A Kinetic Model of Intermediate Formation during Assembly of Cholera Toxin B-subunit Pentamers
2002; Elsevier BV; Volume: 277; Issue: 19 Linguagem: Inglês
10.1074/jbc.m110561200
ISSN1083-351X
AutoresClaire Lesieur, Matthew J. Cliff, Rachel Carter, Roger F.L. James, Anthony R. Clarke, Timothy R. Hirst,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoCholera toxin is the most important virulence factor produced by Vibrio cholerae. The pentameric B-subunit of the toxin can bind to GM1-ganglioside receptors, leading to toxin entry into mammalian cells. Here, the in vitrodisassembly and reassembly of CtxB5 (the B subunit pentamer of cholera toxin) is investigated. When CtxB5 was acidified at pH 1.0 and then neutralized, the B-subunits disassembled and could no longer migrate as SDS-stable pentamers on polyacrylamide gels or be captured by GM1. However, continued incubation at neutral pH resulted in the B-subunits regaining the capacity to be detected by GM1 enzyme-linked immunosorbent assay (t 1/2 ∼ 8 min) and to migrate as SDS-stable pentamers (t 1/2 ∼ 15 min). Time-dependent changes in Trp fluorescence intensity during B-subunit reassembly occurred with a half-time of ∼8 min, similar to that detected by GM1 enzyme-linked immunosorbent assay, suggesting that both methods monitor earlier events than B-pentamer formation alone. Based on the Trp fluorescence intensity measurements, a kinetic model of the pathway of CtxB5 reassembly was generated that depended on trans to cisisomerization of Pro-93 to give an interface capable of subunit-subunit interaction. The model suggests formation of intermediates in the reaction, and these were successfully detected by glutaraldehyde cross-linking. Cholera toxin is the most important virulence factor produced by Vibrio cholerae. The pentameric B-subunit of the toxin can bind to GM1-ganglioside receptors, leading to toxin entry into mammalian cells. Here, the in vitrodisassembly and reassembly of CtxB5 (the B subunit pentamer of cholera toxin) is investigated. When CtxB5 was acidified at pH 1.0 and then neutralized, the B-subunits disassembled and could no longer migrate as SDS-stable pentamers on polyacrylamide gels or be captured by GM1. However, continued incubation at neutral pH resulted in the B-subunits regaining the capacity to be detected by GM1 enzyme-linked immunosorbent assay (t 1/2 ∼ 8 min) and to migrate as SDS-stable pentamers (t 1/2 ∼ 15 min). Time-dependent changes in Trp fluorescence intensity during B-subunit reassembly occurred with a half-time of ∼8 min, similar to that detected by GM1 enzyme-linked immunosorbent assay, suggesting that both methods monitor earlier events than B-pentamer formation alone. Based on the Trp fluorescence intensity measurements, a kinetic model of the pathway of CtxB5 reassembly was generated that depended on trans to cisisomerization of Pro-93 to give an interface capable of subunit-subunit interaction. The model suggests formation of intermediates in the reaction, and these were successfully detected by glutaraldehyde cross-linking. Cholera toxin (Ctx) 1The abbreviations used are: Ctxcholera toxinCtxB5B subunit pentamer of CtxEtx (or LT)heat-labile enterotoxinEtxB5 (or LTB5) Etx B subunit pentamerPBS, phosphate-buffered salineELISAenzyme-linked immunosorbent assayGM1monoganglioside-GM1 (Galβ1–3GalNacβ1-(neu5Acα2–3)-4Galβ1–4Glcβ1-cer) and heat-labile enterotoxin (Etx) are the primary virulence factors produced by Vibrio cholerae and certain toxinogenic strains of Escherichia coli, respectively (1.Hirst T.R. Alouf J.E. Freer J.H. The Comprehensive Sourcebook of Bacterial Protein Toxins. 2nd Ed. Academic Press, Ltd., London1999: 104-129Google Scholar, 2.Kaper J.B. Curr. Opin. Microbiol. 1998; 1: 103-108Crossref PubMed Scopus (89) Google Scholar, 3.Lencer W.I. Hirst T.R. Holmes R.K. Biochim. Biophys. Acta. 1999; 1450: 177-190Crossref PubMed Scopus (222) Google Scholar). Both toxins are heterooligomeric proteins comprising an A-subunit that exhibits ADP-ribosyltransferase activity and five B-subunits that bind with high affinity to the glycolipid receptor, monosialoganglioside GM1, found in the plasma membranes of mammalian cells (4.Holmgren J. Lonnroth I. Mansson J. Svennerholm L. