tailieunhanh - Báo cáo khoa học: Dynamics, stability and iron-binding activity of frataxin clinical mutants

Friedreich’s ataxia results from a deficiency in the mitochondrial protein frataxin, which carries single point mutations in some patients. In the pres-ent study, we analysed the consequences of different disease-related muta-tions in vitro on the stability and dynamics of human frataxin. | ễFEBS Journal Dynamics stability and iron-binding activity of frataxin clinical mutants Ana R. Correia1 Chiara Pastore2 Salvatore Adinolfi2 Annalisa Pastore2 and Claudio M. Gomes1 1 Institute Tecnologia Quimica e Biologica Universidade Nova de Lisboa Oeiras Portugal 2 Nationallnstitute for MedicalResearch MedicalResearch Council London UK Keywords clinicalmutants frataxin Friedreich s ataxia protein dynamics and flexibility protein folding Correspondence C. M. Gomes Instituto Tecnologia Quimica e Biologica Universidade Nova de Lisboa Avenida da Republica 127 2780-756 Oeiras Portugal Fax 351 2144 11277 Tel 351 2144 69332 E-mail gomes@ A. Pastore National institute for Medical Research The Ridgeway Mill Hill London NW7 1AA Tel 44 2088 162629 E-mail apastor@ These authors contributed equally to this work Received 17 April2008 accepted 19 May 2008 doi Friedreich s ataxia results from a deficiency in the mitochondrial protein frataxin which carries single point mutations in some patients. In the present study we analysed the consequences of different disease-related mutations in vitro on the stability and dynamics of human frataxin. Two of the mutations G130V and D122Y were investigated for the first time. Analysis by CD spectroscopy demonstrated a substantial decrease in the thermodynamic stability of the variants during chemical and thermal unfolding wild-type W155R I154F D122Y G130V which was reversible in all cases. Protein dynamics was studied in detail and revealed that the mutants have distinct propensities towards aggregation. It was observed that the mutants have increased correlation times and different relative ratios between soluble and insoluble aggregated protein. NMR showed that the clinical mutants retained a compact and relatively rigid globular core despite their decreased stabilities. Limited proteolysis assays coupled with LC-MS allowed the identification of particularly flexible regions in .