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E London

Publications and source records attributed to E London.

At least 73 records · Page 4Linked to original sources
PubMed1990

Involvement of denaturation-like changes in Pseudomonas exotoxin a hydrophobicity and membrane penetration determined by characterization of pH and thermal transitions. Roles of two distinct conformationally altered states.

Previous investigators have shown that exotoxin A undergoes a conformational switch to a hydrophobic state at low pH. This change appears to play a role in exotoxin A entry into cells by facilitating its penetration of the membranes of acidic organelles. We have examined the effects of pH, temperature, and denaturants in order to define the role of conformational changes in membrane penetration by the exotoxin. We find that two distinct low pH conformations exist. An intermediate low pH state (LI) dominates at pH 3.7-5.4 and is distinguished by blue-shifted fluorescence and weak or no hydrophobicity. The second low pH state (LII) is dominant below pH 3.7 and is characterized by red shifted fluorescence and strong hydrophobicity. LI is a folded state as judged by its spectroscopic properties and the observation that it undergoes distinct and cooperative thermal and denaturant induced unfolding transitions. LII appears to be more like a denatured state, as it shows no cooperative thermal or denaturant induced transitions and has spectroscopic properties very similar to exotoxin A that has been thermally denatured at pH 7. Exotoxin A in the LII state strongly binds detergent micelles and binds and inserts into model membranes. Therefore, denaturation-like conformational changes appear to play an important role in membrane insertion. The pH of the transition to a membrane-inserting state is influenced by the composition of the model membranes and is close to pH 5 in the presence of vesicles containing a phosphatidylglycerol/phosphatidylcholine mixture. These vesicles probably promote formation of the LII state via mass action effects. The implication of these results for membrane penetration and translocation of proteins without apparent hydrophobic regions, such as exotoxin A, is discussed.

ADP Ribose Transferases↗
PubMed1989

Domain-specific bias in arginine/lysine usage by protein toxins.

The content of lysine and arginine residues in a number of A-B type protein toxins has been examined. It is found that the A subunit, or its equivalent, often shows a strong bias in the type of basic amino acid residue used tending towards nearly exclusive use of either arginine or lysine rather than use of both, whereas the B subunit or its equivalent shows no such bias. Although arginine codons are GC-rich and lysine codons are AT-rich, the content of GC and AT in the genes coding for the toxins does not adequately explain this bias. Other explanations are discussed, including the possibility that the bias is linked to catalytic function or membrane interaction. Understanding this bias may yield valuable insights into toxin structure and function. Furthermore, identification of bias in sequences may be a useful tool for identifying new toxins and their domains.

ADP Ribose Transferases↗
PubMed1989

Involvement of nigrotecto-reticulospinal pathways in the iminodipropionitrile (IDPN) model of spasmodic dyskinesias: a 2-deoxy-D-[1-14C]glucose study in the rat.

Chronic administration of iminodipropionitrile (IDPN) to rats causes a persistent behavioral syndrome characterized by lateral and vertical head twitches, random circling, and increased tactile and acoustic startle responses. In order to identify brain areas which are affected in rats manifesting this syndrome, we used the autoradiographic 2-deoxy-D-[1-14C]glucose ([14C]DG) method to map cerebral glucose utilization in IDPN-treated rats. One day after the development of the dyskinetic syndrome, there were significant decreases in local glucose utilization in the substantia nigra pars reticulata (SNr) and compacta (SNc), the dorsal raphe, the superficial and deep layers of the superior colliculus, the inferior colliculi, the interpeduncular nucleus, the medial and dorsolateral geniculate nuclei, and the superior and lateral vestibular nuclei. There were also significant decreases in layer 2 of the cingulate cortex and in the temporal and occipital cortices. In contrast, there were no changes in the motor cortex, the caudate-putamen, the nucleus accumbens, or the median raphe. These findings suggest that deleterious effects of IDPN on the nigrotectal pathways which affect head and neck movements and circling behaviors via the brainstem reticulospinal tracts may play an important role in the IDPN-induced persistent spasmodic dyskinetic syndrome in rats.

