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E London

Publications and source records attributed to E London.

At least 55 records · Page 3Linked to original sources
PubMed1995

Immunochemical analysis of the structure of diphtheria toxin shows all three domains undergo structural changes at low pH.

Diphtheria toxin is a bacterial protein that undergoes a physiologically critical conformational change at low pH. This change involves a partial unfolding event forming a molten globule-like structure, which exposes hydrophobic regions and which allows the toxin to insert into, and translocate across, membranes. In this report, antibody binding was used to examine the regions of the toxin that undergo structural changes at low pH. Monoclonal antibodies specific to the catalytic (C), transmembrane (T), and receptor-binding (R) domains of diphtheria toxin were prepared and isolated. In addition, the binding of anti-peptide antibodies raised against peptides in the C and T domains to toxin was examined. Anti-C monoclonals and antipeptide antibodies were found to bind preferentially to low pH-treated toxin relative to native toxin. Anti-T and anti-R monoclonal binding ranged between preference for native toxin and preference for low pH-treated toxin. These results suggest that the C domain becomes more exposed to solution at low pH, and that both the T and R domains of the B chain undergo major conformational changes at low pH. Based on these results, a model in which low pH induces several coordinated changes in intra- and inter-domain interactions is suggested. The participation of the R domain in these changes is of particular significance because it suggests that the R domain plays a more important role in low pH-induced changes than previously realized.

Animals↗
PubMed1995

Immunochemical analysis shows all three domains of diphtheria toxin penetrate across model membranes.

Diphtheria toxin undergoes membrane insertion and translocation across membranes when exposed to low pH. In this study, the translocation of the toxin has been investigated by the binding of antibodies to two preparations of model membrane-inserted toxin. In one preparation, toxin was added externally to model membrane vesicles and then inserted by exposure to low pH. In the other preparation, toxin was entrapped in the vesicles at neutral pH, and then inserted by decreasing pH. At neutral pH, externally added antibodies could not bind to entrapped toxin, although they could bind to externally added native toxin. However, after low pH exposure, antibodies against all three toxin domains (catalytic (C), transmembrane (T), and receptor-binding (R)) could bind to entrapped toxin, and also to externally added membrane-inserted toxin. The binding to the entrapped toxin shows that all three domains of the toxin translocate to the trans face of the membrane after exposure to low pH. The observation that antibodies bind to both external and entrapped preparations of toxin after low pH exposure shows that toxin inserts in a mixed orientation. A difference in antibody binding to low pH-treated toxin in which the C domain is folded (Lr' conformation) or unfolded (Lr" conformation) was also observed. An increase in antibody binding to C and T domains in the Lr" conformation relative to binding to the Lr' conformation was found for entrapped toxin, suggesting that more of the C and T domains translocate across the bilayer in the Lr" conformation. These results suggest all three toxin domains insert in the membrane bilayer and participate in translocation in vitro. The C and R domains lack classical transmembrane hydrophobic sequences. However, they possess sequences that have the potential to form membrane-inserting beta-sheets.

Animals↗
PubMed1995

Control of the depth of molecules within membranes by polar groups: determination of the location of anthracene-labeled probes in model membranes by parallax analysis of nitroxide-labeled phospholipid induced fluorescence quenching.

The location of anthracene-labeled molecules incorporated into model membranes was measured by fluorescence quenching. The depth of the anthracene group was calculated from the degree of quenching by lipids carrying a nitroxide at different depths, using the parallax analysis (Chattopadhyay & London (1987) Biochemistry 26, 39-45). A series of anthracene derivatives was examined in order to determine what polar functional groups would anchor at the membrane surface, and at what depth anchoring would occur. An anthracene with only a methyl group was not anchored at the membrane surface, but derivatives with polar or charged groups did anchor near the membrane surface as demonstrated by a shallower anthracene depth. Based on anthracene depths, protonated primary amine, secondary amine, and hydroxyl groups appear to be located 15-16 A from the center of the membrane. A quaternary amino locates more shallowly, at 18 A from the bilayer center. A protonated carboxyl group is slightly deeper, at 14 A from the center of the bilayer. Ester groups are found to be weakly anchoring, having a location dependent on the structure of the molecule to which they are attached. In methyl 9-anthracenepropionate, the ester group is located about 13 A from the bilayer center. Anthracene esters attached to cholesterol or cholesterol esters showed various depths. An anthracene ester attached to the tail of cholesterol was located 1-6 A from the center of the bilayer for a cholesterol derivative, but at 12 A from the bilayer center for a cholesterol oleate derivative.(ABSTRACT TRUNCATED AT 250 WORDS)

Anthracenes↗
PubMed1994

Interactions between saturated acyl chains confer detergent resistance on lipids and glycosylphosphatidylinositol (GPI)-anchored proteins: GPI-anchored proteins in liposomes and cells show similar behavior.

