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ABSTRACT: eEF2 (eukaryotic elongation factor 2) occupies an essential role in protein synthesis where it catalyses the translocation of the two tRNAs and the mRNA after peptidyl transfer on the 80 S ribosome. Recent crystal structures of eEF2 and the cryo-electron microscopy reconstruction of its 80 S complex now provide a substantial structural framework for dissecting the functional properties of this factor. The factor can be modified by either phosphorylation or ADP-ribosylation, which results in cessation of translation. We review the structural and functional properties of eEF2 with particular emphasis on the unique diphthamide residue, which is ADP-ribosylated by diphtheria toxin from Corynebacterium diphtheriae and exotoxin A from Pseudomonas aeruginosa.
Biochemical Society Transactions 03/2006; 34(Pt 1):1-6. · 3.71 Impact Factor
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ABSTRACT: Mutagenesis techniques were used to replace two loop regions within the catalytic domain of Pseudomonas aeruginosa exotoxin A (ETA) with functionally silent polyglycine loops. The loop mutant proteins, designated polyglycine Loops N and C, were both less active than the wild-type enzyme. However, the polyglycine Loop C mutant protein, replaced with the Gly(483)-Gly(490) loop, showed a much greater loss of enzymatic activity than the polyglycine Loop N protein. The former mutant enzyme exhibited an 18,000-fold decrease in catalytic turnover number (k(cat)), with only a marginal effect on the K(m) value for NAD(+) and the eukaryotic elongation factor-2 binding constant. Furthermore, alanine-scanning mutagenesis of this active-site loop region revealed the specific pattern of a critical region for enzymatic activity. Binding and kinetic data suggest that this loop modulates the transferase activity between ETA and eukaryotic elongation factor-2 and may be responsible for stabilization of the transition state for the reaction. Sequence alignment and molecular modeling also identified a similar loop within diphtheria toxin, a functionally and structurally related class A-B toxin. Based on these results and the similarities between ETA and diphtheria toxin, we propose that this catalytic subregion represents the first report of a diphthamide-specific ribosyltransferase structural motif. We expect these findings to further the development of pharmaceuticals designed to prevent ETA toxicity by disrupting the stabilization of the transition state during the ADP-ribose transfer event.
Journal of Biological Chemistry 10/2001; 276(37):35029-36. · 4.77 Impact Factor
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ABSTRACT: Previously, we characterized the role of the three naturally occurring Trp residues (W-417, -466, and -558) in the catalytic mechanism of the toxin-enzyme produced by Pseudomonas aeruginosa [Beattie and Merrill (1999) J. Biol. Chem. 274, 15646-15654]. However, the use of intrinsic Trp fluorescence to study toxin-eEF-2 interaction is inherently limited since the spectral properties of the various Trp residues in both proteins cannot easily be distinguished. To facilitate the study of the protein-protein interaction by Trp fluorescence spectroscopy, the Trp residues in the catalytic domain of exotoxin A were replaced with the amino acid analogues 4-fluorotryptophan, 5-fluorotryptophan, 5-hydroxytryptophan, and 7-azatryptophan. The incorporation of analogues was achieved by using a tightly regulated promoter, pBAD, and expressing the protein in a Trp auxotrophic strain of Escherichia coli, BL21, in a minimal medium containing the appropriate tryptophan analogue. Quantitative spectral analysis of the analogue-containing proteins using the Decompose program indicated that we had achieved 87-100% incorporation efficiency depending on the Trp analogue being used. Electrospray mass spectrometry analysis verified that we had achieved nearly total replacement of the L-tryptophan residues within the catalytic domain of exotoxin A with the tryptophan analogues 5-fluorotryptophan and 4-fluorotryptophan. The analogue-substituted proteins showed a variation in their catalytic activities with k(cat) values ranging from 6-fold (4-fluorotryptophan) to 260-fold (5-hydroxytryptophan) lower than the natural enzyme, which was in agreement with previous data using site-directed mutagenesis [Beattie et al. (1996) Biochemistry 35, 15134-15142]. However, the analogue-incorporated enzymes did not show any significant change in their ability to bind NAD(+) as substrate, as determined from a fluorescence-binding assay. The spectral properties of the various analogue-incorporated proteins were evaluated and compared with those of the native protein. Furthermore, selective excitation of the 5-hydroxytryptophan-incorporated toxin was exploited to study its interaction with the elongation factor-2 substrate by fluorescence resonance energy transfer to an acceptor chromophore located on the elongation factor-2 protein. The binding between the toxin-enzyme and elongation factor-2 was shown to be independent of the NAD(+) substrate (983 +/- 63 nM) and showed a small dependence upon the ionic strength of the solution.