Animals↗
PubMed1988

Conformation and model membrane interactions of diphtheria toxin fragment A.

Low pH is believed to play a critical role in the penetration of membranes by diphtheria toxin in vivo. In this report, the pH dependence of the conformation of fragment A of diphtheria toxin has been studied using fluorescence techniques. As pH is decreased, fragment A in solution undergoes a reversible conformational change beginning below pH 5. The conformational change occurs rapidly upon exposure to low pH. It involves both an increase in the exposure of tryptophanyl residues to solution and a switch from a hydrophilic state to a hydrophobic state as judged by fragment A binding to micelles of a mild detergent (Brij 96). At low pH fragment A also rapidly and tightly binds to and penetrates model membranes. Binding is reversed when pH is neutralized. The transition pH, the apparent midpoint of the change between the hydrophilic state and the membrane-penetrating hydrophobic state, occurs at about pH 3.5 in the presence of Brij 96 micelles, pH 4 in the presence of small unilamellar vesicles (SUV) composed of zwitterionic phosphatidylcholine, and pH 5 in the presence of SUV composed of 25 mol % anionic phosphatidylglycerol and 75% phosphatidylcholine. The effects of high temperature provide an important clue as to the nature of the changes at low pH. At neutral pH and high temperature, i.e. in the thermally denatured state, a conformational change similar to that observed at low pH occurs, although fragment A does not become hydrophobic. In addition, the effects of low pH and high temperature on the stability of the native state are cumulative. This indicates that the changes in fragment A both at high temperature and at low pH involve denaturation, although there appears to be only partial unfolding under these conditions. Based on the results of this study, the role of fragment A in diphtheria toxin membrane penetration and translocation is evaluated.

Chromatography, High Pressure Liquid↗
PubMed1988

Localization of the active site of diphtheria toxin.

Information about the location of the active site of diphtheria toxin was derived from proteolysis studies and an analysis of its sequence. It was found that a specific trypsin cleavage within whole diphtheria toxin occurs at Lys-39. Therefore, Lys-39 appears to be a surface residue. Furthermore, protection from proteolysis could be obtained upon binding of either the substrate beta-nicotinamide adenine dinucleotide (oxidized form) (NAD+) or a competing ligand, adenylyl(3'-5')uridine 3'-phosphate (ApUp). The protection by ApUp, which binds to the toxin very tightly, required only stoichiometric levels. The most likely explanation of these results is that both NAD+ binding and ApUp binding block trypsin either through a steric mechanism or through a local conformational change, suggesting Lys-39 may be near the active site. Further evidence supporting this conclusion comes from comparison of the previously determined sequences of diphtheria toxin and of Pseudomonas exotoxin A, a protein that catalyzes an identical reaction. We find a significant degree of homology between the N-terminal halves of the catalytic domains of these two proteins, which apparently represents active-site residues, and that Lys-39 is in the center of the homologous sequence. Furthermore, the location of the amino acid that is the homologue of Lys-39 within the crystal structure of Pseudomonas exotoxin A is also in agreement with a location in or near the active site. Other unusual features in the sequences of diphtheria toxin and Pseudomonas exotoxin A are also described, and on the basis of the experiments presented, a possible function for ApUp is considered.

ADP Ribose Transferases↗
PubMed1988

Interaction of diphtheria toxin with model membranes.

Low pH is believed to trigger membrane penetration by diphtheria toxin in vivo. The effect of pH upon the binding of the toxin to unilamellar model membrane vesicles was determined by using a fluorescence quenching assay. A series of studies were undertaken to determine the effect of lipid composition upon the binding of lipids to the toxin. The binding of toxin to various small unilamellar vesicles of zwitterionic or anionic lipids was similar in extent and was accompanied by deep penetration of the toxin into the fatty acyl chains, in agreement with previous studies. However, the transition pH, which is the pH at and below which toxin binding becomes significant, depended upon the fraction of anionic lipids, being highest with model membranes composed totally of anionic lipids (pH 5.8) and lowest with membranes composed of zwitterionic lipids (pH 5.2). Except for vesicle charge, the transition pH was independent of the nature of the lipid polar groups used. High ionic strength, which had no effect on the transition pH with zwitterionic vesicles, was found to shift the transition pH with totally anionic vesicles to pH 5.2. This suggests that both direct protein-lipid electrostatic interactions and the ionic double layer, which gives rise to a low local pH around anionic vesicles, contribute to the shift in the transition pH. The effect of lipid composition upon the kinetics and strength of binding was also examined. At low pH, binding was rapid and tight. Binding to vesicles containing 20 wt % anionic phosphatidylglycerol was faster and tighter than binding to vesicles of zwitterionic phosphatidylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)