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 2520-2524Crossref PubMed Scopus (278) Google Scholar, 5.Fishman P.H. Moss J. Osborne Jr., J.C. Biochemistry. 1978; 17: 711-716Crossref PubMed Scopus (105) Google Scholar, 6.Fishman P.H. Pacuszka T. Hom B. Moss J. J. Biol. Chem. 1980; 255: 7657-7664Abstract Full Text PDF PubMed Google Scholar). The B pentamer components of both cholera toxin (CtxB5) and E. coli heat-labile enterotoxin (EtxB5) are widely thought of as carrier molecules principally involved in delivering the toxin A-subunit into cells (3.Lencer W.I. Hirst T.R. Holmes R.K. Biochim. Biophys. Acta. 1999; 1450: 177-190Crossref PubMed Scopus (222) Google Scholar). However, more recent studies have revealed that these receptor binding moieties possess striking immunomodulatory properties that can down-regulate inflammatory immune reactions (7.Hirst T.R. Nashar T.O. Pitman R.S. Williams N.A. J. Appl. Microbiol. Sympos. Suppl. 1998; 84: 26-34Crossref Google Scholar, 8.de Haan L. Hirst T.R. J. Nat. Toxins. 2000; 9: 281-297PubMed Google Scholar, 9.Aman A.T. Fraser S. Merritt E.A. Rodigherio C. Kenny M. Ahn M. Hol W.G. Williams N.A. Lencer W.I. Hirst T.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8536-8541Crossref PubMed Scopus (89) Google Scholar). Such findings have prompted renewed interest in the B-subunit pentamers and led to their testing as a potential therapeutic agents for the treatment of inflammatory allergic and autoimmune disorders (10.Bergerot I. Ploix C. Petersen J. Moulin V. Rask C. Fabien N. Lindblad M. Mayer A. Czerkinsky C. Holmgren J. Thivolet C. Proc. Natl. Acad. Sci. U. S. 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Nature. 1983; 306: 551-557Crossref PubMed Scopus (454) Google Scholar, 15.Neill R.J. Ivins B.E. Holmes R.K. Science. 1983; 221: 289-291Crossref PubMed Scopus (34) Google Scholar, 16.Hirst T.R. Randall L.L. Hardy S.J. J. Bacteriol. 1984; 157: 637-642Crossref PubMed Google Scholar). Expression of either CtxB or EtxB in the absence of their corresponding A-subunits results in the formation of highly stable B-subunit pentamers that are devoid of enterotoxic activity. The in vivo pathway of B-subunit pentamerization is poorly understood, chiefly because of the difficulty of investigating such processes in the complex environment of the periplasmic space (17.Mekalanos J.J. Curr. Top. Microbiol. Immunol. 1985; 118: 97-118PubMed Google Scholar, 18.Hardy S.J. Holmgren J. Johansson S. Sanchez J. Hirst T.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7109-7113Crossref PubMed Scopus (104) Google Scholar). The use ofin vitro conditions to study the disassembly and reassembly of the toxins was first reported by Finkelstein et al. (19.Finkelstein R.A. Boesman M. Neoh S.H. LaRue M.K. Delaney R. J. Immunol. 1974; 113: 145-150PubMed Google Scholar) who showed that purified cholera toxin could be denatured in acid urea and subsequently reassembled into active toxin when neutralized. Similarly, when purified CtxB5 or EtxB5 were denatured in acid and subsequently neutralized, the B-subunits were shown to be able to reassemble into stable pentameric complexes (18.Hardy S.J. Holmgren J. Johansson S. Sanchez J. Hirst T.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7109-7113Crossref PubMed Scopus (104) Google Scholar,20.De Wolf M.J.S. Dierick W.S.H. Biochim. Biophys. Acta. 1994; 1223: 285-295Crossref PubMed Scopus (8) Google Scholar, 21.Ruddock L.W. Ruston S.P. Kelly S.M. Price N.C. Freedman R.B. Hirst T.R. J. Biol. Chem. 1995; 270: 29953-29958Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 22.Ruddock L.W. Coen J.J.F. Cheesman C. Freedman R.B. Hirst T.R. J. Biol. Chem. 1996; 271: 19118-19123Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). The intrinsic stability of CtxB5 or EtxB5 in the presence of SDS has meant that pentamer formation can be investigated by use of SDS-PAGE, and this approach has been used to monitor the kinetics of EtxB pentamerization. However, nothing is known of the pathway of assembly intermediates that are formed during the assembly process. Many other bacterial pathogens also produce toxins with complex oligomeric