Detergent binding in trigonal crystals of OmpF porin from Escherichia coli

The structure of the detergent, ocytyl hydroxyethylsufoxide (C8(HE)SO), bound to the OmpF porin from E coli (in the trigonal crystal form) has been determined by neutron crystallography. Due to a dynamic exchange of detergent molecules with their environment they are not ordered on an atomic scale. The structure reported here is therefore at a resolution of approximately 16 A. The X-ray crystallographically determined structure of the protein provides a starting point for the neutron analysis in which the detergent is visualized primarily thanks to its high contrast against D2O. The structure shows the detergent to be located mainly in two areas. It forms toroidal annuli around each OmpF trimer, these annuli fusing to form a detergent belt surrounding a solvent filled column traversing the crystal. Those areas of the protein to which the detergent binds are formed almost exclusively of hydrophobic residues and form a band about 30 A high around the trimer. Its upper and lower bounds are defined by two bands of aromatic residues, tyrosines pointing away from the detergent belt and interacting with the polar headgroups while phenylalanines point inwards. This strongly suggests that the same areas define, in vivo, the location at which protein interacts with lipid. The hydrophobic moiety of detergent is also found mediating the hydrophobic protein-protein interactions at the interface between two trimers on the crystallographic two-fold axis.     

Computer simulations of the OmpF porin from the outer membrane of Escherichia coli

Molecular dynamics simulations were used to study the structure and dynamics of the Escherichia coli OmpF porin, which is composed of three identical 16-stranded beta-barrels. Simulations of the full trimer in the absence of water and the membrane led to significant contraction of the channel in the interior of each beta-barrel. With very weak harmonic constraints (0.005 kcal/mol A2/atom) applied to the main-chain C alpha atoms of the beta-barrel, the structure was stabilized without alteration of the average fluctuations. The resulting distribution of the fluctuations (small for beta-strands, large for loops and turns) is in good agreement with the x-ray B factors. Dynamic cross-correlation functions showed the importance of coupling between the loop motions and barrel flexibility. This was confirmed by the application of constraints corresponding to the observed temperature factors to the barrel C alpha atoms. With these constraints, the beta-barrel fluctuations were much smaller than the experimental values because of the intrinsic restrictions on the atomic motions, and the loop motions were reduced significantly. This result indicates that considerable care is required in introducing constraints to keep proteins close to the experimental structure during simulations, as has been done in several recent studies. Loop 3, which is thought to be important in gating the pore, undergoes a displacement that shifts it away from the x-ray structure. Analysis shows that this arises from the breakdown of a hydrogen bond network, which appears to result more from the absence of solvent that from the use of standard ionization states for the side chains of certain beta-barrel residues.     

Structural relationships in the OmpR family of winged-helix transcription factors

A survey of 108 individuals from a coastal Aboriginal community in north Western Australia revealed that two species of gastrointestinal protozoan parasites (Giardia duodenalis--39.8%, Entamoeba coli--40.7%) and five gastrointestinal helminths (Hymenolepis nana--54.6%, Hookworm [Ancylostoma duodenale]--30.6%, Enterobius vermicularis--6.5%, Trichuris trichiura--2.8%, Strongyloides stercoralis 1.9%) were present. A total of 29 individuals infected with hookworm were offered treatment with either pyrantel pamoate at a single dose rate of 10 mg/kg body weight or albendazole (single 400 mg dose). Seven days after treatment stool samples were examined. Pyrantel had no significant effect against hookworm. In contrast, albendazole cleared hookworm infections completely and reduced the prevalence of Giardia. The former result suggests that locally A. duodenale is resistant to pyrantel and despite its relatively low cost and wide availability, should not be considered a drug of choice at this dose rate in the treatment of hookworm infections (A. duodenale) in endemic regions.     

Mutations in the Res subunit of the EcoPI restriction enzyme that affect ATP-dependent reactions

The Res subunits of the type III restriction-modification enzymes share a statistically significant amino acid sequence similarity with several RNA and DNA helicases of the so-called DEAD family. It was postulated that in type III restriction enzymes a DNA helicase activity may be required for local unwinding at the cleavage site. The members of this family share seven conserved motifs, all of which are found in the Res subunit of the type III restriction enzymes. To determine the contribution, if any, of these motifs in DNA cleavage by EcoPI, a type III restriction enzyme, we have made changes in motifs I and II. While mutations in motif I (GTGKT) clearly affected ATP hydrolysis and resulted in loss of DNA cleavage activity, mutation in motif II (DEPH) significantly decreased ATP hydrolysis but had no effect on DNA cleavage. The double mutant R.EcoPIK90R-H229K showed no significant ATPase or DNA restriction activity though ATP binding was not affected. These results imply that there are at least two ATPase reaction centres in EcoPI restriction enzyme. Motif I appears to be involved in coupling DNA restriction to ATP hydrolysis. Our results indicate that EcoPI restriction enzyme does not have a strand separation activity. We suggest that these motifs play a role in the ATP-dependent translocation that has been proposed to occur in the type III restriction enzymes.     

