title: Microtubule assembly-derived by dimerization of TPPP/p25. Evaluation of thermodynamic parameters for multiple equilibrium system from ITC data.
abstract: 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.
  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.
authors.affiliation: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, H-1113 Budapest, Hungary.
authors.forename: Judit
authors.initials: J
authors.lastname: Oláh
authors.forename: Ágnes
authors.initials: Á
authors.lastname: Zotter
authors.forename: Emma
authors.initials: E
authors.lastname: Hlavanda
authors.forename: Sándor
authors.initials: S
authors.lastname: Szunyogh
authors.forename: Ferenc
authors.initials: F
authors.lastname: Orosz
authors.forename: Krisztián
authors.initials: K
authors.lastname: Szigeti
authors.forename: Judit
authors.initials: J
authors.lastname: Fidy
authors.forename: Judit
authors.initials: J
authors.lastname: Ovádi
mesh_terms: D002151:Calorimetry; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003198:Computer Simulation; D003432:Cross-Linking Reagents / Q000494:pharmacology; D019281:Dimerization; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D008870:Microtubules / Q000378:metabolism*; D008962:Models, Theoretical; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics; D014404:Tubulin / Q000378:metabolism*
chemical_list: D003432:Cross-Linking Reagents; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; C118136:TPPP protein, human; D014404:Tubulin; D006160:Guanosine Triphosphate