The solution structure of a gallium-substituted putidaredoxin mutant: GaPdx C85S

Area of Science:

• Biochemistry
• Structural Biology
• Protein Chemistry

Background:

• Ferredoxins (Pdx) are iron-sulfur proteins crucial for electron transfer.
• The native Fe2S2 cluster in Pdx can be substituted with a single gallium ion.
• This substitution results in a colorless, diamagnetic protein with conserved major structural features.

Purpose of the Study:

• To determine the solution structure of a variant gallium putidaredoxin (C85S GaPdx).
• To investigate the structural and dynamic consequences of replacing the Fe2S2 cluster with gallium.
• To compare the structural dynamics of GaPdx with native Pdx.

Main Methods:

• Multidimensional NMR spectroscopy was employed on uniformly 15N, 13C labeled C85S GaPdx.
• Stereospecific assignments of leucine and valine methyl resonances were achieved using fractional 13C labeling.
• Backbone dihedral angle restraints were derived from 2D and 3D NMR experiments, alongside 1117 NOE-derived distance restraints and angular restraints.

Main Results:

• The solution structure of C85S GaPdx was determined, revealing well-defined regions with a backbone RMSD of 0.525 A (excluding specific loops).
• Increased dynamic behavior was observed in the metal binding loop (residues 34-48), the C-terminus (residues 103-106), and loop 74-76 compared to native Pdx.
• Structural and dynamic differences are discussed in the context of altered charge and packing within the metal binding site.

Conclusions:

• Gallium substitution in Pdx preserves overall protein structure but introduces localized dynamic changes.
• The study provides insights into the role of the metal binding site's charge and packing on protein dynamics.
• NMR methods effectively characterized the structure and dynamics of the GaPdx variant.