Synthesis of Multi-Protein Complexes through Charge-Directed Sequential Activation of Tyrosine Residues

Abstract

Site-selective protein−protein coupling has long been a goal of chemical biology research. In recent years, that goal has been realized to varying degrees through a number of techniques, including the use of tyrosinase-based coupling strategies. Early publications utilizing tyrosinase from Agaricus bisporus(abTYR) showed the potential to convert tyrosine residues into ortho-quinone functional groups, but this enzyme is challenging to produce recombinantly and suffers from some limitations in substrate scope. Initial screens of several tyrosinase candidates revealed that the tyrosinase from Bacillus megaterium (megaTYR) is an enzyme that possesses a broad substrate tolerance. We use the expanded substrate preference as a starting point for protein design experiments and show that single point mutants of megaTYR are capable of activating tyrosine residues in various sequence contexts. We leverage this new tool to enable the construction of protein trimers via a charge-directed sequential activation of tyrosine residues (CDSAT).

Evidence for H-bonding interactions to the μ-η2:η2-peroxide of oxy-tyrosinase that activate its coupled binuclear copper site

“…Finally, in addition to catechol oxidation and monophenol monooxygenation, NspF also catalyzes the monooxygenation of o-aminophenols to o-nitrosophenols in bacterial siderophore biosynthesis. 10 Given the major implications of CBC proteins in both human diseases 11 and critical applications in agriculture (fruit browning 12 ) and biotechnology (protein bioconjugation 13 ), obtaining a mechanistic understanding of their diverse reactivities has been a major goal with broad scientific interest. However, putative differences in the electronic/geometric structures of their Cu(II) 2 O 2 active sites as well as the molecular-level factors that control their reactivity are not fully understood despite extensive research efforts.…”

Chemical and Enzymatic Methods for Post-Translational Protein–Protein Conjugation

“…Later work discovered that the ortho -quinone functional group generated upon tyrosine oxidation undergoes a reaction with the sulfhydryl group of a free cysteine residue. , This approach was used to prepare conjugates of sfGFP with three different proteins: CRISPR-Cas9, a HER2-binding scFv, and nanoluciferase . Further work explored the use of different tyrosinases to expand the scope of tyrosine residues that could be targeted in this manner . A tyrosinase from Bacillus megaterium (megaTYR) was found to be more promiscuous and enabled the oxidation of tyrosine residues in a high number of sequence motifs, as assayed by peptide experiments.…”

“…In traditional bioconjugation reactions between a protein and a small molecule, a common strategy is to use a high stoichiometric excess of the latter in order to increase reaction velocity. This is necessary because proteins are generally present in low concentrations (1–100 μM) and are also large in size, rendering them sterically encumbered coupling partners. , However, in the case of protein–protein coupling reactions, it is generally not practical to use a larger stoichiometric excess of one partner. The protein–protein coupling problem arises because two of these sterically encumbered coupling partners, both present at low concentrations, must come together to form the desired protein–protein conjugate.…”

Putting precision and elegance in enzyme immobilisation with bio-orthogonal chemistry

“…15). 170,178,179 Tyrosinase from Agaricus bisporus (abTYR), catalysed the oxidation of tyrosine to o-quinone intermediates which coupled with N-terminal proline residues in high yield. This coupling strategy was successfully used for the attachment of a variety of phenols to a series of protein substrates, including selfassembled viral capsids, enzymes, and a chitin binding domain (CBD).…”