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

SANTIAGO MARTINEZ; ERNESTO A. ROMAN; F. LUIS GONZÁLEZ FLECHA; MARIANO C. GONZÁLEZ LEBRERO

Tucuman

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

Sociedad Argentina de Biofisica

p { margin-bottom: 0.25cm; direction: ltr; color: rgb(0, 0, 0); line-height: 120%; widows: 2; orphans: 2; }p.western { font-family: "Times New Roman",serif; font-size: 12pt; }p.cjk { font-family: "Times New Roman",serif; font-size: 12pt; }p.ctl { font-family: "Times New Roman",serif; font-size: 12pt; }The copper transport ATPase from Archaeoglobus fulgidus is anintegral membrane protein composed of one transmembrane and fourcytoplasmic domains: the catalytic domain (ATPBD), an actuatordomain, and two metal binding domains. The catalytic domain isresponsible for the enzymatic activity a 75 °C, and includes twosub-domains; one of them binds the nucleotide and the other isinvolved in the catalysis.Theaim of these studies is to characterize the folding mechanism of theATPBD. For this purpose unfoldingand refolding experiments where performed, at equilibriumand time resolved conditions using urea as achaotropic agent.Equilibrium circular dichroism (CD) signals at far UV and theintrinsic fluorescent signal of the unique tryptophan were registeredas function of the urea concentrations. Our results show that theunfolding reaction is reversible and at least one intermediate stateis involved in the folding and mechanism of the ATPBD. Kineticstudies of the unfolding and refolding of the ATPBD were performed byrecording the intrinsic fluorescent signal as a function of time atdifferent urea concentrations. Each time course could be described bya single exponential function of time, obtaining an observed kineticcoefficient ( kobs.). The variations of kobs as function of ureaconcentration also indicate the existence of at least one foldingintermediary. Additionallyunfolding and refolding experiments where performed at differenttemperatures under both equilibrium and time resolved conditions.This allowed us to characterize the free energy barriers involved inof the folding mechanism of the ATPBD. The enthalpic and entropiccomponents of these barriers were determined by fitting a Kramerstype model to the experimental data.