structures (23.Falnes P.O. Sandvig K. Curr. Opin. Cell Biol. 2000; 12: 407-413Crossref PubMed Scopus (244) Google Scholar, 24.Lesieur C. Vecsey-Semjen B. Abrami L. Fivaz M. Gisou van der Goot F. Mol. Membr. Biol. 1997; 14: 45-64Crossref PubMed Scopus (148) Google Scholar). Although proper acquisition of their quaternary structure is also known to be essential for the mode of action of these toxins, no stoichiometric intermediates have yet been isolated. Here, we address the question of intermediate formation by studying the reassembly of CtxB pentamers after acid denaturation and subsequent neutralization at pH 7. The assembly of CtxB was followed using different signals, GM1 ELISA, capturing GM1-bound species, SDS-PAGE, measuring pentamer formation, and tryptophan fluorescence spectroscopy, which was found to monitor a structural transition, consistent with oligomer formation. The latter was subjected to a rigorous analysis by computational modeling. CtxB was purified fromVibrio sp. 60 (pTRH64) as described previously (9.Aman A.T. Fraser S. Merritt E.A. Rodigherio C. Kenny M. Ahn M. Hol W.G. Williams N.A. Lencer W.I. Hirst T.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8536-8541Crossref PubMed Scopus (89) Google Scholar, 25.Richards C.M. Aman A.T. Hirst T.R. Hill T.J. Williams N.A. J. Virol. 2001; 75: 1664-1671Crossref PubMed Scopus (66) Google Scholar) and stored at −80 °C in phosphate-buffered saline, pH 7.2 (150 mm NaCl, 10 mm sodium phosphate, pH 7. 2 (PBS)) at a concentration of 0.34–0.39 mm. The toxin concentration is calculated as the monomeric concentration. SDS-polyacrylamide (13.5%) gel electrophoresis was performed with a Bio-Rad Protean II system using the Laemmli method, as recommended by the manufacturer (Bio-Rad). Either 5 or 2 μg of protein were loaded into each well, and the gels were stained with Coomassie Blue or silver stain, respectively. The buffers used were McIlvaine buffer (0.2 m disodium hydrogen phosphate, 0.1m citric acid, pH 6–9), PBS, or KCl/HCl, pH 1. All buffers were filtered through sterile 0.22-μm filter before use. The conditions used for disassembly and reassembly of CtxB5were adapted from those previously employed for studying the reassembly of E. coli heat-labile enterotoxin B-subunit (EtxB) (22.Ruddock L.W. Coen J.J.F. Cheesman C. Freedman R.B. Hirst T.R. J. Biol. Chem. 1996; 271: 19118-19123Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Briefly, the concentration of purified CtxB5 was adjusted with PBS to 344 μm and then diluted 4 times in 0.1m KCl/HCl, pH 1.0. After specified time intervals in these acidic conditions, the samples were diluted 10 times to a final concentration of 8.6 μm in McIlvaine buffer, pH 7.0, and then incubated for a further 60 min at 23 °C. Both immediately after neutralization and after incubation for specified times at 23 °C samples were removed and diluted 100-fold in PBS to prevent further assembly, followed by analysis using a GM1 ELISA (see below, under “Experimental Procedures”). In addition, samples were also removed from the reaction mixture at specified time points and mixed at a ratio of 4:1 with 5× SDS-PAGE sample buffer. These were kept on ice for up to 1 h before applying them to SDS-polyacrylamide gels without prior heating of the samples. This later procedure permits identification of reassembled CtxB5, since at ambient temperatures CtxB5 is stable in SDS-containing buffers and migrates in SDS-polyacrylamide gels with an electrophoretic mobility characteristic of the B-subunit pentamer. The percentage of reassembled CtxB5 was determined by quantification of the amount of pentamer at each time point relative to the equivalent amount of native CtxB5 as applied on the same gel using the densitometry software (TL TotalLab V1.11) from Phoretix. Reassembly of CtxB was monitored by measurement of Trp fluorescence in a PerkinElmer LS-50B spectrofluorometer. Base-line data collection was initiated in a cuvette containing neutralizing McIlvaine buffer alone into which was added CtxB that had been subjected to a 10-min denaturation in acid as documented above. The toxin concentration during the acidification step was maintained at 86 