SDS-induced hexameric oligomerization of myotoxin-II from Bothrops asper assessed by sedimentation velocity and nuclear magnetic resonance

Abstract

We report the solution behavior, oligomerization state, and structural details of myotoxin-II purified from the venom of Bothrops asper in the presence and absence of sodium dodecyl sulfate (SDS) and multiple lipids, as examined by analytical ultracentrifugation and nuclear magnetic resonance. Molecular functional and structural details of the myotoxic mechanism of group II Lys-49 phospholipase A₂ homologues have been only partially elucidated so far, and conflicting observations have been reported in the literature regarding the monomeric vs. oligomeric state of these toxins in solution. We observed the formation of a stable and discrete, hexameric form of myotoxin-II, but only in the presence of small amounts of SDS. In SDS-free medium, myotoxin-II was insensitive to mass action and remained monomeric at all concentrations examined (up to 3 mg/ml, 218.2 μM). At SDS concentrations above the critical micelle concentration, only dimers and trimers were observed, and at intermediate SDS concentrations, aggregates larger than hexamers were observed. We found that the amount of SDS required to form a stable hexamer varies with protein concentration, suggesting the need for a precise stoichiometry of free SDS molecules. The discovery of a stable hexameric species in the presence of a phospholipid mimetic suggests a possible physiological role for this oligomeric form, and may shed light on the poorly understood membrane-disrupting mechanism of this myotoxic protein class.

Keywords: Analytical ultracentrifugation; Lys-49 phospholipase; Myotoxin-II; Oligomerization; SDS.

Fig. 1

Diffusion-corrected van Holde–Weischet integral sedimentation coefficient distributions of multiple concentrations of mt-II (blue: 2.3 μM, green: 34.9 μM, red: 145.5 μM, cyan: 218.2 μM) generate homogeneous, identical sedimentation coefficient distributions, demonstrating the absence of mass action, reflecting a pure monomeric species

Fig. 2

Diffusion corrected integral sedimentation coefficient distributions from SDS-mt-II titrations at 24 μM mt-II concentration (measured at 280 nm) and SDS concentrations variable between 0.001 and 0.5% (0.03–17.35 mM). Red: 0.001% (0.03 mM), green: 0.002% (0.07 mM), magenta: 0.003% (0.10 mM), purple: 0.004% (0.14 mM), dark red: 0.008% (0.28 mM), yellow 0.05% (1.73 mM), blue: 0.1% (3.47 mM), cyan: 0.5% (17.35 mM)

Fig. 3

Diffusion corrected integral sedimentation coefficient distributions from SDS-mt-II titrations at 1.1 μM protein concentration (measured at 220 nm) and SDS concentrations variable between 0.0 and 0.0016%. Red: 0.0% (0 mM), green: 0.001% (0.03 mM), magenta: 0.0015% (0.05 mM), purple: 0.0016% (0.06 mM), black: 1% (34.69 mM) SDS control, showing 0.9 s micelle sedimentation at 220 nm, explaining s values below 1.0 s in samples with SDS

Fig. 4

Diffusion corrected integral sedimentation coefficient distributions from SDS-mt-II titrations at 3.2 μM protein concentration (measured at 225 nm) and SDS concentrations variable between 0.0008 and 0.5%. Red: 0.0% (0 mM), green: 0.0008% (0.03 mM), dark red: 0.0013% (0.05 mM), yellow 0.05% (1.73 mM), blue: 0.1% (3.47 mM), cyan: 0.5% (17.35 mM)

Fig. 5

Molar mass as a function of oligomeric size and SDS:protein ratio in the complex. Diagonal lines: The stoichiometry (black: 0.5:1, red: 0.75:1, green: 1:1 and blue: 1.25:1) has very little influence on the molar mass. Calculated molar masses based on the hydrodynamic measurements of the complex as a function of partial specific volume and stoichiometry (black: 0.7265 ml/g, red: 0.7272 ml/g, green: 0.7280 ml/g, blue: 0.7287 ml/g). These lines intersect very close to the hexameric configuration, proving that the complex is hexameric

Fig. 6

700 MHz ¹H NMR spectra with water suppression of mt-II in phosphate buffer at 37, 22, and 10 °C and pH 7.9

Fig. 7

700 MHz ¹H NMR spectra with water suppression of mt-II with 0.2% (wt) SDS in phosphate buffer at 22 °C and pH 7.9

Fig. 8

Comparison between the 700 ¹H NMR spectra of mt-II in buffer (top), mt-II with 0.2% (wt) SDS in buffer (middle) and SDS in buffer (bottom) at 22 °C and pH 7.9 over the region containing the SDS signals

Fig. 9

NMR analysis of mt-II in the presence of 0.2% (wt) SDS at pH 7.9. A The T₁ values of mt-II in buffer at 22 °C; B The 2D ¹H DOSY spectra of mt-II in Buffer at 22 °C; C The T₁ values of mt-II with SDS in buffer at 22 °C; D The 2D ¹H DOSY spectra of mt-II with SDS in Buffer at 22 °C