[SCAN] Directed evolution for tweaking with protein unfolding pathways

Scan Seminar

Title: Directed evolution for tweaking with protein unfolding pathways: improving thermostability of PpAzoR, an azoreductase from Pseudomonas putida

Speaker: Vânia Brissos

From: Microbial & Enzyme Technology Lab, ITQB

Abstract:

Protein stability arises from a combination of factors which are often difficult to rationalise. Therefore its improvement is better addressed through directed evolution than by rational design approaches. PpAzoR is a FMN-dependent NADPH:quinone/dye oxidoreductase from Pseudomonas putida MET94, showing broad substrate specificity from natural or synthetic quinones to azo synthetic dyes. However its low thermal stability with a half life of 13 min at 50°C decreases its potential for applications. Five rounds of mutagenesis/recombination followed by high-throughput screening (≈ 10,000 clones) yielded a variant enzyme showing 300-fold higher half life at 50°C than that of homodimeric PpAzoR. The characterization using fluorescence, calorimetry and light scattering shows that this variant has a folded state slightly less stable than the wild type (with lower melting and optimal temperatures) but in contrast is more resistant to irreversible denaturation. Its superior kinetic stability was therefore related to an increased resistance of the unfolded monomers to aggregation through the introduction of mutations that disturbed hydrophobic patches and increased the surface net charge of the protein. Variants with increased thermodynamic stability (10 to 20°C higher melting temperature than wild type) were also examined showing the distinctive nature of mutations that lead to improved structural robustness: these occur in residues that are mostly involved in strengthening the solvent-exposed loops or the inter-dimer interactions of the folded state. Overall, this work suggests that protein charge can be exploited to impart robust resistance to protein aggregation with implications on de novo protein design efforts, where unpredictable protein properties, including aggregation, remain a significant challenge. From the point of view of protein evolvability mutations that prevent the aggregation of the unfolded state, increasing thus the level of soluble protein, contribute to protein fitness, and therefore variant 1B6 is potentially a good candidate to be evolved for new functions of PpAzoR.