Diphtheria Toxin↗
PubMed1988

Spectroscopic and ionization properties of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-labeled lipids in model membranes.

The spectroscopic and ionization properties of various lipids labeled with the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group have been studied in model membranes using fluorescence, absorbance and electrophoretic mobility measurements. Electrophoretic measurements show that the NBD group is uncharged at neutral pH. However, at high pH, hydroxyl addition or deprotonation occurs with a pKa, depending upon conditions, of 11.5-11.8 for the NBD group of headgroup-labeled phosphatidylethanolamine (NBD-PE) and 11.1-11.5 for NBD labels placed at the end of one fatty acyl chain of a phosphatidylcholine (6-NBD-PC and 12-NBD-PC). This type of behavior is not observed in the case of a methylated NBD label placed in the flexible 'tail' of cholesterol (NBD-cholesterol). The similarity in pKa for NBD-PE and NBD-PCs suggests that in these cases the NBD group is at a similar depth in the membrane. This was examined further by comparison of the fluorescence emission maximum of the NBD group in model membranes with that in solvents of varying polarity. The apparent polarity experienced by NBD groups in model membranes indicates that for NBD-PE and 12-NBD-PC they are located at the polar region whereas the NBD group of NBD-cholesterol is deeply buried in a nonpolar region of the membrane. This conclusion is supported further by fluorescence quenching experiments measuring NBD exposure to the aqueous quencher Co2+. The results of this study confirm the tentative conclusions of our previous fluorescence quenching studies on the location of NBD groups in model membranes.

4-Chloro-7-nitrobenzofurazan↗
PubMed1987

Parallax method for direct measurement of membrane penetration depth utilizing fluorescence quenching by spin-labeled phospholipids.

This report describes a method suitable for determining the depth of a wide variety of fluorescent molecules embedded in membranes. The method involves determination of the parallax in the apparent location of fluorophores detected when quenching by phospholipids spin-labeled at two different depths is compared. By use of straightforward algebraic expressions, the method allows calculation of depth in angstroms. Furthermore, the analysis can be extended to quenching by energy-transfer acceptors or brominated probes under appropriate conditions. Application of the method to quenching of 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD)-labeled lipids by spin-labeled lipids located at three different depths is demonstrated in model membranes. It is shown that the calculated depths of the NBD groups are self-consistent to the extent that they are the same no matter which two spin-labels have been used in a particular experiment. In addition, the calculated depth is independent of spin-label concentration in the membrane within +/- 1 A, ruling out major effects due to spin-label perturbation. The quenching experiments show that the location of the NBD group in head-group-labeled phosphatidylethanolamine is at the polar/hydrocarbon interface and that of an NBD label on the "tail" of cholesterol is deeply buried, as expected. Unexpectedly, NBD labels placed at the end of fatty acyl chains of phosphatidylcholines are also near the polar/hydrocarbon interface. Presumably, the polarity of the NBD group results in "looping" back to the surface of the NBD groups attached to flexible acyl chains.

4-Chloro-7-nitrobenzofurazan↗
PubMed1986

A fluorescence-based detergent binding assay for protein hydrophobicity.

