Microtubule assembly-derived by dimerization of TPPP/p25. Evaluation of thermodynamic parameters for multiple equilibrium system from ITC data

Judit Oláh, Ágnes Zotter, Emma Hlavanda, Sándor Szunyogh, Ferenc Orosz, Krisztián Szigeti, Judit Fidy, Judit Ovádi

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10 Citations (Scopus)


Background: The disordered Tubulin Polymerization Promoting Protein/p25 (TPPP/p25) modulates the dynamics and stability of the microtubule system. In this paper the role of dimerization in its microtubule-related functions is established, and an approach is proposed to evaluate thermodynamic constants for multiple equilibrium systems from ITC measurements. Methods: For structural studies size exclusion chromatography, SDS-PAGE, chemical cross-linking, circular dichroism, fluorescence spectroscopy and isothermal titration calorimetry were used; the functional effect was analyzed by tubulin polymerization assay. Numerical simulation of the multiple equilibrium was performed with Mathematica software. Results: The dimerization of TPPP/p25 is promoted by elevation of the protein concentration and by GTP addition. The dimeric form displaying enhanced tubulin polymerization promoting activity is stabilized by disulfide bond or chemical cross-linking. The GTP binding to the dimeric form (Kd-GTP = 200 μM) is tighter with one order of magnitude than to the monomeric one leading to the enrichment of the dimers. A mathematical model elaborated for the multiple equilibrium of the TPPP/p25-GTP system was validated by fitting the GTP-dependent changes of ellipticity and fluorescence signal in the course of TPPP/p25 titrations. The evaluation of the equilibrium constants rendered it possible to determine the thermodynamic parameters of the association of different TPPP/p25 forms with GTP from ITC measurements. Conclusions/General Significance: The dimerization of TPPP/p25 with favorable physiological functional potency is proposed to play significant role in the fine tuning of TPPP/p25-mediated microtubule assembly; the unfolded monomers might be involved in the formation of pathological inclusions characteristic for Parkinson's disease and other synucleinopathies.

Original languageEnglish
Pages (from-to)785-794
Number of pages10
JournalBiochimica et Biophysica Acta - General Subjects
Issue number7
Publication statusPublished - Jul 1 2012


  • GTP binding
  • Mathematical modeling for multiple equilibrium
  • Oligomerization-dependent function
  • Tubulin Polymerization Promoting Protein/p25

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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