Direct Measurement of the Nanomechanical Stability of a Redox Protein Active Site and Its Dependence upon Metal Binding

• Published in: The journal of physical chemistry. B 2015-09, Vol.119 (36), p.12050-12058
• Main Authors: Giannotti, Marina I, Cabeza de Vaca, Israel, Artés, Juan M, Sanz, Fausto, Guallar, Victor, Gorostiza, Pau
• Format: Article
• Language: English
• Subjects: Apoproteins - chemistry; Apoproteins - metabolism; Azurin; Azurin - chemistry; Azurin - metabolism; Binding; Catalytic Domain; Computer simulation; Copper; Copper - metabolism; Copper - pharmacology; Cupredoxins; Enginyeria mecànica; Force spectroscopy; Impacte ambiental; Mechanical Phenomena; Models, Molecular; Nanomechanical stability; Nanostructure; Nanotechnology; Oxidation-Reduction; Physical chemistry; Protein; Protein Binding; Protein Conformation; Protein Stability - drug effects; Protein Unfolding - drug effects; Proteins; Proteïnes; Pseudomonas aeruginosa; Residues; Single molecule; Stability; Àrees temàtiques de la UPC
• ISSN: 1520-6106; 1520-5207
• DOI: 10.1021/acs.jpcb.5b06382

The structural basis of the low reorganization energy of cupredoxins has long been debated. These proteins reconcile a conformationally heterogeneous and exposed metal-chelating site with the highly rigid copper center required for efficient electron transfer. Here we combine single-molecule mechanical unfolding experiments with statistical analysis and computer simulations to show that the metal-binding region of apo-azurin is mechanically flexible and that high mechanical stability is imparted by copper binding. The unfolding pathway of the metal site depends on the pulling residue and suggests that partial unfolding of the metal-binding site could be facilitated by the physical interaction with certain regions of the redox protein.