Exploring amino-acid radical chemistry: protein engineering and de novo design

Abstract

Amino-acid radical enzymes are often highly complex structures containing multiple protein subunits and cofactors. These properties have in many cases hampered the detailed characterization of their amino-acid redox cofactors. To address this problem, a range of approaches has recently been developed in which a common strategy is to reduce the complexity of the radical-containing system. This work will be reviewed and it includes the light-induced generation of aromatic radicals in small-molecule and peptide systems. Natural redox proteins, including the blue copper protein azurin and a bacterial photosynthetic reaction center, have been engineered to introduce amino-acid radical chemistry. The redesign strategies to achieve this remarkable change in the properties of these proteins will be described. An additional approach to gain insights into the properties of amino-acid radicals is to synthesize de novo designed model proteins in which the redox chemistry of these species can be studied. Here we describe the design, synthesis and characteristics of monomeric three-helix bundle and four-helix bundle proteins designed to study the redox chemistry of tryptophan and tyrosine. This work demonstrates that de novo protein design combined with structural, electrochemical and quantum chemical analyses can provide detailed information on how the protein matrix tunes the thermodynamic properties of tryptophan.

Electrochemical and Structural Properties of a Protein System Designed To Generate Tyrosine Pourbaix Diagrams

“…These types of residues form the hydrophobic packing layers above, below and at the level of W32 in the α 3 W structure. 18,37 Depending on the pH, the α 3 Y-K29H aromatic ring-protons exhibit at least 12–26 additional inter-residue NOEs to aliphatic protons with resonances in the 1.40 to 4.17 ppm region (Figs. 6A and B).…”

“…Its folding is driven by the hydrophobic effect. 18,35,37 Its unfolding/folding transition is reversible and cooperative (e.g. Fig.…”

Decarboxylation involving a ferryl, propionate, and a tyrosyl group in a radical relay yields heme b

“…Four aromatic amino acids that could potentially harbor radical electron density (20) surround the pair of reactive propionates: Y145 (propionate 2), and W198, W157, and Y113 (propionate 4) (Fig. 1).…”

Formal Reduction Potential of 3,5-Difluorotyrosine in a Structured Protein: Insight into Multistep Radical Transfer

“…The α 3 X family of well-structured proteins was developed to address these concerns (10, 21). This model protein system yields thermodynamic information that is uncompromised by electrochemical irreversibility and radical side reactions (22-24).…”