophobic than that of Salmonella typhimurium or Pseudomonas aeruginosa. Furthermore, treatment with low concentrations of surfactant caused cell lysis. These results suggest that the cell surface hydrophobicity in B. fragilis plays an important role in the efficient transmembrane penetration of hydrophobic compounds. This efficiency explains the susceptibility of B. fragilis to hydrophobic antimicrobial agents.