Biochemistry 09/2001; 40(34):10273-83. · 3.42 Impact Factor
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ABSTRACT: Pseudomonas aeruginosa exotoxin A (ETA) is a member of the family of bacterial ADP-ribosylating toxins that use NAD(+) as the ADP-ribose donor. The reaction catalyzed by ETA involves the nucleophilic attack of the diphthamide residue on the anomeric carbon of the nicotinamide ribose forming a new glycosidic bond. A fluorometric assay involving the use of etheno-beta-nicotinamide adenine dinucleotide (epsilon-NAD(+)), an analog of NAD(+), has been found to provide a rapid, reliable, and sensitive procedure for assessing the kinetic parameters of this class of enzymes including ETA and its C-terminal fragment, PE24. Furthermore, application of this new assay facilitated the determination of the kinetic parameters for the protein substrate of ETA, elongation factor, which has previously been difficult to characterize. These findings provide new insights into catalytic mechanism of dipthamide-specific ribosyltransferases. In addition, this assay should also prove valuable for the study of NADases or NAD(+)-glycohydrolase enzymes (B. Weng, W. C. Thompson, H. J. Kim, R. L. Levine, and J. Moss, 1999, J. Biol. Chem. 274, 31797-31803; Y. S. Cho, M. K. Han, O. S. Kwark, M. S. Phoe, Y. S. Cha, N. H. An, and U. H. Kim, 1998, Comp. Physiol. B: Biochem. Mol. Biol. 120, 175-181) and the poly-ADP-ribosyltransferases (A. A. Pieper, A. Verma, J. Zhang, S. H. Snyder, 1999, Trends Pharmacol. Sci. 20, 171-181; M. K. Jacobson and E. L. Jacobson, 1999, Trends Biochem. Sci. 24, 415-417).
Analytical Biochemistry 06/2001; 292(1):26-33. · 3.00 Impact Factor
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ABSTRACT: The molecular aggregate size of the closed state of the colicin E1 channel was determined by fluorescence resonance energy transfer experiments involving a fluorescence donor (three tryptophans, wild-type protein) and a fluorescence acceptor (5-(((acetyl)amino)ethyl)aminonaphthalene-1-sulfonic acid (AEDANS), Trp-deficient protein). There was no evidence of energy transfer between the donor and acceptor species when bound to membrane large unilamellar vesicles. These experiments led to the conclusion that the colicin E1 channel is monomeric in the membrane-bound closed channel state. Experiments were also conducted to study the membrane topology of the closed colicin channel in membrane large unilamellar vesicles using acrylamide as the membrane-impermeant, nonionic quencher of tryptophan fluorescence in a battery of single tryptophan mutant proteins. Furthermore, additional fluorescence parameters, including fluorescence emission maximum, fluorescence quantum yield, and fluorescence decay times, were used to assist in mapping the topology of the closed channel. Results suggest that the closed channel comprises most of the polypeptide of the channel domain and that the hydrophobic anchor domain does not transverse the membrane bilayer but nonetheless is deeply embedded within the hydrocarbon core of the membrane. Finally, a model is proposed which features at least two states that are in rapid equilibrium with each other and in which one state is more heavily populated than the other.
