tailieunhanh - báo cáo khoa hoc : Cu(I)- and proton-binding properties of the first N-terminal soluble domain of Bacillus subtilis CopA

CopA, a P-type ATPase transporter involved in copper detoxification in Bacillus subtilis, contains two soluble Atx1-like domains separated by a short linker at its N-terminus, an arrangement that occurs widely in copper transporters from both prokaryotes and eukaryotes. Both domains were previously found to bind Cu(I) with very high affinity. | IFEBS Journal Cu I - and proton-binding properties of the first N-terminal soluble domain of Bacillus subtilis CopA Liang Zhou Chloe Singleton Oliver Hecht Geoffrey R. Moore and Nick E. Le Brun Centre for Molecular and StructuralBiochemistry Schoolof Chemistry University of East Anglia Norwich UK Keywords Bacillus subtilis copper exchange copper trafficking cysteine thiol P-type ATPase Correspondence N. E. Le Brun School of Chemistry University of East Anglia Norwich NR4 7TJ UK Fax 44 1603 592003 Tel 44 1603 592699 E-mail Received 1 September 2011 revised 25 October 2011 accepted 7 November 2011 doi CopA a P-type ATPase transporter involved in copper detoxification in Bacillus subtilis contains two soluble Atx1-like domains separated by a short linker at its N-terminus an arrangement that occurs widely in copper transporters from both prokaryotes and eukaryotes. Both domains were previously found to bind Cu I with very high affinity. Above a level of 1 Cu I per CopAab dimerization occurred leading to a highly luminescent multinuclear Cu I species Singleton C Le Brun NE 2009 Dalton Trans 688-696 . To try to understand the contributions of each domain to the complex Cu I -binding behaviour of this and related proteins we purified a wild-type form of the first domain CopAa . In isolation the domain bound Cu I with very high affinity K 1 X 1018 M 1 and underwent Cu I -mediated protein association resulting in a mixture of dimer and tetramer species. Addition of further Cu I up to 1 Cu I per CopAa monomer led to a weakly luminescent species whereas further additions 2 Cu I per CopAa monomer resulted in protein unfolding. Analysis of the MTCAAC binding motif Cys residue acid-base properties revealed pKa values of and consistent with the pH dependence of Cu I binding and with the proposal that low proton affinity is associated with high Cu I affinity. Finally Cu I exchange between CopAa and the chelator .