The solution structure of a gallium-substituted putidaredoxin mutant: GaPdx C85S.
The Fe2S2 cluster of the ferredoxin putidaredoxin (Pdx) can be replaced by a single gallium ion, giving rise to a colorless, diamagnetic protein in which, apart from the metal binding site, the major structural features of the native ferredoxin are conserved. The solution structure of the C85S variant of gallium putidaredoxin (C85S GaPdx), in which a non-ligand cysteine is replaced by a serine, has been determined via multidimensional NMR methods using uniformly 15N, 13C labeled samples of C85S GaPdx. Stereospecific assignments of leucine and valine methyl resonances were made using 13C, 1H HSQC spectra obtained with fractionally 13C-labeled samples, and backbone dihedral angle restraints were obtained using a combination of two-dimensional J-modulated 15N, 1H HSQC and three-dimensional (HN)CO(CO)NH experiments. A total of 1117 NOE-derived distance restraints were used in the calculations, including 454 short range (i-j < or = 3), 456 long range (i-j > or = 4) interresidue restraints and 207 non-trivial intraresidue restraints. 97 phi and 55 chi 1 angular restraints were also included in the calculation of a family of 20 structures using a combined distance geometry-simulated annealing protocol. Most regions of the protein are well defined in the calculations, with an RMSD of 0.525 A for backbone atoms excluding the metal binding loop (residues 34-48) and the last three C-terminal residues (residues 103-106). Where comparison is possible, these regions show an increase in dynamic behavior over the native protein, as does the loop containing residues 74-76. Structural and dynamic differences between native Pdx and GaPdx are discussed in relation to charge and packing of the metal binding site.
Pochapsky
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Department of Chemistry, Brandeis University, Waltham, MA 02254-9110, USA.
Kuti
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Kazanis
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D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D005288:Ferredoxins / Q000737:chemistry* / Q000235:genetics; D005708:Gallium; D007501:Iron / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012996:Solutions
D001426:Bacterial Proteins; D005288:Ferredoxins; D012996:Solutions; C100137:putidaredoxin; D005708:Gallium; D007501:Iron