μm, whereas after dilution in McIlvaine buffer at appropriate pH levels, the reassembly reaction was monitored over a range of toxin concentrations from 4.3 to 26 μm. Excitation was at 295 nm, with emission recorded at 354 nm, and slit widths of 5 and 8 nm, respectively. When emission spectra were recorded, the Raman contribution for water was removed by subtraction of a buffer blank. The amount of B-subunit in a reassembly mixture that had acquired the ability to bind to GM1 receptors was determined using a GM1 ELISA (21.Ruddock L.W. Ruston S.P. Kelly S.M. Price N.C. Freedman R.B. Hirst T.R. J. Biol. Chem. 1995; 270: 29953-29958Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 22.Ruddock L.W. Coen J.J.F. Cheesman C. Freedman R.B. Hirst T.R. J. Biol. Chem. 1996; 271: 19118-19123Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 26.Amin T. Hirst T.R. Protein Expression Purif. 1994; 5: 198-204Crossref PubMed Scopus (44) Google Scholar). Samples of the reassembly mixture were taken at specified time points, diluted 100-fold to a toxin concentration of 86 nm and then added to ELISA plates that had previously been coated with 200 ng of GM1 and subsequently blocked with 1% Marvel in PBS. Samples were serially diluted 2-fold in PBS, and bound B-subunits were detected using a polyclonal mouse anti-CtxB5 antiserum (α12) used at a 1/10000 dilution. All other steps of the GM1 ELISA were as reported previously (26.Amin T. Hirst T.R. Protein Expression Purif. 1994; 5: 198-204Crossref PubMed Scopus (44) Google Scholar). For quantification of the amount of CtxB bound to the ELISA plates for each test sample, optical density readings corresponding to dilutions located on the linear part of the curve were compared with the dilution of a CtxB5 standard (1 μg/ml, diluted 2-fold), giving the same optical density reading. All the fluorescence spectroscopic data were fitted using the Scientist program (MicroMath Scientific Software) to perform a numerical simulation based on the kinetic pathway proposed in the model (Fig. 6 A). The numerical simulation was performed using Euler integration, and least squares fitting was performed using the modified Powell method within the Scientist program (version 2.01, Micromath 1995). The dependent variables of the equations are M, D, T, TE, and P for the molar concentration of monomer, dimer, trimer, tetramer, and pentamer with the proline in a cis conformation and U, M2, M3, M4, M5 for the molar concentration of monomer, dimer, trimer, tetramer, and pentamer with the proline in trans conformation. The kinetic equations are described as follows. U′=ktrans·M+kdiss·(M2+M3+M4+M5)Equation 1 −U·(kcis+kass·(M+D+T+TE))M′=kcis·U+kdiss·(M2+M3+M4+M5)+2·kdiss·(D+T+TE)Equation 2 −M·(ktrans+kass·(U+M2+M3+M4+2·(M+D+T+TE)))M2′=kass·U·M+ktrans·D+kdiss·(M3+M4+M5)−M2·(kdiss+kcis+kass·(M+D+T))Equation 3 D′=kass·M2+kcis·M2+kdiss·(M3+M4+M5+2·T+2·TE)Equation 4 −D·(kdiss+ktrans+kass·(U+M2+M3+2·(M+D+T)))M3′=kass·(U·D+M·M3)+ktrans·T+kdiss·(M+M5)Equation 5 −M3·(2·kdiss+kcis+kass·(M+T))T′=2·kass·M·D+kcis·M3+kdiss·(M4+M5+2·TE)Equation 6 −T·(2·kdiss+ktrans+kass·(U+M2+2·(M+T)))M4′=kass·(U·T+M·M3+M2·D)+ktrans·TEEquation 7 −kdiss·M5−M4·(3·kdiss+kcis+kass·M)TE′=kass·(2·M·T+D2)+kcis·M4+kdiss·M5Equation 8 −TE·(3 kdiss+ktrans+kass·(U+2·M)) M5′=kass·(TE·U+M2·T+D·M3+M·M4)−M5·(4·kdiss+kcis)Equation 9 P′=2·kass·(M·TE+D·T)+kcis·M5Equation 10 In the above equations a prime (′) represents the rate of change of concentration (e.g. P′ represents d[P]/dt). It is assumed that the fluorescence signal (SIG) for the formation of each interface is equal and therefore varies according to the following relation.SIG=F·(U+M+R·(M2+2·M3+3·M4Equation 11 +4·M5+D+2·T+3·Te+5·P))where F is the fluorescence intensity at time 0, and R is the ratio of the fluorescence intensity of tryptophan at a properly formed interface by that at a free interface. The fluorescence signal was carried by the pentamer as well as intermediates. The data for all protein concentrations were fitted globally. Differential equations were derived for each partial reaction and weighted statistically before being compiled into a Scientist equation file. All four rate constants, plus base line and the signal of fluorescence were allowed to vary in the fitting procedure. CtxB5 was acidified and neutralized at a final toxin concentration of 26 μm. The reassembly reaction was performed at 23 °C, and aliquots were taken at discrete times (0, 2, 4, 10, and 30 min) after neutralization and mixed for 2 min with glutaraldehyde at a final concentration of 4% (v/v). 