Past Results

2019

Damry AM et al. (2019) Communications Biology 2, 433

Here, we study a key question in protein biophysics: how do novel modes of conformational exchange (i.e. dynamics) arise in globular proteins. To do so, we investigated the contribution of individual mutations in DANCER-3, a pentamutant of streptococcal protein G domain β1 (Gβ1), which we previously designed to spontaneously switch between two conformations that have not been previously observed for this fold. Using a combination of solution NMR and molecular dynamics simulations, we demonstrate that only two of the five mutations are necessary to create this conformational exchange, and that these mutations work synergistically when introduced into the wild-type background, with one destabilizing the native Gβ1 structure and the other allowing two new conformational states to be accessed on the energy landscape. Based on these results, we propose a biophysical model for the molecular evolution of conformational dynamics in globular proteins in which a permissive mutation that silently reshapes unexplored regions of the protein energy landscape could spontaneously emerge, followed by a destabilizing mutation that would give rise to a new dynamic process by making the native state less stable than the alternate conformations now made accessible by the permissive mutation.

2019

St-Jacques AD et al. (2019) ACS Catalysis 9, 5480-5485

Here, we developed a multistate computational protein design methodology named multichemical state analysis (MCSA), which can be used to optimize enzyme sequences for productive binding of multiple target substrates. We used MCSA to redesign the active site of branched-chain amino acid aminotransferase to accept both α-ketoglutarate and the non-native substrate L-histidine. Screening of a designed combinatorial library comprising 32 mutants for enhanced L-histidine transamination activity yielded four variants displaying up to ≈200-fold improvements to kcat/KM with the non-native substrate (12.5% hit rate), a library size several orders of magnitude smaller than was required to obtain similar activity enhancements in other aminotransferases with a single round of directed evolution. MCSA opens the door to the rational design of broad-specificity biocatalysts and multi-substrate enzymes displaying tailored specificity.

2019

Parmeggiani F et al. (2019) ACS Catalysis 9, 3482–3486.

In this collaborative project with the group of Nicholas Turner (U. Manchester), we used rational design to engineer the first reported aminotransferase displaying native-like catalytic activity towards D-tryptophan (kcat/KM = 700 M-1 s-1). This engineered variant enabled us to develop a one-pot biocatalytic process that combines asymmetric synthesis of substituted L-tryptophans from indoles by tryptophan synthase (TrpS), with a stereoinversion cascade based on L-amino acid deaminase (LAAD) and our engineered aminotransferase (DAAT). We show that this biocatalytic process can be used for the synthesis of 12 D-tryptophan derivatives containing electron-donating or withdrawing substituents at all benzene-ring positions on the indole group, with high conversion rate (84% to >99%) and enantiomeric excess (91% to >99%) starting from commercially-available materials. We also demonstrate that our process is applicable to preparative-scale synthesis of all 12 D-tryptophans (isolated yields of 63% to 79%), many of which are highly-valuable building blocks of pharmaceuticals and natural products.

2017

Davey et al. (2017) Nature Chemical Biology 13, 1280-1285.

This manuscript presents the first demonstration of the rational design of proteins that can spontaneously exchange between two predefined conformational states in the absence of an external stimulus, and on a timescale relevant to function. To achieve this result, we developed a broadly-applicable computational method to engineer protein dynamics that we term meta-multistate design (meta-MSD). We used meta-MSD to design spontaneous exchange between two novel conformations introduced into the global fold of protein G domain β1 (Gβ1). The designed proteins, named DANCERs, for Dynamic And Native Conformational ExchangeRs, are stably folded and exchange between predicted conformational states on the millisecond timescale. Our new method for design of conformational exchange paves the way to the design of proteins with a more versatile range of functions than was previously possible, such as those that must adopt more than one conformational state (e.g., enzymes, molecular rotors, biosensors).

2016

Pandelieva AT et al. (2016) ACS Chemical Biology 11, 508-517.

2016/09/01. We recently used rational design to increase the quantum yield and brightness of red fluorescent proteins (RFPs) by rigidifying their chromophore via the creation of a triple-decker motif of aromatic rings (see figure below). The best mutant identified displayed an over three-fold improvement relative to the parent RFP and was isolated following the screening of only 48 mutants, a library size that is several orders of magnitude smaller than those previously used to achieve equivalent gains in quantum yield in other RFPs.