INVESTIGADORES
GONZALEZ FLECHA Francisco Luis
congresos y reuniones científicas
Título:
Free energy barriers for catalysis in the ATP binding domain of the thermophilic Cu(I) transport ATPase from Archaeoglobus fulgidus
Autor/es:
LURDES SABECKIS; SANTIAGO MARTINEZ; LUIS M. BREDESTON; ERNESTO A. ROMAN; MARIANO C. GONZÁLEZ LEBRERO; F. LUIS GONZÁLEZ FLECHA
Lugar:
Tucuman
Reunión:
Congreso; III Latin American Federation of Biophysical Societies (LAFeBS) Congress. IX IberoAmerican Congress of Biophysics. XLV Reunion Anual Sociedad Argentina de Biofisica.; 2016
Institución organizadora:
Sociedad Argentina de Biofisica
Resumen:
p { margin-bottom: 0.25cm; direction: ltr; font-variant: normal; color: rgb(0, 0, 0); line-height: 120%; text-align: left; page-break-inside: auto; widows: 2; orphans: 2; text-decoration: none; page-break-after: auto; }p.western { font-family: "Times New Roman",serif; font-size: 12pt; font-style: normal; font-weight: normal; }p.cjk { font-family: "Times New Roman",serif; font-size: 12pt; font-style: normal; font-weight: normal; }p.ctl { font-family: "Times New Roman",serif; font-size: 12pt; }The thermophilic Cu(I) transportATPase from Archaeoglobusfulgidus (Af-CopA)is an membrane protein that couples the energy from ATP hydrolysis tothe translocation of Cu(I) across plasma membranes. The ATPhydrolysis takes place within the globular domain of CopA(Af-CopA-ATPBD)which is located in the intracellular portion of the cell. It iscomposed by two subdomains: a nucleotide binding or N-domain and aphosphorylation or P -domain1.The aim of this work is to study andcharacterize the hydrolysis of ATP catalyzed by the A-fCopA-ATPBDusing a combined experimental and computational approach. For this purpose the ATPBD was cloned,expressed in E. Coliand purified by Ni-NTA chromatography. The ATPase activity wasmeasured as a function of ATP concentration determining kcatand kcat/kMat different temperatures. By fitting a Kramerstype model to the kineticcoefficients, it was obtained ∆G#,∆H#and ∆S#,following the analysis described in previous reports3.In addition, computational analysiswas performed using AMBER and a self-developed code named LIO2to optimize the selected quantum subsystem calculus. Crystalstructure of Af-ATPBD(PDB 3A1C) was used as initial structure. It was solvated with TIP3Pwater box, energy-minimized and equilibrated with classical moleculardynamics simulation protocol. Free energy calculations were performedusing Hybrid QM/MM methods beginning with different initialstructures, changing the protonation state of Asp424, or atoms of thesphere of Mg2+.ΔG#values for each coordinate were obtained using Jarzynski´s equality.Comparisons between experimental andtheorical kinetic parameters allow us to obtain valuable informationto better understand the catalytical mechanism of Af-ATPBDand to compare it with that corresponding to the complete membraneprotein3.