5× sample buffer was added at a ratio of 1:4 to quench the reaction, and the samples were analyzed by SDS-PAGE and silver-stained. CtxB pentamers disassemble in acidic conditions, giving rise to monomeric B-subunits that can reassemble if placed in buffers of neutral pH (18.Hardy S.J. Holmgren J. Johansson S. Sanchez J. Hirst T.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7109-7113Crossref PubMed Scopus (104) Google Scholar, 22.Ruddock L.W. Coen J.J.F. Cheesman C. Freedman R.B. Hirst T.R. J. Biol. Chem. 1996; 271: 19118-19123Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). To investigate the reaction mechanism of reassembly, we analyzed the time course of CtxB oligomerization after acid denaturation and neutralization. First, we monitored the extent of disassembly and reassembly using a GM1 ELISA technique that captures assembled B-subunits on GM1-immobilized microtiter plates. CtxB5 was incubated for various times ranging from 0.3 to 60 min in HCl/KCl buffer, pH 1.0, then neutralized in McIlvaine buffer, pH 7.0, diluted and immediately tested in a GM1 ELISA. Short periods of acidification, e. g. 0.3 min was insufficient to achieve full disassembly of CtxB5, since ∼15% of the B-subunits were detected in the GM1 ELISA immediately after neutralization (Fig. 1 A). However, it was found that acidification for a period of 10–60 min resulted in full disassembly of the B-subunits, since no protein could be detected in the GM1 ELISA (Fig. 1 A; 0 min of neutralization). If after neutralization, the samples were maintained at 23 °C for 60 min before being diluted and then tested in the GM1 ELISA, it was evident that the B-subunits had reassembled and could now be detected by this technique (Fig. 1 A). It was noted that the extent of reassembly declined if the B-subunits were acidified for longer time periods. When CtxB5 was acidified for 10 min then neutralized and incubated for 60 min, ∼75% of the B-subunits regained the ability to be detected in the GM1 ELISA. Because extended periods of acidification reduced the overall yield of assembled CtxB, all subsequent reassembly reactions reported were performed with a 10-min acidification step followed by neutralization. To confirm that assembly of CtxB5 occurs during the 60-min incubation period, samples were taken both immediately and 60 min after neutralization and analyzed by SDS-PAGE without prior heating of the samples. As can be seen in Fig. 1 B, after 60 min CtxB pentamers are clearly present, whereas immediately after neutralization they were absent (compare lanes 2 and 3). The electrophoretic mobility of reassembled CtxB5 was identical to that of native CtxB5 (lane 1). Analysis of the kinetics of B-subunit reassembly was monitored by both GM1 ELISA and SDS-PAGE by sampling at various time points after neutralization (Fig. 2). In this experiment CtxB5 was acidified for 10 min and then neutralized to give a final concentration of B-monomers of 8.6 μm. By both techniques a time-dependent increase in formation of reassembled B-subunits was observed, but the half-times for reassembly were different, corresponding to ∼8 or ∼15 min by GM1 ELISA and SDS-PAGE, respectively. In addition the amount of assembled CtxB detectable by the two methods at each time point was also different. These discrepancies may be explained if the GM1 ELISA technique detects intermediate species in addition to CtxB5, since SDS-PAGE monitors the amount of the pentamer alone. In this regard, the GM1 binding site is located at the interface between two subunits, and it is thus reasonable that a dimer, a trimer, or a tetramer as well as a pentamer may bind to GM1. To investigate the kinetics of the assembly process further, Trp fluorescence was employed as a continuous probe to yield data that could be used to model the reassembly reaction