Proteins anchored by GPI are poorly solubilized from cell membranes by cold nonionic detergents because they associate with detergent-resistant membranes rich in cholesterol and sphingolipids. In this study, we demonstrated that cholesterol and sphingolipid-rich liposomes were incompletely solubilized by Triton X-100. GPI-anchored placental alkaline phosphatase incorporated in these liposomes was also not solubilized by cold Triton X-100. As sphingolipids have much higher melting temperatures (Tm) than cellular phospholipids, a property correlated with Tm might cause detergent inextractability. In support of this idea, we found that the low-Tm lipid dioleoyl phosphatidylcholine (DOPC) was efficiently extracted from detergent-resistant liposomes by Triton X-100, whereas the high-Tm lipid dipalmitoyl phosphatidylcholine (DPPC) was not. The fluorescence polarization of liposome-incorporated diphenylhexatriene was measured to determine the "fluidity" of the detergent-resistant liposomes. We found that these liposomes were about as fluid as DPPC/cholesterol liposomes, which were present in the liquid-ordered phase, and much less fluid than DOPC or DOPC/cholesterol liposomes. These findings may explain the behavior of GPI-anchored proteins, which often have saturated fatty acyl chains and should prefer a less-fluid membrane. Therefore, we propose that acyl chain interactions can influence the association of GPI-anchored proteins with detergent-resistant membrane lipids. The affinity of GPI-anchored proteins for a sphingolipid-rich membrane phase that is not in the liquid crystalline state may be important in determining their cellular localization.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1994

Method for efficient pelleting of small unilamellar model membrane vesicles.

Separation of model membrane vesicles from external solution is an important step in quantification of vesicle-associated materials. Ordinarily, such experiments cannot be done with small unilamellar vesicles because they are too small to be pelleted by centrifugation. A method is described that overcomes this difficulty by use of brominated phospholipids. It is shown that efficient pelleting of small unilamellar vesicles (greater than 98%) can be obtained by ultracentrifugation.

Bromine↗
PubMed1993

Extension of the parallax analysis of membrane penetration depth to the polar region of model membranes: use of fluorescence quenching by a spin-label attached to the phospholipid polar headgroup.

The parallax method is a method by which the depth of fluorescent molecules within a membrane is calculated from the ratio of quenching induced by two spin-labeled phospholipids at different depths. In this report, the method is extended to measurements of depth in the polar headgroup region of the membrane through use of a lipid with a spin-label attached to the polar choline moiety. Quenching data indicate that the choline-attached nitroxide is close to 19.5 A from the bilayer center, in good agreement with the choline location previously determined by diffraction measurements. By using quenching results obtained with this polar headgroup-labeled phospholipid, depths more accurate than those measured previously can be obtained for fluorophores in the polar region of the membrane. It appears that the most reliable results are obtained when depth is calculated from the quenching of the two spin-labels that quench a specific fluorophore most strongly. Applying this approach to a series of anthroyloxy-labeled fatty acids indicates that the depth of the anthroyloxy group is almost linearly related to the number of carbon atoms between it and the carboxyl group. The fatty acid carboxyl group itself is close to 18.6 A from the bilayer center in the ionized form and 16 A from bilayer center in the protonated form. This is close to the depth of the carboxyl groups on phospholipid fatty acyl chains. More accurate depths have also been obtained for 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD) labeled phospholipids using the quenching of the choline-attached spin-label.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Chloro-7-nitrobenzofurazan↗
PubMed1993

Simple centrifugation method for efficient pelleting of both small and large unilamellar vesicles that allows convenient measurement of protein binding.

Separation of unilamellar model membrane vesicles from external solution is often an important step in quantitation of vesicle bound or entrapped materials. An efficient method that allows pelleting of both small and large model membrane vesicles by centrifugation is described in this report. In this method streptavidin is added to vesicles containing a trace amount of biotinylated lipid. The resulting aggregation allows pelleting of the vesicles using an ordinary high-speed centrifuge. Control experiments show that the addition of streptavidin does not induce substantial vesicle fusion or leakage of substances trapped in the internal aqueous compartment of the vesicles. The method can accommodate different phospholipid compositions and lipid concentrations. Experiments with proteins that switch between hydrophilic and hydrophobic states show that the method can readily be used to monitor protein binding to vesicles.

Adenosine Triphosphatases↗
PubMed1992

Deep penetration of a portion of Escherichia coli SecA protein into model membranes is promoted by anionic phospholipids and by partial unfolding.