Journal of Biological Chemistry 09/1999; 274(35):24539-49. · 4.77 Impact Factor
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ABSTRACT: The X-ray structure of the catalytic domain of Pseudomonas aeruginosa exotoxin A (PE24) has recently been solved to high resolution, facilitating studies on the interaction of PE24 with its target substrate, eukaryotic elongation factor-2 (eEF-2). PE24 exhibits mono-ADP-ribosyltransferase (ADPRT) activity in a mechanism that has been proposed to feature a nucleophilic attack by the diphthamide residue (nucleophile) of eEF-2 on the C-1 of the nicotinamide ribose of NAD(+). The interaction of wheat germ eEF-2 with PE24 was studied by employing an enzyme-linked immunosorbent assay (ELISA), devised to assess protein-protein interactions. It was shown that the proteins associate with each other only in the presence of the enzyme's nucleotide substrate, NAD(+), and exhibit a dose-dependent association that is saturable. The apparent dissociation constant (K(d)) for this protein-protein interaction is 50 nM and is salt-dependent. The association is maximal at low ionic strength and is progressively weaker at higher salt concentrations, which corroborates previous findings on the salt dependence of ADPRT activity for this toxin. This finding suggests that the sensitivity of ADPRT activity toward high salt resides in the interaction between the catalytic domain of the toxin and eEF-2. A major product of the glycohydrolase activity of PE24, nicotinamide, inhibits the binding between PE24 and eEF-2 with an ID(50) of 20 microM. The naturally occurring, noncatalytic mutant of PE24, H426Y, did not bind eEF-2 in the ELISA, verifying that His 426 is located at the center of the eEF-2 binding site within ETA.
Analytical Biochemistry 09/1999; 272(2):216-23. · 3.00 Impact Factor
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ABSTRACT: Unfolding of the soluble colicin E1 channel peptide was examined with the use of urea as a denaturant; it was shown that it unfolds to an intermediate state in 8.5 M urea, equivalent to a dimeric species previously observed in 4 M guanidinium chloride. Single tryptophan residues, substituted into the peptide at various positions by site-directed mutagenesis, were employed as fluorescent probes of local unfolding. Unfolding profiles for specific sites within the peptide were obtained by quantifying the shifts in the fluorescence emission maxima of single tryptophan residues on unfolding and plotting them against urea concentration. Unfolding reported by tryptophan residues in the C-terminal region was not characteristic of complete peptide denaturation, as evidenced by the relatively blue-shifted values of the fluorescence emission maxima. Unfolding was also monitored by using CD spectroscopy and the fluorescent probe 2-(p-toluidinyl)-naphthalene 6-sulphonic acid; the results indicated that unfolding of helices is concomitant with the exposure of protein non-polar surface. Unfolding profiles were evaluated by non-linear least-squares curve fitting and calculation of the unfolding transition midpoint. The unfolding profiles of residues located in the N-terminal region of the peptide had lower transition midpoints than residues in the C-terminal portion. The results of unfolding analysis demonstrated that urea unfolds the peptide only partly to an intermediate state, because the C-terminal portion of the channel peptide retained significant structure in 8.5 M urea. Characterization of the peptide's global unfolding by size-exclusion HPLC revealed that the partly denatured structure that persists in 8.5 M urea is a dimer of two channel peptides, tightly associated by hydrophobic interactions. The presence of the dimerized species was confirmed by SDS/PAGE and intermolecular fluorescence resonance energy transfer.
Biochemical Journal 07/1999; 340 ( Pt 3):631-8. · 4.90 Impact Factor
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ABSTRACT: Single tryptophan mutant proteins of a catalytically active domain III recombinant protein (PE24) from Pseudomonas aeruginosa exotoxin A were prepared by site-directed mutagenesis. The binding of the dinucleotide substrate, NAD+, to the PE24 active site was studied by exploiting intrinsic tryptophan fluorescence for the wild-type, single Trp, and tryptophan-deficient mutant proteins. Various approaches were used to study the substrate binding process, including dynamic quenching, CD spectroscopy, steady-state fluorescence emission analysis, NAD+-glycohydrolase activity, NAD+ binding analysis, protein denaturation experiments, fluorescence lifetime analysis, steady-state anisotropy measurement, stopped flow fluorescence spectroscopy, and quantum yield determination. It was found that the conservative replacement of tryptophan residues with phenylalanine had little or no effect on the folded stability and enzyme activity of the PE24 protein. Dynamic quenching experiments indicated that when bound to the active site of the enzyme, the NAD+ substrate protected Trp-558 from solvent to a large extent but had no effect on the degree of solvent exposure for tryptophans 417 and 466. Also, upon substrate binding, the anisotropy of the Trp-417(W466F/W558F) protein showed the largest increase, followed by Trp-466(W417F/W558F), and there was no effect on Trp-558(W417F/W466F). Furthermore, the intrinsic tryptophan fluorescence exhibited the highest degree of substrate-induced quenching for the wild-type protein, followed in decreasing order by Trp-417(W466F/W558F), Trp-558(W417F/W466F), and Trp-466(W417F/W558F). These data provide evidence for a structural rearrangement in the enzyme domain near Trp-417 invoked by the binding of the NAD+ substrate.
