Manganous ion binding to tubulin

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Tubulin self-assembly into microtubules is essential for cellular functions.
• Microtubule assembly is typically regulated by microtubule-associated proteins and divalent cations.
• Understanding the role of specific cations in tubulin polymerization is crucial for cell biology.

Purpose of the Study:

• To investigate the interaction of manganese (Mn(II)) with purified tubulin.
• To determine the mechanism by which Mn(II) induces tubulin assembly.
• To characterize the binding sites and affinity of Mn(II) on tubulin.

Main Methods:

• Phosphocellulose chromatography for tubulin purification.
• Electron paramagnetic resonance (EPR) spectroscopy to study Mn(II) binding.
• Atomic absorption spectroscopy to quantify metal ion interactions.

Main Results:

• Mn(II) induces the assembly of pure tubulin into temperature-sensitive microtubules.
• Tubulin binds Mn(II) at one high-affinity site (KD = 1.6 ± 0.3 μM) and 8 ± 2 low-affinity sites (KD = 0.38 ± 0.18 mM).
• Mn(II) binding displaces Mg(II) from tubulin, indicating substitution at the high-affinity site.

Conclusions:

• Mn(II) acts as a substitute for Mg(II) in promoting tubulin assembly.
• The binding of Mn(II) to tubulin is specific, with other divalent cations like Co(II) and Zn(II) able to displace it, but not Ca(II).
• EPR spectral analysis suggests the bound Mn(II) at the high-affinity site is accessible to the solvent.