8 research outputs found
Structural parameters for the CtxB<sub>5</sub> and LTB<sub>5</sub> complexes.
a<p>Number of intermolecular donor-acceptor pairs within 5 Ã… in the crystal structure of the complex. The number of independent donor-acceptor pairs is given in parentheses.</p>b<p>Relative translational diffusion coefficient.</p>c<p>Rotational diffusion coefficient of protein 1.</p>d<p>Rotational diffusion coefficient of protein 2.</p
pH-dependence of the association rates determined by SDA (simulation of diffusional Association).
<p>Rates of association were computed at different pH for the wild-type toxin and for the histidine mutants by Brownian Dynamics simulation. <b>A. Rates of association of CtxB subunits.</b> For the sake of clarity only mutants H57N and H18Y-H94N are indicated on the figure. H13N and H13N-H57N gave similar results than H57N and H18Y and H94N gave similar results than H18Y-H94N. <b>B. Rates of association of LTB subunits.</b> The rates of association of wild-type and the two histidine mutants H13N and H57N are shown. The rates of association are calculated for two native independent contacts at an intermolecular distance of 6 Ã….</p
Mid-point pH values determined by SDA for CtxB<sub>5</sub> wild-type and histidine mutants.
a<p>MP stands for mid-point pH.</p
Formation of CtxB intermediates as function of pH.
<p>CtxB<sub>5</sub> was treated for 15 min at pH 1.0 and subsequently diluted to a final concentration of 8.6 µM in McIlVaine buffers at indicated pH. The samples were incubated for 30 min at 23°C. <b>A. </b><b>Determination of the CtxB intermediates by cross linking and SDS PAGE.</b> Immediateley after neutralization or 30 min later, the samples were cross-linked with glutaraldehyde for 2 min prior mixing with 4× sample buffer and subsequent analysis by SDS-PAGE. Cross-linked CtxB Pentamer (P<sub>CL</sub>) and stoechiometric intermediates (D, dimer; T, trimer; Te, tetramer) as well as cross-linked CtxB monomer (M<sub>CL</sub>) were identified on the gel. Native CtxB<sub>5</sub> cross-linked (P<sub>CL</sub>) and not cross-linked (P<sub>NCL</sub>) were also loaded on the gel to identify their respective position. L stands for the apparent molecular weights of standards, indicated in kDa. <b>B.</b> Time-dependent changes in the intensity of the Trp-fluorescence maxima of the samples were measured, immediately after dilution into the cuvette containing McIlVaine buffer at indicated pH, and for 30 min. The ratio of the fluorescence intensity at‘t’ by the fluorescence intensity at t = 0 (just after addition of the sample into the cuvette) was plotted against time for comparison purpose. Reassembly is monitored for pH 7.0 (□), 6.7 (◊); 6.2 (▴) and 5.9 (Δ). <b>C.</b> Half-times of the reassembly reactions at indicated pH, calculated from the increase of the Trp-fluorescence intensity were plotted against pH.</p
Reassembly of CtxB as function of pH.
<p><b>A. Reassembly of CtxB into SDS stable pentamer.</b> Equal amount of reassembled CtxB was applied on each lane of a SDS-PAGE. CtxB reassembled for 0 min and 30 min at pH 7.0 (lanes 2 and 3), or for 30 min at pH 6.0 (lane 4) or and at pH 5.0 (lane 5). Lane 1 is the native CtxB<sub>5</sub>. Molecular weight standards are indicated in kDa on the left of the gel. The respective apparent positions of the native CtxB pentamer and of the CtxB monomer are indicated on the right of the gel. <b>B. Reassembly of CtxB into species capable of recognizing GM1.</b> CtxB<sub>5</sub> (○) was treated at pH 1.0 for 15 min and subsequently diluted to a final concentration of 8.6 µM in McIlVaine buffers at indicated pH. The samples were incubated for 30 min at 23°C and analyzed by GM1-Elisa. The results of three independent experiments are shown as a mean ± S.D.</p
Scenari of the steps possibly inhibited by the low pH during the reassembly of CxtB.