Journal of Biological Chemistry 06/1999; 274(22):15646-54. · 4.77 Impact Factor
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ABSTRACT: In vitro, the channel-forming domain of colicin E1 requires activation by acidic pH (<4.5) or detergents. The activation of this domain to its insertion-competent state results in an increased ability of the protein to dock onto and to form channels in artificial membranes. Fluorescence methods were used to characterize the conformational changes occurring in a channel-forming peptide of colicin E1 in solution with pH. The 178-residue thermolytic fragment of colicin E1 contains three Trp residues, W-424, W-460, and W-495. In order to study the structural and dynamic requirements for activation of the C-terminal domain of colicin E1, single-Trp-containing peptides were prepared by site-directed mutagenesis. All of the mutant peptides displayed in vitro channel activity and cellular cytotoxicity similar to the those of wild-type peptide. Two Trp residues, W-413 and W-424, exhibited pH-sensitive fluorescence parameters. Upon acidification (pH 6.0 --> 3.5), the fluorescence quantum yield of W-413 and W-424 increased 50% and 80%, respectively, indicating a significant change in the local environment of the peptide segment containing these two Trp residues. The fluorescence decay of W-413 and W-424 was best fit by three fluorescence decay components, two of which were sensitive to pH. However, only small changes in spectral shape and position were observed for W-424 fluorescence, whereas there were larger changes in these fluorescence parameters for W-413. The quantum yields for the Trp residues in the seven other single-Trp mutant peptides and the wild-type peptide were distinct but only slightly affected by changes in pH. Time-resolved fluorescence measurements showed that W-460, -484, and -495 each had two fluorescence decay components with similar decay times, with one component dominating the fluorescence decay behavior. Furthermore, the individual fluorescence decay times for all the single-Trp peptides, except for W-413 and W-424, were insensitive to pH changes. At pH 3.5, the fluorescence of the wild-type peptide was fit by three decay time components, with the two longer decay times being quite different from the fluorescence decay times of the single-Trp mutant proteins (W-424, -460, and -495, the naturally occurring Trp residues). In contrast, at pH 6.0, the wild-type peptide showed double-exponential decay kinetics. Time-resolved fluorescence anisotropy decay measurements of the three single-Trp mutant proteins, containing a naturally occurring Trp residue, suggest that local segmental motion of the peptide as reported by each of the three tryptophans is highly restricted and largely insensitive to changes in pH. On the other hand, the anisotropy decay profiles of the wild-type protein were consistent with energy transfer occurring between Trp residues, likely between W-460 and W-495. These steady-state and time-resolved fluorescence results show that W-413 and W-424 report conformational changes which may be associated with the insertion-competent state and reside on the protein segment(s) which form the pH-activated trigger of the channel peptide.
Biochemistry 06/1997; 36(23):6874-84. · 3.42 Impact Factor
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ABSTRACT: The equilibrium unfolding pathway of the colicin E1 channel peptide was shown in a previous study to involve an unfolding intermediate, stable in approximately 4 M guanidine hydrochloride, which comprised primarily the C-terminal hydrophobic alpha-helical hairpin segment of the peptide [Steer, B. A., & Merrill, A. R. (1995) Biochemistry 34, 7225-7233]. In this study, the structural nature of this unfolding intermediate was investigated further, and it was found that the intermediate primarily consists of a dimer species and is comprised of two partially denatured monomeric peptides, which appear to be associated by hydrophobic interactions. The dimerized structure was detected by size-exclusion high-performance liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, chemical cross-linking, and intermolecular fluorescence energy transfer. Using stopped-flow fluorescence spectroscopy, the kinetics of the denaturation and dimerization of the colicin E1 channel peptide in 4 M guanidine hydrochloride were examined. Denaturation kinetics were also investigated by wild-type peptide Trp fluorescence and 1-anilinonaphthalene-8-sulfonic acid binding. The kinetics of dimer formation were examined by monitoring the time dependence of intermolecular Trp to 5-[[2-[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid fluorescence resonance energy transfer upon denaturation in 4 M guanidine hydrochloride. In addition, single Trp mutant peptides were employed as site-specific fluorescent probes of unfolding kinetics and reported diverse and characteristic unfolding kinetics. However, it was shown that following a rapid and major unfolding transition the peptide's core residues cluster slowly, by hydrophobic association, forming an intermediate species which is a prerequisite to dimerization. These equilibrium and kinetic unfolding data describe a unique unfolding mechanism where the channel peptide forms a partially unfolded dimerized structure in 4 M guanidine hydrochloride.