Anaerobic regulation of the Escherichia coli dmsABC operon requires the molybdate-responsive regulator ModE

The 9804 gene, which encodes a human Ly-6 protein most similar to mouse differentiation Ag TSA-1/Sca-2, has also been called RIG-E. Like mouse TSA-1, it has a broad tissue distribution with varied expression levels in normal human tissues and tumor cell lines. Like some members of the murine Ly-6 family, the 9804 gene is responsive to IFNs, particularly IFN-alpha. Overlapping genomic fragments spanning the 9804 gene (5543 bp) have been isolated and characterized. The gene organization is analogous to that of known mouse Ly-6 genes. The first exon, 2296 bp upstream from exon II, is entirely untranslated. The three coding exons (II, III, and IV) are separated by short introns of 321 and 131 bp, respectively. Primers were developed for specific amplification of 9804 gene fragments. Screening of human-hamster somatic cell hybrids and yeast artificial chromosomes (YACs) indicated that the gene is distal to c-Myc, located in the q arm of human chromosome 8. No positives were detected from the Centre d'Etude du Polymorphisme Humain mega-YAC A or B panels, nor from bacterial artificial chromosome libraries; two positive cosmids (c101F1 and c157F6) were isolated from a human chromosome 8 cosmid library (LA08NC01). Fluorescence in situ hybridization of metaphase spreads of chromosome 8, containing hybrid cell line 706-B6 clone 17 (CL-17) with cosmid c101F1, placed the 9804 gene close to the telomere at 8q24.3. This mapping is significant, since the region shares a homology with a portion of mouse chromosome 15, which extends into band E where Ly-6 genes reside. Moreover, the gene encoding E48, the homologue of mouse Ly-6 molecule ThB, has also been mapped to 8q24.     

Mutations in the nucleotide-binding sites of P-glycoprotein that affect substrate specificity modulate substrate-induced adenosine triphosphatase activity

The amino- and carboxy-terminal nucleotide-binding domains (NBD1 and NBD2) of P-glycoprotein (P-gp) share over 80% sequence identity. Almost all of NBD1 can be exchanged by corresponding NBD2 segments with no significant loss of function, except for a small segment around the Walker B motif. Within this segment, we identified two sets of residues [ERGA --> DKGT (522-525) and T578C] that, when replaced by their NBD2 counterparts, cause dramatic alterations of the substrate specificity of the protein [Beaudet, L., and Gros, P. (1995) J. Biol. Chem. 270, 17159-17170]. We wished to gain insight into the molecular basis of this defect. For this, we overexpressed the wild-type mouse Mdr3 and variants bearing single or double mutations at these positions in the yeast Pichia pastoris. P-gp-specific ATPase activity was measured in yeast plasma membrane preparations after detergent solubilization and reconstitution in Escherichia coli proteoliposomes. P-gp proteoliposomes from P. pastoris showed a strong verapamil- and valinomycin-stimulated ATPase activity, with characteristics (KM, Vmax) similar to those measured in mammalian cells. Mutations did not appear to affect the KM for Mg2+ATP ( approximately 0.4 mM), but maximum velocity (Vmax) of the drug-stimulated ATPase activity was severely affected in a substrate/modulator-specific fashion. Indeed, all mutants showed complete loss of verapamil-induced ATPase, while all retained at least some degree of valinomycin-induced ATPase activity. Photolabeling studies with [125I]iodoarylazidoprazosin, including competition with MDR drugs and modulators, suggested that drug binding was not affected in the mutants. The altered drug resistance profiles of the ERGA --> DKGT(522-525) and T578C mutants in vivo, together with the observed alterations in substrate-induced ATPase activity of these proteins, suggest that the residues involved may form part of a signal pathway between the membrane regions (substrate binding) and the ATP binding sites.     

A molecular dynamics study of the pores formed by Escherichia coli OmpF porin in a fully hydrated palmitoyloleoylphosphatidylcholine bilayer

In this paper we study the properties of pores formed by OmpF porin from Escherichia coli, based on a molecular dynamics simulation of the OmpF trimer, 318 palmitoyl-oleoyl-phosphatidylethanolamine lipids, 27 Na+ ions, and 12,992 water molecules. After equilibration and a nanosecond production run, the OmpF trimer exhibits a C-alpha root mean square deviation from the crystal structure of 0.23 nm and a stable secondary structure. No evidence is found for large-scale motions of the L3 loop. We investigate the pore dimensions, conductance, and the properties of water inside the pore. This water forms a complicated pattern, even when averaged over 1 ns of simulation time. Around the pore constriction zone the water dipoles are highly structured in the plane of the membrane, oriented by the strong transversal electric field. In addition, there is a net orientation along the pore axis pointing from the extracellular to the intracellular side of the bilayer. The diffusion coefficients of water inside the pore are greatly reduced compared to bulk. We compare our results to results from model pores (Breed et al., 1996. Biophys. J. 70:1 643-1 661; Sansom et al. 1997. Biophys. J. 73:2404-241 5) and discuss implications for further theoretical work.     

