Reversible unfolding of the catalytic domain of the Cu(I) transport ATPase from the hyperthermophilic Archaeoglobus fulgidus

S. MARTINEZ GACHE; EA. ROMAN; RM. GONZÁLEZ LEBRERO; FL. GONZÁLEZ FLECHA

San Miguel de Tucuman

Congreso; III Latin American Federation of Biophysical Societies (LAFeBS) IX IberoAmerican Congress of Biophysics. XLV Reunion Anual SAB 2016.; 2016

P { margin-bottom: 0.21cm; }The copper transportATPase from Archaeoglobus fulgidus is an integral membrane proteincomposed of seven transmembrane segments and four globular domains:the catalytic (ATPBD), an actuator and two metal binding domains. TheATPBD is responsible for the hydrolysis of ATP which occurs with amaximum activity at 70 ºC. This domain is composed by twosub-domains (N and P); the N-subdomain binds the nucleotidic part ofATP and the P-subdomain is phosphorylated in D424 during the reactioncycle. The aim of this work isto characterize the folding mechanism of the ATPBD. For this purposeunfolding and refolding experiments were performed at pre-equilibriumand equilibrium conditions using urea as a denaturing agent. In equilibrium conditionscircular dichroism (CD) signals at far UV and the intrinsicfluorescent signal of the unique tryptophan were registered asfunction of the urea concentrations. Unfolding plots obtained for CDand intrinsic fluorescence differ one from each other, indicatingdifferent unfolding paths for the reversible unfolding of the ATPBD.As consecuence it is impossible to postulate a two state mechanismfor these process. Additionally, time courses of unfolding andrefolding of ATPBD, by measuring tryptophan fluorescence as afunction of time were obtained in the presence of different ureaconcentration. Even though, the experimental data could be describedby a single exponential function of time, the dependence of thekinetic coefficients (kobs) with the urea concentrationindicated the existence of, at least, one intermediate state.Finally, equilibrium and time courses experiments were performed atdifferent temperatures. This results, were analysed according toKramer?s theory and thermodynamic activation parameters (∆G?,∆H? y ∆S?) were obtained.