<p>Each monomer is represented by a circle. The deprotonated and the protonated CtxB monomers are indicated in white and in black, respectively. In scenari 1, 2 and 3, the β-strands constituting the two subunit interfaces (25–33 a.a. and 97–103 a.a.) are indicated by a line only when they are capable of associating. If the association is impaired by the low pH, the strands of the interfaces are not represented. The native CtxB<sub>5</sub> is represented as a ring of five monomers according to the x-ray crystallographic structure (<i>10</i>). <b>2A. Scenario 1.</b> The folding of the CtxB monomer is inhibited by the low pH. The protonated (black square) and the deprotonated CtxB (white circle) monomers have two different folds, and only the deprotonated CtxB monomer persue the assembly process. The protonated CtxB monomer misfolds irreversibly. <b>2B. CtxB<sub>5</sub> interfaces and histidine residues.</b> For simplicity, out of the five CtxB monomers that composed the native pentamer, only three are shown in strands. Each monomer has two interfaces (Interfaces 1 and 2) involving two different β-strands. The strand number 3 of M (residues 25 to 33) associates with the C-terminal end of the β-strand number 6 of monomer M+1 (residues 97 to 103) to form the interface 1 (I<sub>1</sub>). The C-terminal end of the β-strand number 6 of monomer M associates with the strand number 3 of monomer M-1 to form the interface 2 (I<sub>2</sub>). The four histidine residues are indicated as balls and sticks, histidines 18 and 94 which are located upstream the two β-strands of the interfaces are colored in black. The figure was made using rasmol and using the coordinates from the x-ray structure of CtxB<sub>5</sub> (<i>10</i>). <b>2C. Scenario 2.</b> The formation of both the interfaces 1 and 2 is inhibited by the low pH. The protonated CtxB monomer is association-incompetent. Only the deprotonated CtxB monomer can associate. <b>2D. Scenario 3.</b> The formation of either interface 1or 2 is inhibited by the low pH. The CtxB protonated can form only one of the two interfaces and is association-deficient. <b>2E. Scenario 4.</b> An intramolecular rearrangement (folding) within the CtxB pentamer is inhibited by the low pH. Both the protonated and the deprotonated CtxB monomers can associate together (black and white) or separately (white-white or black-black). The formation of the native pentamer is pH-dependent. In each scenario, the steps which involve deprotonation/protonation of the CtxB subunit are indicated by a star (*). The native pentamer is considered the most stable species of the reaction and therefore its formation is assumed irreversible.</p
Conformational changes in the structure of the CtxB monomer as function of pH.
<p>CtxB<sub>5</sub> was treated for 15 min at pH 1.0 and subsequently diluted to a final concentration of 8.6 µM, or otherwise specified, in McIlVaine buffers at indicated pH <b>A. CtxB monomeric state.</b> After acidification and just after dilution in McIlVaine buffer at pH 7.0, CtxB was cross-linked (0′) with glutaraldehyde for 2 min prior mixing with 4× sample buffer and subsequent analysis by SDS-PAGE. The same treatment was performed on native CtxB<sub>5</sub>, to indicate the position of cross-linked CtxB monomer (M<sub>CL</sub>), dimer (D), trimer (T) tetramer (Te) and pentamer (P<sub>CL</sub>) (<i>23</i>). Native, not cross-linked CtxB<sub>5</sub>, was also loaded on the gel to indicate the position of the native pentamer without cross-linking (P<sub>NCL</sub>). Some of the native CtxB<sub>5</sub>, not cross-linked, did not resist the SDS treatment after boiling and dissociated into monomer, indicating the apparent molecular weight of the non cross-linked monomer (M<sub>NCL</sub>). L stands for the apparent molecular weights of standards, indicated in kDa. Only the cross-linked samples were boiled prior loading. <b>B.</b> pH dependences of the fluorescence intensity of CtxB monomers just after dilution in McIlVaine buffer at indicated pH, for reassemblies at 8.6 µM (□) and at 35 µM (▪). a.u. stands for arbitrary unit. The data for the toxin concentration at 35 µM were divided by four to allow comparison with the data for the toxin concentration at 8.6 µM on the same graph. <b>C. Far-UV CD spectra.</b> Native CtxB<sub>5</sub> at pH 7.0 (•) and at pH 5.0 (♦). Native CtxB<sub>5</sub> was acidified for 15 min at pH 1.0 and diluted into McIlVaine buffers at pH 7.0, just after dilution (—) (CtxB monomer), or 30 min later (○) (reassembled CtxB) or just after dilution at pH 5.0 (◊). (□) acidified CtxB at pH 1.0 for 15 min. The toxin concentration is 8.6 µM.</p
Effect of the toxin concentration on the pH-dependence of the CtxB reassembly.
<p><b>A.</b> Yields of reassembly determined by GM1-Elisa after 30 min incubation at 8.6 µM (○) and at 35 µM (•) at indicated pH. The data for a reassembly at 8.6 µM are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015347#pone-0015347-g001" target="_blank">figure 1B</a>. Each sample was assayed in triplicate and the results of three independent experiments were shown as a mean ± S.D. <b>B.</b> CtxB<sub>5</sub> was treated for 15 min at pH 1.0 and subsequently diluted to a final toxin concentration of 35 µM in McIlVaine buffer at pH 7.0 or at pH 6.0 for 30 min at 23°C. The presence of CtxB pentamer and of CtxB intermediates was determined by cross-linking combined to SDS-PAGE (15%). The position of the native cross-linked pentamer (P<sub>CL</sub>) was identified by loading a native CtxB<sub>5</sub> sample after cross-linking. M<sub>CL</sub> stands for cross-linked CtxB monomer. <b>C.</b> The presence of pentamers reassembled at pH 7.0 and at pH 6.0, after a pre-treatment at pH 1.0 for 15 min, and capable of resisting SDS-treatment was monitored by SDS-PAGE (12%). The incubation time and the pH are indicated at the top and at the bottom of the gel, respectively. After 30 min incubation at pH 7.0 or at pH 6.0, 0.1 M Tris buffer at pH 8.0 (+ Tris) was added to the samples for 15 more min (45′). Native CtxB<sub>5</sub> was also loaded on the gel as a marker of the pentamer position. In the gels, 1 µg of sample was loaded on each well and the gels were silver stained. L stands for the apparent molecular weight of standards, indicated in kDa.</p