Biochemistry 04/1997; 36(10):3037-46. · 3.42 Impact Factor
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ABSTRACT: The role of the tryptophan residues in the substrate-binding and catalytic mechanism of an enzymatically active C-terminal fragment of Pseudomonas aeruginosa exotoxin A was studied by individually or jointly replacing these residues with phenylalanine. Substitution of W-466 decreased the ADP-ribosyltransferase and NAD(+)-glycohydrolase activities by 20- and 3-fold, respectively. In contrast, substitution of W-417 or W-558 with phenylalanine both resulted in a 3-fold decrease in ADP-ribosyltransferase activity with, however, only a decrease by 40% and 70% in NAD(+)-glycohydrolase activity, respectively. Simultaneous replacement of W-466 and W-558 resulted in a 200-fold decrease in ADP-ribosyltransferase and an 6-fold decrease in NAD(+)-glycohydrolase activities, suggesting that W-466 may play a minor role in the transfer of ADP-ribose to the eEF-2 protein. Chemical modification of the tryptophan residues in the wild-type toxin fragment by N-bromosuccinimide revealed the presence of a single residue important for enzymatic activity, W-466, with a minor contribution from W-558. Additionally, tryptophan residues, W-305 and W-417, were refractory to oxidation by N-bromosuccinimide, which likely indicated the buried nature of these residues within the protein structure. Titration of the wild-type toxin fragment with NAD+ resulted in the quenching of the intrinsic tryptophan fluorescence to 58% of the initial value. Titration of the various single and a double tryptophan replacement mutant protein(s) indicated that W-558 and W-466 are responsible for the substrate-induced fluorescence quenching, with the former being responsible for the largest fraction of the observed quenching in the wild-type toxin. Consequently, a molecular mechanism is proposed for the substrate-induced fluorescence quenching of both W-466 and W-558. Furthermore, molecular modeling of the recent crystal structures for both exotoxin A (domain III fragment) and diphtheria toxin, combined with a variety of previous results, has led to the proposal for a catalytic mechanism for the ADP-ribosyltransferase reaction. This mechanism features a SN1 attack (instead of the previously purported SN2 mechanism) by the diphthamide residue (nucleophile) of eukaryotic elongation factor 2 on the C-1 of the nicotinamide ribose of NAD+, which results in an inversion of configuration likely due to steric constraints within the NAD(+)-toxin-elongation factor 2 complex.
Biochemistry 01/1997; 35(48):15134-42. · 3.42 Impact Factor
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ABSTRACT: Pseudomonas aeruginosa exotoxin A(ETA) and its C-terminal, enzymatically active fragment (PE40, 375 residues) were studied by high-performance size-exclusion chromatography, steady-state and stopped-flow fluorescence spectroscopy, and circular dichroism spectroscopy. Both proteins have been overexpressed and purified by high-performance liquid chromatography. The effect of various activation conditions (pH, urea, and DTT) on enzymatic activity was studied. Upon enzymatic activation, structural changes induced within both proteins' structures were monitored, and these changes were correlated with concomitant alterations in the catalytic activity of the proteins. The pH optimum of enzymatic activity for both ETA and PE40 was between 7.0 and 8.0, decreasing to nearly zero at acidic (pH 5.0) and basic (pH 11-12) values. Analysis of the pH titration data revealed the presence of two distinct pKa values which implicate a His residue(s) (likely His-440 and -426) and a Tyr or Lys residue (possibly Tyr-481). The identity and possible role of an active site Lys residue is not known. Additionally, a significant increase in the Stokes radii of both proteins was detected when the pH was lowered from 8.0 to 6.0. The enzymatic activity of PE40 was not affected by urea or DTT, and its Stokes radius decreased monotonically with increasing urea concentration in the presence of DTT. In contrast, the enzymatic activity of ETA peaked when the protein was preincubated with 4.0 M urea, and this coincided with a large transition (increase) in the protein's Stokes radius between 3 and 5 M urea. Furthermore, loss of helical secondary structure of both PE40 and ETA commenced at approximately 2 M urea and progressively diminished at higher denaturant concentrations. The unfolding of both proteins in urea (and DTT) was reversible, and the free energies of unfolding were determined by both circular dichroism and fluorescence spectroscopy and were found to be 13.7 +/- 2.9 and 9.8 +/- 3.4 kJ/mol, respectively, for ETA and were 17.8 +/- 6.8 and 7.5 +/- 3.6 kJ/mol, respectively, for PE40. The refolding rate of PE40 was relatively rapid [t 1/2(1) = 27 s, t 1/2(2) = 624 s], which was in stark contrast to the refolding rate of ETA (t 1/2 = several hours). The relative refolding rates of PE40 and ETA help to explain the mechanism of in vitro enzyme activation and assay.
