Related Experiment Video
Updated: Aug 23, 2026
Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD
Published on: December 30, 2016
Structure of the azurin mutant Phe114Ala from Pseudomonas aeruginosa at 2.6 A resolution
1Department of Inorganic Chemistry, Chalmers University of Technology and The University of Göteborg, Sweden.
Abstract:
The crystal structure of azurin mutant Phe114Ala from Pseudomonas aeruginosa has been solved by molecular replacement. The final crystallographic R value is 0.185 for 9832 reflections to a resolution of 2.6 A. The root-mean-square deviation for main-chain atom positions is 0.020 A between the four independent monomers in the asymmetric unit. The mutant Ala114 crystallized from PEG 4000 in a new crystal form and the crystals are monoclinic, P2(1), a= 51.0, b = 83.6, c= 66.4 A and beta = 110.5 degrees. The four molecules in the asymmetric unit are packed as a dimer of dimers and are related by an approximate twofold axis. The dimer packing and the dimer contact region are very similar to that of the Alcaligenes denitrificans azurin dimer. The mutation was performed at residue Phe114, which exhibits a pi-electron overlap with the copper ligand His117, to investigate its suggested role in the electron self-exchange reaction. Removal of steric constrains from the phenylalanine side chain created a somewhat different geometry around the copper site with an increased mobility of His117 resulting in an enlarged Cu-N length which may be responsible for the slight differences obtained in the spectral properties of the mutant versus the wild-type protein.
Insights
This study determined the crystal structure of a Pseudomonas aeruginosa azurin mutant (Phe114Ala). The mutation altered copper site geometry, affecting electron transfer properties.
Area of Science:
- Biochemistry
- Structural Biology
- Crystallography
Background:
- Azurin is a small blue copper protein involved in electron transfer.
- The Phe114 residue in Pseudomonas aeruginosa azurin potentially influences electron self-exchange via pi-electron overlap with the copper ligand His117.
Purpose of the Study:
- To elucidate the structural and functional impact of mutating phenylalanine at position 114 to alanine (Phe114Ala) in Pseudomonas aeruginosa azurin.
- To investigate the role of Phe114 in the protein's electron self-exchange mechanism.
Main Methods:
- X-ray crystallography was employed to solve the crystal structure of the Phe114Ala azurin mutant.
- Molecular replacement was used for structure determination.
- The crystal structure was refined to a resolution of 2.6 Å.
Main Results:
- The crystal structure of Pseudomonas aeruginosa azurin Phe114Ala mutant was determined.
- The mutant crystallized in a monoclinic P2(1) space group, forming a dimer of dimers.
- The mutation resulted in altered copper site geometry, including increased mobility of His117 and an enlarged Cu-N bond length.
Conclusions:
- The removal of steric constraints at Phe114 affects the copper site's geometry and the mobility of the histidine ligand.
- These structural changes may explain observed differences in spectral properties between the mutant and wild-type azurin.
- The study provides insights into the structural basis of electron transfer in azurin proteins.