Protein hydrophobicity is often detected by binding of protein to micelles of a mild detergent. A new fluorescence method for detection of this binding is presented. The method is based on a long-range quenching of tryptophan fluorescence by energy transfer to a pyrene-labeled phospholipid probe incorporated into micelles of Brij 96. The method is rapid, simple, and requires only a few micrograms of protein. Strongest quenching is obtained when both pyrene probe and brominated Brij 96, a short-range quencher, are combined. To define the best assay conditions the physical properties and quenching behavior of micelles with or without these probes have been compared. It is shown that both quenchers accurately measure binding of model compounds and protein toxins to micelles. Comparison of quenching by the different probes can be used to derive information on tryptophan location relative to the micelle core.

Bromine↗
PubMed1986

Similarity of the conformation of diphtheria toxin at high temperature to that in the membrane-penetrating low-pH state.

At high temperature, nicked free monomers of diphtheria toxin undergo a transition to a thermally denatured state, with a midpoint of 45-50 degrees C. In this report, the high-temperature (60 degrees C) conformation has been compared to the native (neutral pH) and low-pH (pH less than 5) conformations. The low-pH and high-temperature conformations are similar although not identical. As in the conformation at low pH, and unlike the toxin in its native conformation, the protein in its conformation at high temperature is hydrophobic, has low fluorescence intensity, and has increased exposure of tryptophan to aqueous solution. As at low pH, at high temperature the circular dichroism spectrum shows at most only partial unfolding of secondary structure. In contrast, the conformation of the toxin in guanidinium chloride is much closer to a random coil. The effects of high temperature and low pH interact in the sense that sensitivity of the native conformation to one is increased by the other. That is, the transition temperature between native and thermally denatured states is decreased as pH is decreased, and the transition pH between neutral-pH and low-pH states is increased as temperature is increased. This implies that there is some region on the protein where high temperature and low pH can disrupt conformation in a similar manner. Taken together, these results indicate that the low-pH and high-temperature conformations can both be defined as partially denatured states, even though unfolding may not be extensive at low pH. Similar behavior may occur in other proteins that undergo functionally important conformational disruption at low pH.

Chemical Phenomena↗
PubMed1986

Effects of opioid analgesics on local cerebral glucose utilization.

The autoradiographic 2-deoxy-D-[1-14C]glucose ([14-C]DG) method was used to map the effects of morphine, oxymorphone and nalbuphine on local cerebral glucose utilization (LCGU), an index of local brain function. The mu agonists (morphine, oxymorphone) decreased LCGU in thalamic nuclei, including some which have been implicated in somatosensory processing. Nalbuphine did not produce these effects, but stimulated LCGU in nuclei of the spinal trigeminal tract. The findings suggest that different supraspinal mechanisms mediate the actions of mu vs. kappa opioids, and that the [14-C]DG procedure is helpful in elucidating the anatomical areas involved.

Animals↗
PubMed1985

Effect of pH on the conformation of diphtheria toxin and its implications for membrane penetration.

The pH-triggered change in diphtheria toxin conformation and the physical properties of the toxin above and below the transition pH have been examined. Exposure to low pH (less than or equal to 5 at 23 degrees C, less than or equal to 5.3 at 37 degrees C) triggers a rapid (t1/2 less than 30 s) change in toxin conformation; the transition occurs over a narrow pH range (0.2 unit). Below the transition pH, buried tryptophans become exposed, and the toxin becomes hydrophobic, binding very tightly to detergent. Aggregation is observed at low pH, probably due to this extreme hydrophobicity. Circular dichroism and fluorescence properties show that the low-pH conformation is not extensively unfolded. Therefore, the toxin "opens" at low pH without becoming a random coil. The conformation change is partly irreversible, and the degree of irreversibility parallels the degree of aggregation. Reduction of the disulfide bonds does not increase hydrophobicity at neutral pH. Furthermore, none of the structural variants of toxin (monomer or dimer, bound to ApUp or free, and nicked between subunits or intact) are hydrophobic at neutral pH or differ in transition pH markedly. Therefore, these factors do not mimic the effect of low pH. These observations are consistent with a functional role for the pH-triggered changes during penetration of the membranes of acidic organelles. The toxin may have adapted a conformational change similar to partial denaturation for a critical role in function. The possible nature of the pH-sensitive interactions and the effects of aggregation are discussed briefly.

