Protein Science, Vol 6, Issue 2 383-390, Copyright © 1997 by Cold Spring Harbor Laboratory Press

ARTICLE

Complete (1)H, (13)C, and (15)N NMR resonance assignments and secondary structure of human glutaredoxin in the fully reduced form

C. SUN, A. HOLMGREN and J. H. BUSHWELLER
Department of Chemistry, Dartmouth College, Hanover, New Hampshire 03755

Human glutaredoxin is a member of the glutaredoxin family, which is characterized by a glutathione binding site and a redox-active dithiol/disulfide in the active site. Unlike Escherichia coli glutaredoxin-1, this protein has additional cysteine residues that have been suggested to play a regulatory role in its activity. Human glutaredoxin (106 amino acid residues, M(r) = 12,000) has been purified from a pET expression vector with both uniform (15)N labeling and (13)C/(15)N double labeling. The combination of three-dimensional (15)N-edited TOCSY, (15)N-edited NOESY, HNCA, HN(CO)CA, and gradient sensitivity-enhanced HNCACB and HNCO spectra were used to obtain sequential assignments for residues 2-106 of the protein. The gradient-enhanced version of the HCCH-TOCSY pulse sequence and HCCH-COSY were used to obtain side chain (1)H and (13)C 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 helices extending approximately from residues 4-10, 24-36, 53-64, 83-92, and 94-104. The secondary structure also shows four {beta}-strands comprised of residues 15-19, 43-48, 71-75, 78-80, which form a {beta}-sheet almost identical to that found in E. coli glutaredoxin-1. Complete (1)H, (13)C, and (15)N assignments and the secondary structure of fully reduced human glutaredoxin are presented. Comparison to the structures of other glutaredoxins is presented and differences in the secondary structure elements are discussed.
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