Title: Introduction of an Artificial Cu Binding Site at the Surface of CA II: Pitfalls of Rational Design Finally Scooped by Serendipity
Volume: 5
Issue: 1
Author(s): Johannes Schulze Wischeler, Andreas Heine and Gerhard Klebe
Affiliation:
Keywords:
Carbonic anhydrase II, copper-ion, cysteine, metal-center, protein crystallography, site-directed mutagenesis, Artificial Cu, Rational Design, zinc metalloenzyme, catalyzes, hydration, carbon dioxide, catalytic reaction, protons, crystallographic study, density, binding pocket, Zn2+ ion, active site, equilibrium rate, hypothesis, Huisgen reaction, triazole, binding site, novel residues, artificial metal, high-affinity metal, helix-helix interactions, amino acids, target protein, enzyme, histidines, cysteine residue, trigonal planar geometry, axial position, mutagenesis, artificial Cu2+, carbonyl groups, stoichiometry, isothermal titration calorimetry, protein monomers, metal chelation, Design of Mutants, Structural Investigation of CA II, thiole group, high flexibility, trigonal-planar coordination, Pseudomonas aeruginosa, linear coordination site, disulfide bond, wild-type structures, melting temperature, X-ray crystallography, p-chloromercuribenzoic acid, anaerobic conditions, Cambridge Structural Database, disulfide bridge, Stedium Biotech, Cloning, Healthcare, glutathione-S-transferase, Lysis, inhibitor cocktail tablet, Protein Purification, biohybrid materials
Abstract: The aim of this study was the introduction of an artificial copper center at the surface of carbonic anhydrase II (CA II) which subsequently might be used by azide and alkyne building blocks to catalyze triazole formation following 2+3 dipolar cycloaddition. Several mutants were produced to introduce favorable copper coordinating residues taking model copper-proteins from nature as reference templates. Unfortunately, the embarked rational design strategy remained unsuccessful which underlines that our still rather limited understanding of amino acid exchanges on the architecture of proteins might provoke unpredictable effects while tampering with complex biological systems such as a highly functionalized proteins. Finally, more by serendipity than design the formation of an artificial Cu center at the surface of CA II could be achieved. The metal ion is coordinated by two histidines and a serine residue in square planar coordination geometry.