Cell Membrane↗
PubMed1984

Fluorescence characterization of the low pH-induced change in diphtheria toxin conformation: effect of salt.

We have examined the effect of pH on diphtheria toxin conformation using intrinsic protein fluorescence and a new fluorescence quenching method. In aqueous solutions, fluorescence indicates toxin conformation undergoes a drastic change at low pH. This conformational change is closely associated with a switch from a hydrophilic conformation to a hydrophobic one, as judged by quenching-detected detergent binding. In the absence of NaC1 these changes occur around pH 4-4.5. However, in 150 mM NaCl the conformational change occurs in the pH 5-5.5 range, close to the pH the toxin is expected to encounter in endosomes and lysosomes. Therefore, the conformational change observed at low pH is likely to be physiologically significant.

Diphtheria Toxin↗
PubMed1984

Fluorimetric determination of critical micelle concentration avoiding interference from detergent charge.

Upon mixing detergent solutions with the neutral fluorescent molecule 1,6-diphenyl-1,3,5-hexatriene a large increase in fluorescence is observed if detergent exceeds the critical micelle concentration. This property has been used to determine the critical micelle concentration of anionic, uncharged, zwitterionic, and cationic detergents. Regardless of detergent charge, the critical micelle concentrations obtained agree with the values obtained by other methods. This fluorescence assay is both sensitive and rapid, and should provide a simple and general method for determination of critical micelle concentration of any detergent.

Colloids↗
PubMed1983

Regeneration of the native bacteriorhodopsin structure from two chymotryptic fragments.

Chymotrypsin cleaves bacteriorhodopsin to two fragments: C-1, amino acids 72-248, and C-2, amino acids 1-71. Denaturation and renaturation of these fragments have been studied. Following denaturation in sodium dodecyl sulfate, both C-1 and C-2 regain secondary structure in phospholipid/cholate/sodium dodecyl sulfate mixed micelles. When combined, they form a complex with a secondary structure resembling that of bacterio-opsin. However, on further incubation in phospholipid/cholate/sodium dodecyl sulfate, separate fragments as well as the C-1 and C-2 complex denature again. Retinal binds tightly to the C-1 and C-2 complex (Kb greater than 10(7) M-1) and stabilizes the folded conformation. The formation of the complex of C-1, C-2, and retinal is maximal at pH 6.0. The ternary complex contains two species: one which absorbs similarly to the light-adapted purple membrane and a second with a lambda max between 450 and 500 nm. The formation of the latter species is favored at higher temperatures and is reversible. Vesicles formed from the ternary complex of C-1, C-2, and retinal translocate protons at a level close to that of intact bacteriorhodopsin.

Bacteriorhodopsins↗
PubMed1982

The site of attachment of retinal in bacteriorhodopsin. A resonance Raman study.

The retinal chromophore of bacteriorhodopsin is attached as a Schiff's base with the epsilon-amino group of a lysine residue. The site of attachment has now been investigated by the use of resonance Raman spectroscopy which has previously been shown to be sensitive to 15N isotope substitution at the Schiff's base. Bacteriorhodopsin samples obtained from bacteria grown in a medium containing either [epsilon-14N]- or [epsilon-15N]lysine were cleaved with chymotrypsin to give, in each case, the two fragments C-1 (amino acids 72-248) and C-2 (amino acids 1-71). The fragments were recombined in different combinations into lipid/detergent mixtures and retinal was added to regenerate the chromophore. Resonance Raman spectroscopy showed that, in both the light-adapted (BR 570) and the M 412 intermediate forms, the chromophore is attached to the large C-1 fragment. This result eliminates Lys-41 as the attachment site in these forms of bacteriorhodopsin. Together with the accompanying report, which demonstrates that the epsilon-amino group in Lys-41 is not required for regeneration of the native chromophore or for proton translocation, these results provide strong evidence that the chromophore remains attached as a Schiff's base to Lys-216 during the entire photocycle.

Bacteriorhodopsins↗