SecA protein, a principal component of the protein export machinery of Escherichia coli, is found both in the cytoplasm and inner membrane of cells. Previous in vitro and in vivo studies demonstrated that the interaction of SecA with the inner membrane requires the presence of physiological levels of anionic (acidic) phospholipids. In this report the degree of SecA insertion into model membranes and the conformational changes associated with this event have been examined. The extent of association of SecA with model membranes was determined by photolabeling with a hydrophobic reagent, and the depth of insertion of the protein into the phospholipid bilayer was determined by the amount of quenching of SecA fluorescence by both brominated and spin-labeled phospholipids. These methods demonstrated that SecA penetrates deep within the acyl chain region of the phospholipid bilayer. It was also found that SecA penetration into vesicles was associated with a major conformational change in the protein. This change can be induced by higher temperatures and involves a partial unfolding event as judged by differential scanning calorimetry, SecA fluorescence and increased sensitivity to proteolysis. These properties suggest the induction of a molten-globule-like conformation in a portion of the SecA polypeptide. This change was also induced at lower temperatures by the presence of membranes containing a physiological amount of the anionic phospholipid, phosphatidylglycerol. The partial unfolding and concomitant deep insertion of SecA into membranes may aid in the insertion of precursor proteins into the inner membrane and may influence possible interactions between SecA and the integral membrane export machinery components.

Adenosine Triphosphatases↗
PubMed1992

Calibration of the parallax fluorescence quenching method for determination of membrane penetration depth: refinement and comparison of quenching by spin-labeled and brominated lipids.

We previously introduced the "parallax" method, which uses fluorescence quenching by spin-labeled lipids in order to measure the depth of molecules within a membrane [Chattopadhyay, A., & London, E. (1987) Biochemistry 26, 39-45]. In this report the accuracy of this method is established by comparison of spin-label quenching to that obtained using brominated lipids. To accomplish this, the fluorescent molecules used were a fatty acid labeled with a carbazole buried deeply within the acyl chain region of the membrane, an acyl-Trp with the Trp residue residing near the polar membrane region, and cytochrome b5, which has Trp residues in its membrane-inserted region. The depths calculated from the amount of bromine quenching agreed with those determined using parallax analysis. This indicates that the depth reported by parallax analysis is accurate and that the spin labels residue very close to their predicted locations in the membrane. Furthermore, there was good agreement when parallax analysis was applied both to quenching by brominated and spin-labeled molecules, suggesting that the analysis is valid in both cases. The effect that different distributions and motions of fluorophores and quenchers would have on parallax analysis was also examined. For uniform distributions of quenchers or fluorophores over a range of depths, it was found that the analysis reports the average fluorophore depth. In addition, experimental data suggest that motional effects do not significantly alter the measured depths. This is consistent with the motions during the short excited state lifetime of the fluorophores being relatively small and/or relatively isotropic.

Bromine↗
PubMed1992

Determination of the location of fluorescent probes attached to fatty acids using parallax analysis of fluorescence quenching: effect of carboxyl ionization state and environment on depth.

In this report, parallax analysis of fluorescence quenching (see the preceding paper in this issue) was used to determine the location (depth) of anthroyloxy and carbazole probes attached to model membrane inserted fatty acids. A monotonic increase in depth was found as the number of carbon atoms between the attachment site of the probe and the fatty acyl carboxyl group is increased. It was also found that depth is sensitive to pH, with an increase in probe depth upon protonation of the fatty acid carboxyl group of around 0.5-2.5 A, depending on probe location and identity. This result shows that carboxyl protonation causes an increase in depth all along a fatty acid chain. In addition, it indicates that parallax analysis is very sensitive to small changes in depth. At a given pH, no significant change in probe depth was observed in vesicles containing anionic phospholipid or at various ionic strengths, suggesting these parameters do not strongly regulate fatty acyl chain location. It was also found that there is a decrease of the apparent depth of each of the fatty acyl attached probes both at longer excitation wavelengths and at longer emission wavelengths. This is consistent with there being a distribution of depth for each fluorophore, with shallower fluorophore dominating the fluorescence at red-shifted wavelengths. Solvent relaxation effects also appear to contribute to this wavelength dependence.

Electrochemistry↗
PubMed1992

Hsp85 conformational change within the heat shock temperature range.

One of the major mammalian heat shock proteins, hsp85, aggregates extensively when heated in the presence of non-ionic detergents (J Cell. Physiol. 140: 601-607, 1989). The present study used intrinsic fluorescence and susceptibility to tryptic proteolysis to probe hsp85 conformation within the physiological and heat shock temperature ranges. Fluorescence intensity decreased and the emission spectrum was red-shifted (2.5 nm) as hsp85 was heated from 15 degrees to 50 degrees C. Upon heating in the absence of detergent, the red shift, monitored by the ratio of fluorescence emission at 330 nm to that at 350 nm, began at 38 degrees-45 degrees C with a transition midpoint at 45 degrees-50 degrees C, depending on the rate of temperature increase. This transition was masked by 1% n-octyl-O-glucoside - a detergent previously shown to promote aggregation. The spectral changes were not reversible upon cooling to 15 degrees C. Susceptibility to proteolysis in the absence of detergent, measured by the degradation of characteristic large fragments, increased sharply between 40 degrees C and 45 degrees C. These findings suggest that hsp85 undergoes a major conformational change within the range of temperatures known to induce hsp synthesis. This change is consistent with partial unfolding which exposes additional sites to the aqueous environment and influences detergent binding.