Biochemistry 08/1996; 35(28):9042-51. · 3.42 Impact Factor
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ABSTRACT: The soluble colicin E1 channel peptide has a roughly spherical, highly alpha-helical, compact structure. The structural unfolding properties of the colicin E1 channel peptide were analyzed using fluorescence techniques. The guanidine hydrochloride-induced unfolding pattern of the wild-type channel peptide was examined by monitoring intrinsic tryptophan fluorescence. Additionally, peptide unfolding was examined with the fluorophore, 1-anilinonaphthalene-8-sulfonic acid. In order to probe the unfolding of local segments, single-tryptophan channel peptides were constructed by site-directed mutagenesis. Shifts in fluorescence emission maxima of the single tryptophan residues were used to monitor site-specific unfolding events, in the presence of guanidine hydrochloride. The unfolding patterns reported by tryptophans in different regions of the peptide were diverse. The concentration of guanidine hydrochloride at the unfolding transition midpoint for each mutant peptide and the free energy of unfolding were calculated in order to estimate local segment stabilities. Also, secondary structure unfolding was monitored using circular dichroism spectroscopy. The results of unfolding analysis showed that the channel peptide's unfolding mechanism involves an intermediate structure stabilized by the C-terminal hydrophobic core of the peptide. Knowledge of the unfolding pattern of the soluble channel peptide will aid in the understanding of the secondary and tertiary structural interactions within the channel peptide and the mechanism of colicin E1 activation.
Biochemistry 06/1995; 34(21):7225-33. · 3.42 Impact Factor
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ABSTRACT: Integrated light-scattering (ILS) spectroscopy was used to monitor the binding of the colicin E1 channel peptide to POPC:POPG large unilamellar vesicles (LUV; 60:40, mol:mol) at acidic pH (3.5). Binding conditions were chosen such that nearly all of the channel peptide was bound to the vesicles with little free peptide remaining in solution. The increase in vesicle size upon the insertion of the channel peptide was measured by performing a discrete inversion technique on data obtained from an ILS spectrometer. Vesicle size number distributions were determined for five different systems having peptide/vesicle ratios of approximately 0, 77, 154, 206, and 257. The experiment was repeated four times (twice at two different vesicle concentrations) to determine reproducibility. The relative changes in vesicle radius upon peptide binding to the membrane vesicles was remarkably reproducible even though these changes represented only a few nanometers. A comparison of vesicle size number distributions in the absence of bound peptide was made between ILS and dynamic light scattering (DLS) data and showed similar results. However, DLS was incapable of detecting the small changes due to peptide-induced vesicle swelling. The membrane-bound volume of the colicin E1 channel peptide was approximately 177 +/- 22 nm3. These data indicate that in the absence of a membrane potential (closed channel state) the colicin E1 channel peptide inserts into the membrane resulting in a significant displacement of the lipid bilayer as evidenced from the dose-dependent increase in the vesicle radius. These results indicate that ILS spectroscopy is a sensitive sizing technique that is capable of detecting relatively small changes in membrane vesicles and may have a wide application in the determination of peptide binding to membrane vesicles.
