COMPLETE H-1, C-13, AND N-15 NMR RESONANCE ASSIGNMENTS AND SECONDARY STRUCTURE OF HUMAN GLUTAREDOXIN IN THE FULLY REDUCED FORM

Human glutaredoxin is a member of the glutaredoxin family, which is ch aracterized by a glutathione binding site and a redox-active dithiol/d isulfide in the active site. Unlike Escherichia coli glutaredoxin-1, t his protein has additional cysteine residues that have been suggested to play a regulatory role in its activity. Human glutaredoxin (106 ami no acid residues, M(r) = 12,000) has been purified from a pET expressi on vector with both uniform N-15 labeling and C-13/N-15 double labelin g. The combination of three-dimensional N-15-edited TOCSY, N-15-edited NOESY, HNCA, HN(CO)CA, and gradient sensitivity-enhanced HNCACB and H NCO spectra were used to obtain sequential assignments for residues 2- 106 of the protein. The gradient-enhanced version of the HCCH-TOCSY pu lse sequence and HCCH-COSY were used to obtain side chain H-1 and C-13 assignments. The secondary structural elements in the reduced protein were identified based on NOE information, amide proton exchange data, and chemical shift index data. Human glutaredoxin contains five helic es extending approximately from residues 4-10, 24-36, 53-64, 83-92, an d 94-104. The secondary structure also shows four beta-strands compris ed of residues 15-19, 43-48, 71-75, 78-80, which form a beta-sheet alm ost identical to that found in E. coli glutaredoxin-1. Complete H-1, C -13, and N-15 assignments and the secondary structure of fully reduced human glutaredoxin are presented. Comparison to the structures of oth er glutaredoxins is presented and differences in the secondary structu re elements are discussed.