Animals↗
PubMed1992

How bacterial protein toxins enter cells; the role of partial unfolding in membrane translocation.

Bacterial protein toxins translocate across membranes by processes that are still mysterious. Studies on diphtheria toxin have shown that partial unfolding processes play a major role in toxin membrane insertion and translocation. Similar unfolding behaviour is seen with other bacterial toxins. The lessons gained from this behaviour allow us to propose novel mechanisms for toxin translocation.

Bacterial Proteins↗
PubMed1991

Self-translocation of diphtheria toxin across model membranes.

To understand the mechanism of diphtheria toxin membrane translocation, the toxin was entrapped within lipid vesicles, and its low pH-induced translocation across the lipid bilayer was measured. Proteolysis and resistance to guanidinium chloride denaturation were used to demonstrate that the toxin molecules were entrapped. Low pH-induced movement of entrapped toxin to the outer (trans) face of the bilayer was assayed by the binding of external streptavidin to biotin-labeled entrapped toxin. Complete translocation was quantified by the amount of protein released into the external medium. Using whole toxin, it was found that the A fragment was efficiently translocated, but the B fragment was not. This was true both in the low temperature (A domain folded) and high temperature (A domain unfolded) toxin conformations previously identified [Jiang J. X., Abrams, F. S., and London, E. (1991) Biochemistry 30, 3857-3864]. Remarkably, even isolated fragment A appeared to self-translocate under some conditions. Toxin-induced translocation may partly result from formation of a nonspecific toxin-induced pore. This idea is supported by the toxin-induced release of fluorescent dextrans coentrapped within the vesicles. However, low pH-induced exposure of entrapped toxin on the outside of the membrane was conformation dependent. Exposure was greatest for the high temperature conformation. This suggests the existence of a more specific translocation process. The nature and relationship of these processes, and their relative roles in translocation in vivo are discussed.

Binding Sites↗
PubMed1991

Folding changes in membrane-inserted diphtheria toxin that may play important roles in its translocation.

Diphtheria toxin membrane penetration is triggered by the low pH within the endosome lumen. Subsequent exposure to the neutral pH of the cytoplasm is believed to aid in translocation of the catalytic A domain of the toxin into the cytoplasm. To understand the effects of low pH and subsequent exposure to neutral pH on translocation, we studied toxin conformation in solution and in toxin inserted in model membranes. Two conformations were found at low pH. One form, L', predominates below 25-30 degrees C, and the other, L", predominates above 25-30 degrees C and is formed from the L' state by an unfolding event. Both forms are hydrophobic and penetrate deeply into membranes. After pH neutralization, the L' and L'' conformations give rise to two new conformations, R' and R'', respectively. The R' and R" conformations differ from each other in that in the R' state the A domain remains folded, whereas in the R" state the A domain is unfolded. This is confirmed by the finding that only the R' state possesses the capacity to bind and hydrolyze NAD+. It is also supported by the finding that the R'' state can also be formed by thermal unfolding of the R' state. The R conformations differ from the low-pH L conformations in that although they remain largely membrane-inserted, it appears that a large portion of the toxin is no longer in contact with the hydrophobic core of the bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)

Diphtheria Toxin↗
PubMed1990

The effect of high pH upon diphtheria toxin conformation and model membrane association: role of partial unfolding.

Penetration of membranes by diphtheria toxin in vivo is at least partially triggered by a low pH-induced conformational change occurring within the lumen of an acidic organelle. In order to gain insight into the nature of this change the behavior of the toxin at high pH was characterized and compared to that previously determined at low pH. We find that near pH 10.5 a major conformational change occurs. This change is accompanied by a marked decrease in fluorescence intensity, a red shift in fluorescence emission maximum, and increased susceptibility of protein fluorescence to acrylamide quenching. Differential scanning calorimetry shows that the high pH conformational change involves a cooperative endothermic unfolding transition. These changes at high pH are very similar to those induced by low pH, supporting the conclusion that the changes at low pH also involve a denaturation-like process. In addition, at high pH the toxin gains the ability to bind to model membranes, again similar to its behavior at low pH. On the basis of these studies we conclude that exposure of hydrophobic sequences due to partial unfolding is one dominating component in inducing hydrophobic behavior at both high and low pH, but that at low pH Asp/Glu protonation also contributes to hydrophobicity.

Calorimetry, Differential Scanning↗