Biophysical Journal 02/1995; 68(1):131-6. · 3.65 Impact Factor
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ABSTRACT: The lipid requirement for the binding of wild-type Pseudomonas aeruginosa exotoxin A (ETA) to model endosomal membrane vesicles was evaluated using a fluorescence quenching technique. The binding of toxin to monodisperse model membrane vesicles (0.1 micron diameter) composed of various molar ratios of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS) prepared by an extrusion method [Hope, M. J., et al. (1986) Chem. Phys. Lipids 40 89-107] was pH-dependent, with maximal binding observed at pH 4.0. Analysis of the binding curves indicated that the interaction of ETA with the membrane bilayer is dominated by a set of high-affinity binding sites (Kd = 2-8 microM; 60:40 (mol:mol) POPC/POPS large unilamellar vesicles (LUV)). The binding of toxin to membrane vesicles was highly pH-dependent, but was ionic strength-independent. Toxin-induced pore formation in the lipid bilayer, as measured by the release of the fluorescent dye, calcein, from LUV was pH-dependent, with optimal dye release occurring at pH 4.0. The rate of dye release from membrane vesicles decreased rapidly with increasing pH and approached zero at pH 6.0 and higher. The pKa for this process ranged over 4.3-4.5. Calcein release from LUV was also sensitive to changes in the ionic strength of the assay buffer, with maximal release occurring at 50 mM NaCl. Higher ionic strength medium resulted in a dramatic reduction in the rate of dye release from vesicles, indicating that the toxin-induced pore is modulated by ionic interactions.(ABSTRACT TRUNCATED AT 250 WORDS)
Biochemistry 12/1994; 33(44):12981-9. · 3.42 Impact Factor
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ABSTRACT: The membrane-associated closed channel state of the colicin E1 thermolytic peptide was studied by the Parallax Method of depth-dependent fluorescence quenching. A number of single Trp-containing peptides of colicin E1 were prepared to facilitate the use of Trp as a probe for the topography of the channel peptide in the membrane-bound state. The bound form of the channel peptide was studied by binding channel peptide to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine/1-pal-mitoyl -2-oleoyl-sn- glycero-3-phosphatidylglycerol large unilamellar vesicles (60:40, mol/mol, approximately 0.1 microns diameter vesicles prepared by an extrusion technique) at low pH (pH 3.5). Depth-dependent fluorescence quenching studies using two nitroxide-labeled phospho-lipids (1-palmitoyl-2-(5-doxylstearoyl)-sn-glycero-3-phosphatidylcho++ +-line and 1-palmitoyl-2-(12-doxylstearoyl)-sn-glycero-3-phosphatidylcholine) were conducted to determine the membrane location of each Trp residue for the vesicle-bound peptide. The three naturally occurring Trp residues in the colicin channel peptide, Trp-424, Trp-460, and Trp-495, were found to reside at membrane depths (from the C-2 carbon of the fatty acyl chain) of 7.4, 3.1, and 8.4 A, respectively. Three Trp residues (Trp-355, Trp-460, and Trp-507) in the channel peptide were classified as shallow (0-5.0 A from C-2 carbon). The remaining 9 Trp residues were classified as moderately buried (5.1-10.0 A). None of the dozen tryptophyls were classified as deeply buried in the membrane bilayer (10.1-15.0 A). A model for the colicin E1 channel based on these measurements along with previous data obtained from proteolysis, chemical labeling, ESR quenching, and mutagenesis experiments is proposed. This model for the closed state of the channel has as its central feature of the presence of only two trans-membrane segments. The membrane-associated portion of the channel includes the hydrophobic membrane anchor domain, Ala-474 to Ile-508. Furthermore, the fluorescence quenching data are consistent with the NH2-terminal helices (helices 1-7) lying on the surface of the membrane with the helical axis being oriented parallel to the membrane plane.
