Thermodynamic and kinetic characterization of a psichrophile frataxin variant

ERNESTO A. ROMAN

Santiago de Chile

Simposio; Biophysics at the End of the World; 2016

Universidad de Chile

p { margin-bottom: 0.25cm; direction: ltr; color: rgb(0, 0, 0); line-height: 120%; text-align: left; }p.western { font-family: "Arial",sans-serif; font-size: 11pt; }p.cjk { font-family: "Arial",sans-serif; font-size: 11pt; }p.ctl { font-family: "Arial",sans-serif; font-size: 10pt; }Here,we will study the stabilization mechanisms and dynamics of frataxinscaffold using a variant from Pyschromonasingrahamii asa model (pFXN). We previously reported that pFXN stability is highlymodulated by pH in the range 6-8 (Roman EA at al. Biochim. Biophys.Acta, 2013). Here, we will discuss our results on the thermodynamicand kinetic stability of pFXN by rapid mixing experiments. Foldingand unfolding dependence on pH behave differently leading to twoapparent pKa for native and unfolded states. Models with singleprotonation sites for the native state were discarded. This impliesthat at least different group of residues or different conformationsexists in the native state. Apparent pKa for native and unfoldedstates around 8.4 and 5.0, respectively. This suggests that a strongshift in the pKa of a residue/s upon folding is associated with thepH stabilization. In addition, temperature equilibrium chemicalunfolding experiments at different pHs showed a bell-shapeddependence of the Gibbs Free Energy between 4 and 26 Celsius degrees.The thermodynamic analysis suggests that the increase in stabilitycomes mainly from a change in the entropy energy term. This may beexplained by residual structure in the unfolded state, localunfolding in the native state, but also from difference in thesolvation shells due to charge states in both ensembles. Constant pHsimulations showed that besides histidine residues there are someglutamic acid residues located in the main loop that have their pKashifted towards pH 6-7. This result is in agreement with the factthat His point mutants are not able to revert the stabilitydependence on pH. Preliminary NMR results show that a complex networkof hydrogen bonded interactions are strongly regulated by pH in inthe range between 6 and 8.