and, thus, explain the apparent discrepancy in the assembly kinetics observed by GM1 ELISA and SDS-PAGE. Each CtxB subunit contains a single Trp residue at position 88 that is located at the subunit interface in native CtxB5and should therefore be a useful probe for studying CtxB assembly. Emission scans of native CtxB5 and of CtxB just after neutralization (referred to as CtxB (0 min)) are shown in Fig. 3 A. The fluorescence intensity of CtxB immediately after neutralization was found to be 4-fold lower than that of native CtxB5 when measured at a concentration 8.6 μm (Fig. 3 A). This indicates that upon dissociation/unfolding and neutralization of CtxB, the environment around Trp-88 changes. Taking advantage of this, we investigated the time-dependent change in fluorescence intensity during a reassembly reaction (Fig. 3 B). The experiment was initiated by recording the signal of McIlvaine pH 7 buffer alone, which was then followed by the addition of acidified toxin (arrow in Fig. 3 B). The trace shows that there was an abrupt increase in the initial fluorescence intensity due to the addition of the toxin. Thereafter, a slower increase in fluorescence intensity was observed during the next 60 min. Such a slow increase in fluorescence is unlikely to be monitoring a conformational change associated with the early stages of folding (for review see Refs. 27.Ptitsyn O.B. Curr. Opin. Struct. Biol. 1995; 5: 74-78Crossref PubMed Scopus (225) Google Scholar, 28.Widmann M. Christen P. J. Biol. Chem. 2000; 275: 18619-18622Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 29.Galzitskaya O.V. Ivankov D.N. Finkelstein A.V. FEBS Lett. 2001; 489: 113-118Crossref PubMed Scopus (72) Google Scholar). To assess whether the slow increase in fluorescence intensity was attributable to an intrinsic slow folding process in the CtxB monomer or to the oligomerization event, the influence of CtxB concentration on the kinetics of fluorescence changes was investigated. CtxB5 was acidified at 86 μm for 10 min and diluted at concentrations ranging from 4.3 to 26 μm in buffer of appropriate pH values to give a final reaction of pH of 7. As can be seen in Fig. 4 the rate of increase in fluorescence intensity increased with CtxB concentration. This demonstrates that the fluorescence measurements monitor a concentration-dependent process. A first-order reaction such as folding of the monomer or intramolecular rearrangement of the pentamer would be concentration-independent. We therefore conclude that the changes in Trp fluorescence monitor the progress of a multi-molecular reaction in which association events were rate-limiting. Interestingly, the half-time of the oligomerization reaction obtained from fluorescence measurements at a CtxB concentration of 8.6 μm was 7.6 ± 0.5 min, which was virtually identical to the half-time of reassembly determined by GM1 ELISA (Fig. 2). This suggests that both techniques are reporting the same reaction. To test if this is the case, the half-time for reassembly at various CtxB concentrations was determined by GM1 ELISA and compared with the half-times obtained using Trp fluorescence (Table I). A striking concurrence of half-times for assembly was obtained using the two techniques. We therefore conclude that the GM1 ELISA and changes in Trp fluorescence measure the same reaction.Table IHalf-times of CtxB5 reassembly as measured by Trp fluorescence and GM1-ELISAMethodsToxin Concentration26 μm17.2 μm8.6 μm4.3 μmminGM1-ELISA2.1 ± 0.53.4 ± 0.77.7 ± 1.015.0 ± 4.0Trp fluorescence2.6 ± 0.23.6 ± 0.57.6 ± 0.516.0 ± 3.0The half-times are given in minutes for toxin reassembly at four different toxin concentrations. All the experiments were done at 23 °C for 60 min. Open table in a new tab The half-times are given in minutes for toxin reassembly at four different toxin concentrations. All the experiments were done at 23 °C for 60 min. Because of the accuracy of the kinetic measurements of CtxB assembly obtained by Trp fluorescence, a kinetic model of the reaction mechanism was generated. Given that the changes in Trp fluorescence appear to be reporting the same events as those involved in B-subunit captured by GM1, we speculated that