Journal of Biological Chemistry 03/1994; 269(6):4187-93. · 4.77 Impact Factor
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ABSTRACT: The single cysteine residue (Cys-505) located in the hydrophobic membrane anchor domain in the colicin E1 COOH-terminal channel peptide was labeled with the thiol-specific fluorescent reagent IAEDANS [5-[[[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid]. The labeling stoichiometry was nearly 1:1 [AEDANS: peptide (mol:mol)]. Eleven single Trp mutants of the channel peptide were prepared, and the FRET efficiency for each Trp residue (donor) and the AEDANS chromophore (acceptor), covalently attached to Cys-505, was measured. The FRET efficiencies for the various donor-acceptor pairs ranged from 15% to approximately 100% for the native peptide in solution (pH6.0). The FRET efficiency for the W-507 channel peptide-AEDANS adduct approached 100% since this adduct showed no detectable Trp fluorescence. Activation of the channel peptide to the insertion-competent state upon addition of the nonionic detergent octyl beta-D-glucoside [10,000:1 detergent: peptide (mol:mol)] resulted in decreased FRET efficiencies. The detergent-activated colicin E1 channel peptide-AEDANS adducts possessed significant in vitro channel activity at pH 6.0. The relative changes in the FRET efficiencies upon peptide activation ranged from -1% (W-495 channel peptide-AEDANS adduct) to 48% (W-355 channel peptide-AEDANS adduct). A direct correlation existed between the relative change in FRET efficiency upon channel peptide activation and the position of the Trp (donor) residue within the channel peptide primary sequence (higher relative delta E the closer the Trp donor was to the NH2 terminus), except for the W-484 channel peptide-AEDANS adduct, which showed a higher relative delta E than either W-443 or W-460 channel peptide-AEDANS adducts.(ABSTRACT TRUNCATED AT 250 WORDS)
Biochemistry 03/1994; 33(5):1108-15. · 3.42 Impact Factor
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ABSTRACT: Colicin E1 or any of its COOH-terminal channel peptides can be activated in vitro by acidic (< 4.5) pH or detergents. In its activated or insertion-competent state, the colicin E1 thermolytic (178 residue) channel peptide demonstrated an increased ability to bind and form channels in artificial membranes. An earlier report [Merrill et al. (1990) Biochemistry 29, 5829-5836] indicated that the structural change occurring in the channel peptide upon activation was not a large unfolding but seemingly involves a more subtle conformational change. To probe the solution structure of the colicin channel peptide and the structural changes occurring upon activation, 12 single-tryptophan-containing mutant peptides have been prepared. All of the peptides displayed cellular cytotoxicity comparable to the wild-type peptide. Fluorescence quenching by acrylamide of each Trp residue genetically engineered into the channel peptide indicated that tryptophyls located at positions 355, 367, 393, 413, and 443 report significant conformational changes which are associated with the insertion-competent state. Calculation of the bimolecular quenching constants for each single-Trp peptide showed that there are three classes of Trp residues found in the native colicin E1 channel peptide. None of the Trp residues were found to be completely inaccessible to acrylamide (buried). The NH2-terminal region near Trp-355 and -367 along with the COOH-terminal hydrophobic domain, including Trp-484, -495, and -507, was largely buried in the channel peptide soluble structure. Two peptide segments, one containing Trp-393, -404, and -413 and a second encompassing Trp-431 and -443, were moderately to very exposed regions in the soluble channel peptide.(ABSTRACT TRUNCATED AT 250 WORDS)
Biochemistry 07/1993; 32(27):6974-81. · 3.42 Impact Factor
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ABSTRACT: Colicin E1 immunity protein encoded by the plasmid colicin E1 was overproduced in Escherichia coli from an expression plasmid which was constructed by placing the immunity protein-encoding sequence downstream of the tac promoter and by properly positioning the ribosome binding site on a run-away replication vector. The immunity protein was solubilized with 0.5% Brij 58 from the membrane fraction and purified to homogeneity by a simple batch procedure with hydroxyapatite gel and reverse-phase chromatography. A 15-residue NH2-terminal amino acid sequence was determined to be the same as that deduced from the DNA sequence. The effect of the purified immunity protein on membranes was tested in vitro using solute-loaded liposomes. The immunity protein added to the liposomes showed a small but significant channel or lytic activity that is an indicator of its hydrophobic nature.
Plasmid 06/1993; 29(3):236-40. · 1.52 Impact Factor
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ABSTRACT: The mechanism of channel formation and action of channel-forming colicins is a paradigm for the study of dynamic aspects of membrane-protein interactions. The following experimental results concerning interaction of the colicin E1 channel domain with target membranes, in vitro and in vivo, are discussed: (1) the nature of the translocation-competent state of the channel-forming domain; (2) unfolding of the colicin channel peptide during in vitro binding and anchoring of the channel to liposome membranes at acidic pH; (3) reversal of channel peptide binding to liposomes by an alkaline-directed pH shift; (4) voltage-driven translocation and gating of the ion channel, discussed in the context of a four-helix model for a monomeric channel; (5) rescue of colicin-treated cells by high levels of external K+; (6) trypsin rescue of cells depolarized by the colicin ion channel; and (7) interaction of the channel domain with its immunity protein.
FEMS microbiology immunology 10/1992; 5(1-3):71-81.
