Thiol-disulphide interchange in tubulin: kinetics and the effect on polymerization

Abstract

All 20 cysteine residues are accessible to disulphide reagents in the tubulin dimer, whereas only four are accessible in taxol-stabilized microtubules. Reaction rates with disulphide reagents are a function of the reagent, are decreased by G nucleotides, and increased with increase in pH and urea. With transient (stop-flow) kinetics, DTNB [5,5'-dithiobis-(2-nitrobenzoic acid)] and 2,2'-dithiodipyridine progress curves cannot be fitted by the sum of exponential terms based only on classes of cysteines. The mixed disulphide products react further to form both intra- and intermonomer disulphide bonds that can be reversed by reducing agents. With MMTS (methyl methanethiosulphonate) or ODNB (n-octyl-dithio-2-nitrobenzoate), virtually no protein-protein disulphide bonds are formed and the ODNB reaction can be given as the sum of three exponential terms with pseudo-first-order rate constants of 0.206, 0.069 and 0.010 s(-1) at pH 6.5, suggesting three classes of thiol reactivities. Limited cysteine substitution leads to only small changes in tryptophan or CD spectra, whereas complete substitution leads to loss of the helix content. MMTS-induced loss of SH groups leads to progressive increases in the critical concentration and loss of polymerization competence that can be reversed by assembly promoters such as higher protein concentration, taxol or high ionic strength. Under such conditions, the substituted tubulin forms protofilament-based structures such as microtubules, open tubules, sheets and/or bundles.

Scheme 1. Thiol–disulphide (‘disulfide’) reactions used in the present study

Figure 1. Reaction kinetics of ODNB, DTNB and 2-DTP with tubulin-SH at 25 °C

Tubulin (1.0 μM=20.0 μM SH) in Mes a.b. (pH 6.5) was treated with 0.5 mM ODNB (curve 1), DTNB (curve 2), 2-DTP (curve 3) or 0.5 mM reagents in the presence of 4 M urea (curve 4). With DTNB and 2-DTP, reactions were 97±1.5% (19.4±0.3 SH) complete, and with ODNB, the reaction was 90±1.5% (18.0±0.3 SH) complete; with 4 M urea, the reaction was 98±1.0% complete within the mixing time. Curve 5, microtubules were assembled in taxol buffer (20 μM taxol and 10% DMSO in Mes a.b.) at 37 °C for 30 min, pelleted, washed twice, resuspended in taxol buffer and then treated with DTNB for different time periods at a DTNB/SH molar ratio of 50:1. Samples were removed at different time points, pelleted and supernatant solutions were assayed at 412 nm.

Figure 2. Denaturation and SH reactivity

(A) Absorption measurements were performed at 4 °C for 5 μM tubulin and 186 μM DTP concentrations in Mes a.b. and results are expressed as initial rates determined graphically. Fluorescence (φ) was measured at room temperature with excitation at 295 nm and 3 mm light path. Open symbols depict absorbance and closed symbols represent fluorescence emission maximum. The inset shows the time course of the DTP reaction in 4 M urea. Reactions were started by tubulin addition and were recorded 20 s later. (B) CD spectra of tubulin in 50 mM phosphate buffer (pH 7.0) at room temperature. ○, control; +, fully modified with ODNB; ———, fully modified with MMTS;········, tubulin in 50 mM Tris/HCl (pH 8.5).

Figure 3. Reaction kinetics of DTNB with tubulin-SH in the presence of GTP, GDP and ATP in Mes a.b. at 25 °C

Tubulin at 0.5 μM (10.0 μM SH) was reacted with 1.0 mM DTNB in the absence of added nucleotides (curve 1), 30.0 μM GDP (curve 2A), 200 μM GDP (curve 2B) and 1.0 mM GDP (curve 2C); 30.0 μM GTP (curve 3A), 200 μM GTP (curve 3B) and 1.0 mM GTP (curve 3C), or 1.0 mM ATP (curve 4). Tubulin was incubated with nucleotides for 20 min at 20 °C before the addition of DTNB addition.

