Accessing to Helix/coil transition events involved in the catalytic cycle of E. coli thiol peroxidase

DIEGO SEBASTIAN VAZQUEZ; AGUDELO, WILLIAM A.; FERRER-SUETA, GERARDO; JAVIER SANTOS

Santa Fe

Workshop; III Taller de Resonancia Magnética NMR and EPR at the Forefront of Research; 2016

Universidad Nacional del Litoral

Peroxiredoxins (PRDX) are ubiquitous enzymes that reduce different kind of peroxides via two catalytic cysteines [1]. All PRDX shows conformational changes during the catalytic cycle restricted to the region that contain the cysteines. The active fully folded (FF) conformation of the E. coli thiol peroxidase (EcTPX) requires a key peroxidatic cysteine (CP, located in the αH2). First, CP is oxidized to sulfenic acid and then, after acritically local unfolding step (LU), C P forms a disulfide bond with the resolving cysteine (CR, located in the αH3, see Figure 1AB) [2]. It is clear that the energetic cost to unfold both helix, has a direct impact to the catalytic ratio of substrate reduction, but the contribution has not been clarified yet. In this study, we performed a microsecond time-scale common molecular dynamic simulations of the WT and 3 point-mutants of αH3 to glycine in order to destabilize the helix and a conformational searching profile though several potential-biased accelerated molecular dynamic simulations (aMD) followed by essential mode analysis, in an attempt to determine the energetic cost of the FF→LU event; and the experimental determination of the ΔΔGN→U of unfolding. Preliminary results suggest that: I) the destabilization of the αH3 promote the local unfolding of αH3 in two of the three mutants but not in the wild-type EcTPX, II) The aMD simulations capture the FF→LU transition in the WT protein characterized by an intermediate with rotameric and secondary structure changes that involve αH2 and αH3.