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Updated: Jan 30, 2026

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
Published on: March 16, 2011
Fine Tuning of Functional Features of the CuA Site by Loop-Directed Mutagenesis
Ulises A Zitare1, Jonathan Szuster1, María C Santalla1
1Departamento de Química Inorgánica, Analítica y Química Física, Facultad de Ciencias Exactas y Naturales , Instituto de Química Física de los Materiales, Medio Ambiente y Energía (INQUIMAE) , Universidad de Buenos Aires and CONICET, 1428 Buenos Aires , Argentina.
Abstract:
Here we report the spectroscopic and electrochemical characterization of three novel chimeric CuA proteins in which either one or the three loops surrounding the metal ions in the Thermus thermophilus protein have been replaced by homologous human and plant sequences while preserving the set of coordinating amino acids. These conservative modifications mimic basic differences between CuA sites from different organisms and allow for fine tuning the energy gap between alternative electronic ground states of CuA.. This results in a systematic modulation of thermodynamic and kinetic electron transfer (ET) parameters and in the selection of one of two possible redox-active molecular orbitals, which differ in the ET reorganization energy by a factor of 2. Moreover, the ET mechanism is found to be frictionally controlled, and the modifications introduced into the different chimeras do not affect the frictional activation parameter.
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