Cloning and expression of the major porin protein gene opcP of Burkholderia (formerly Pseudomonas) cepacia in Escherichia coli

OBJECTIVES: We sought to assess the effects of aging on the endothelial physiology of a group of Chinese adults. BACKGROUND: Several studies have documented an association between aging and progressive arterial endothelial dysfunction in white subjects. We hypothesized that age-related endothelial dysfunction, an important event in atherosclerosis, might be less marked in southern Chinese subjects, in whom the prevalence of coronary heart disease is only approximately 20% of that in industrialized countries. METHODS: We studied endothelial function in 76 healthy adults aged 16 to 70 years: 38 Chinese from a village of 3,000 people in southern China and 38 white subjects from Sydney, Australia. In each ethnic group, there were 19 younger persons (16 to 40 years) and 19 older adults (55 to 70 years). None had evidence of diabetes, hypertension or clinical vascular disease or had ever been regular cigarette smokers. With the use of high resolution external vascular ultrasound, brachial artery diameter was measured at rest, after flow increase (causing endothelium-dependent dilation) and after sublingual nitroglycerin (an endothelium-independent dilator). RESULTS: Endothelium-dependent dilation was similar in young Chinese (mean +/- SD 8.3 +/- 2.5%), young whites (7.9 +/- 2.0%) and older Chinese (6.8 +/- 2.9%), but it was significantly impaired in older whites (1.8 +/- 2.5%, p < 0.001 by analysis of variance). On multivariate analysis, older age was associated with impaired endothelium-dependent dilation (p < 0.001) (independent of the effects of serum cholesterol, gender and vessel size) in the white but not in the Chinese subjects (p = 0.83). Nitroglycerin-induced dilation was not significantly different with aging in either ethnic group. CONCLUSIONS: Endothelium-dependent dilation is similar in the arteries of healthy young Chinese and white adults. With older age, however, Chinese subjects are less susceptible to impaired endothelial function.     

Interdomain interactions between the hydrophilic domains of the mannitol transporter of Escherichia coli in the unphosphorylated and phosphorylated states

Interdomain interactions in the mannitol-specific enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli play a key role in the mechanism of mannitol transport across the membrane [Boer et al. (1995) Biochemistry 34, 3239-3247; Loikema et al. (1991) Biochemistry 30, 6716-6721]. In this study, we focus on the interaction between the hydrophilic A and B domains and try to determine those as a function of the phosphorylation state of the enzyme. To this end, unfolding studies on the subcloned domains IIAmtl and IIBmtl, as well as on the binary combination IIBAmtl, were performed, both in the unphosphorylated and in the phosphorylated states, using GuHCl and heat as the denaturant. It is shown that IIAmtl and IIBmtl, as well as P-IIAmtl and P-IIBmtl, unfold according to a two-state mechanism but that IIBAmtl and P2-IIBAmtl do not exhibit such behavior. Two transitions are observed instead, indicating a lack of strong positive cooperative interactions. DSC studies of the unphosphorylated proteins showed a destabilization of the B domain in IIBAmtl with respect to the free IIBmtl as indicated by a lowereing of the melting temperature and a lower enthalpy of unfolding. Furthermore, it is shown that phosphorylation has a destablilizing effect on both IIAmtl and IIBAmtl but not on IIBmtl. Possible explanations for this behavior and the biological relevance of the destabilizing forces in IIBAmtl are discussed.     

Iron regulates transcription of the Escherichia coli ferric citrate transport genes directly and through the transcription initiation proteins

Water compartments were studied in 72 black and 128 white women, aged 20 to 70 years. Total body water (TBW) was measured by tritiated water dilution, and extracellular water (ECW) was measured by using delayed gamma neutron activation for the determination of total body chloride. Intracellular water (ICW) was assessed as the difference between TBW and ECW. Fat-free mass (FFM) was estimated by the measurement of total body potassium (TBK) and total body fat (TBF) by measurement of total body carbon (TBC). Total body protein was calculated from total body nitrogen (TBN). TBW was found to decline with age and to also be significantly influenced by body size. The extracellular water space was 41.5% of TBW. The decline of TBW with age is due primarily to a decline in ICW. The hydration of the FFM increased with age for the overall population because of a decline in TBK and TBN and an increase in ECW. Body composition models that have assumed constancy of hydration should be adjusted not only for gender but for age and body size.