Figure 4. SDS/PAGE analysis on non-reducing 7% NuPAGE gels (Tris-acetate buffer) of tubulin modified by disulphide reagents

Coomassie Blue staining. (A) Tubulin (10 μM=200 μM SH) was treated with 2-DTP or DTNB at reagent/SH molor ratios of 0.2, 0.3, 0.5 and 20 in Mes a.b., at pH 6.5 and 25 °C for 60 min; 4 μg of sample/lane was loaded. Lane 1, molecular-mass standards (kDa); lanes 2–5, DTNB-modified tubulin; lanes 6–9, 2-DTP-modified tubulin; lane 10, unmodified tubulin; M, monomer; D, dimer. The broken line and/or * indicates the ideal dimer size. The molar ratios of reagent/SH are listed below the lanes. (B) DTNB-induced mobility changes in monomers as a function of tubulin concentration. Tubulin (0.5, 1 and 2 μM) was treated with 20:1 and 50:1 molar ratios of DTNB/SH in Mes a.b. (pH 6.5) at 25 °C for 60 min and separated. Lane 1, molecular-mass standards; lane 2, untreated control; lanes 3–8, increasing concentrations of tubulin; 0.56 μg of tubulin/lane was loaded. Triplet monomers are labelled 1–3. The arrow indicates control monomer.

Figure 5. Kinetics of ODNB reaction with tubulin-SH at 25 °C

Tubulin (1.69 μM=34 μM SH) was mixed with 0.5 mM ODNB in the molar ratio 15:1, in a 4 mm path length cell. The kinetic trace at 412 nm was extracted from scans between 395 and 545 nm. (A) Stop-flow analysis in Mes a.b. (pH 6.5). (B) Stop-flow analysis in 0.1 M Tris/HCl, pH 8.5 (note the change in time scale). Data were fitted to eqn (2) using Olis RSM software; residuals are shown in the insets. Note that the apparently greater noise level is due to the expanded time scale and greater number of points/s.

Figure 6. Comparison of monomer mobility shifts caused by ODNB and DTNB and their reversal by DTT

Coomassie Blue staining. Tubulin (2 μM) was treated with 2.0 mM ODNB or DTNB in Mes a.b. at 25 °C for 90 min. (A) ODNB-modified tubulin without (lanes 1–2) or with (lanes 5–9) 5 mM DTT boiled for 0, 1, 3, 5 and 10 min, by loading 2 μg/lane. Lane 1, control; lane 2, ODNB only; lane 3, molecular-mass standards; lane 4, control with DTT. (B) Conditions are the same as for (A) but with DTNB in lanes 2 and 5–9. Large arrows indicate control monomers and small arrows indicate different monomers.

Figure 7. Changes in the critical concentrations produced by MMTS

Tubulin (TB) was treated with 1–5 mol of MMTS/dimer for 90 min at 4 °C and then polymerized at 37 °C as described in the Methods subsection. (A) Effect of MMTS on tubulin polymerization in 0.1 M Mes a.b. The inset shows typical time courses: A, control; B, 1 MMTS/dimer; C, 2 MMTS/dimer; and D, 3 MMTS/dimer. (B) Effect of polymerization promoters on unmodified tubulin in 0.1 M Mes a.b. without or with 20 μM taxol or 0.6 M Mes a.b. at the critical concentration. (C) Effect of MMTS on tubulin polymerization in 0.1 M Mes a.b. +20 μM taxol. (D) Effect of MMTS on tubulin polymerization in 0.6 M Mes a.b. Note the changes in scale.

Figure 8. Critical concentrations for polymerization of MMTS-modified tubulin

Data obtained from experiments like those of Figure 9 extrapolated to a D₃₅₀ of 0.01. Topmost curve, 0.1 M Mes a.b.; middle curve, 0.6 M Mes a.b.; bottom curve, 20 μM taxol in 0.1 M Mes a.b.

Figure 9. Effect of polymerization promoters on microtubule assembly of MMTS-modified tubulin

(A, C, E) unmodified tubulin; (B, D, F) MMTS-modified tubulin. (B) 2 MMTS/dimer; (D) 4 MMTS/dimer; (F) 3 MMTS/dimer. (A, B) In 0.1 M Mes a.b.; (C, D) in 20 μM taxol in 0.1 M Mes a.b.; (E, F) in 0.6 M Mes a.b. All magnifications are×123000.