Electrochemical and structural properties of a protein system designed to generate tyrosine Pourbaix diagrams.
Academic Article
Overview
Research
Identity
Additional Document Info
Other
View All
Overview
abstract
This report describes a model protein specifically tailored to electrochemically study the reduction potential of protein tyrosine radicals as a function of pH. The model system is based on the 67-residue (3)Y three-helix bundle. (3)Y contains a single buried tyrosine at position 32 and displays structural properties inherent to a protein. The present report presents differential pulse voltammograms obtained from (3)Y at both acidic (pH 5.6) and alkaline (pH 8.3) conditions. The observed Faradaic response is uniquely associated with Y32, as shown by site-directed mutagenesis. This is the first time voltammetry is successfully applied to detect a redox-active tyrosine residing in a structured protein environment. Tyrosine is a proton-coupled electron-transfer cofactor making voltammetry-based pH titrations a central experimental approach. A second set of experiments was performed to demonstrate that pH-dependent studies can be conducted on the redox-active tyrosine without introducing large-scale structural changes in the protein scaffold. (3)Y was re-engineered with the specific aim to place the imidazole group of a histidine close to the Y32 phenol ring. (3)Y-K29H and (3)Y-K36H each contain a histidine residue whose protonation perturbs the fluorescence of Y32. We show that these variants are stable and well-folded proteins whose helical content, tertiary structure, solution aggregation state, and solvent-sequestered position of Y32 remain pH insensitive across a range of at least 3-4 pH units. These results confirm that the local environment of Y32 can be altered and the resulting radical site studied by voltammetry over a broad pH range without interference from long-range structural effects.