oligomer formation (i.e. dimer, trimer, tetramer, and pentamer) is the event being monitored. As can be seen on the x-ray crystallographic structure of CtxB5 reported by Zhanget al. (30.Zhang R.G. Scott D.L. Westbrook M.L. Nance S. Spangler B.D. Shipley G.G. Westbrook E.M. J. Mol. Biol. 1995; 251: 563-573Crossref PubMed Scopus (300) Google Scholar, 31.Zhang R.G. Westbrook M.L. Westbrook E.M. Scott D.L. Otwinowski Z. Maulik P.R. Reed R.A. Shipley G.G. J. Mol. Biol. 1995; 251: 550-562Crossref PubMed Scopus (110) Google Scholar), the subunit interface between adjacent monomers is formed primarily through hydrogen bond interactions between β-strand number 3 of one subunit (i.e. amino acids Phe-25 to Ala-32) and the terminal portion of β-strand number 6 of the adjacent subunit (i.e. amino acids Ala-97 to Asn-103) (30.Zhang R.G. Scott D.L. Westbrook M.L. Nance S. Spangler B.D. Shipley G.G. Westbrook E.M. J. Mol. Biol. 1995; 251: 563-573Crossref PubMed Scopus (300) Google Scholar, 31.Zhang R.G. Westbrook M.L. Westbrook E.M. Scott D.L. Otwinowski Z. Maulik P.R. Reed R.A. Shipley G.G. J. Mol. Biol. 1995; 251: 550-562Crossref PubMed Scopus (110) Google Scholar). For these β-strands to align, proline at position 93 must be in a cis configuration, so that β-strand number 6 orients toward the adjacent subunit to form the inter-subunit interface (Fig. 5). In generating a kinetic model of CtxB assembly we have thus assumed that acis proline conformation is required for formation of the inter-subunit interface. Therefore in this model (Fig. 6 A), the monomer exists in two states, a trans configuration (filled circles) and a cis configuration (open circles), and the rate-limiting step in the oligomerization reaction is the rate of proline isomerization. These two states of the monomer have different physical properties with respect to their ability to interact with another monomer and to assemble into oligomeric complexes. Each monomer has two surfaces that are able to interact with adjacent monomers. Thus, if one considers monomer M, a stable subunit interface can be formed through a hydrogen bond network between β-strand number 3 of M and β-strand number 6 of subunit M + 1 or it can be formed between β-strands number 6 of M and β-strand number 3 of subunit M-1. However, if M is in a trans configuration, only one of its surfaces can interact with another monomer since in this case β-strand number 6 is not in the proper orientation to interact with β-strand number 3 of M-1. Thus, if M is in a transconfiguration it can only associate with M + 1, which must be in acis configuration, whereas if M is in a cisconfiguration, both of its surfaces are available for interaction with M + 1 (in a cis configuration) and M − 1 (incis or trans states). Hence only one subunit in the trans configuration can be present in any oligomeric complex, with proline isomerization to a cis configuration required for further association of other monomers. The equilibrium between trans and cis states in any complex formed during the assembly reaction is represented by thevertical arrows in Fig. 6 A. Thehorizontal and the diagonal arrows represent all possible association and dissociation reactions with monomer in atrans or cis configuration (upper pathway), whereas association and dissociation with monomer only in a cis configuration is represented by thehorizontal arrows of the lower pathway. Apart from the monomer in a trans state that cannot associate on one side before proline isomerization, all pathways to the pentamer are allowed, and the rate constants forcis-trans isomerization (k trans for cis-to-transand k cis fortrans-to-cis), association (k ass), and dissociation (k diss) are the same at any stage in the pathway. It is assumed that the dissociation of pentamers is infinitely slow. The fluorescence data were fitted based on the model shown in Fig. 6 A. The equations describing how each species appears and disappears in time are given under “Experimental Procedures.” Based on those equations, a numerical simulation of all the fluorescence data was undertaken, as described under “Experimental Procedures,” and a
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