4. Expanding new-to-nature reactions beyond heme proteins
4.7 Adaptation of PsEFE for high-throughput screening
set up co-crystallization experiments, in which PsEFE VMM, NOG, manganese chloride, and either the sulfonyl azide or sultam were incubated with the protein, followed by centrifugal filtration to remove any precipitated protein. The purified protein solution was used to set up drop-volume ratio crystallization refinement screens with streak-seeding, and crystals grew with the same clustered needle morphology over the same time frame. These crystals were cryoprotected with PEG 400 in place of ethylene glycol in the attempt to prevent ethylene glycol binding in the active site. While ethylene glycol was no longer found in the active site, there was also no electron density supporting the binding of either the sulfonyl azide or sultam product. The lid loop also remained open in the structures collected from crystals in the co-crystallization attempts. It is possible that the crystallization conditions prevent the lid-loop region from closing, though similar conditions are used by Schofield to crystallize PsEFE WT. It is also possible that the substrate or product binding do not induce the protein to adopt the closed conformation, or that the co-crystallization conditions were not conducive to substrate or product binding.
To investigate whether the lid loop still closes, nitrene-transfer reactions using PsEFE variants could be performed in the presence of different amounts of L-Arg. This would provide insight into whether L-Arg binding has been impacted in the evolved variants. If L- Arg inhibits PsEFE nitrene-transfer activity, co-crystallization of PsEFE VMM with L-Arg would show whether the lid closing mechanism has been disrupted and whether L-Arg’s binding mode has been affected by the mutants as rationalized above.
clear that expanding the diversity of protein variants with activity for new-to-nature reactions has led to the discovery of starting points for new reactions for which the wild-type protein exhibited no activity.42
Looking to new PsEFE library generation, I noted that many active-site residues have already been targeted for mutagenesis during the evolution of the aziridination enzyme variant PsEFE MLHMM, but only 7% of the sites within PsEFE have been targeted. Expanding the sequence diversity targeted in future rounds of evolution will improve the probability of finding beneficial mutations. Using the tile-based mutagenesis strategy laid out in Chapter 5, I designed and ordered a mutagenesis library consisting of a total of 127 amino-acid sites targeted over three tiles in PsEFE VMM (Figure 4-27). The tiles include almost all active- site residues (excluding residues whose side chains comprise the metal’s primary coordination sphere), the flexible lid-loop region, and the C-terminus, which is highly flexible based on crystallographic B-factors and could contribute to the protein’s instability.
The goal of this library is to expand the sequence breadth targeted in the next rounds of PsEFE evolution on new-to-nature reactions and the identification of residues which stabilize or generally activate the PsEFE scaffold. The tiles were also chosen such that three additional tiles covering complementary sequence space could be ordered and used to generate libraries targeting most of the remaining sequence space.
Figure 4-27. PsEFE VMM 127-site-saturation library. Green regions show amino-acid residues targeted for site-saturation mutagenesis in the Twist library for PsEFE VMM.
With any large library design, it is important to have a screening method to effectively search the library’s sequence space for the desired fitness enhancements. As protein stability is a potential issue for future PsEFE engineering, developing a rapid screen for protein expression and stability would enable the enrichment of stable PsEFE variants, which could then be screened for new-to-nature activity. Unlike heme proteins, which can be quantified in lysate via carbon monoxide binding assays,50 spectrophotometric assays developed for αKG-dependent enzymes typically rely on succinate formation. As my initial goal was to assay protein expression and stability, it was only necessary to determine whether the full- length protein is present and soluble. To this end, I employed a split-fluorescent protein system, in which the protein of interest is tagged with a short (16 amino acid) fragment of a fluorescent protein. When the tagged protein of interest and the complementary fluorescent
protein fragment are co-expressed, the two fluorescent protein fragments self-assemble, and the protein’s fluorescence is enhanced. This approach was developed and used to assay soluble protein expression51 and has more recently been used to normalize enzyme concentrations in biocatalytic reactions to enable separately analyzing protein expression and specific activity.52
mNeonGreen2(1-10) (mNG2(1-10), complemented by mNG2(11)) was chosen for the fluorescent protein system as it was engineered for decreased background fluorescence in the absence of the complementary 11th β-strand, and was readily available through Addgene.53 The construct from Addgene expressed mNG2(1-10) under IPTG induction control; to allow for orthogonal induction control, I subcloned the mNG2(1-10) encoding gene into the pBAD33 vector under arabinose induction control. Separately, pET22b constructs containing IPTG-inducible PsEFE VMM with and without C-terminal polyhistidine tags were C-terminally tagged with mNG2(11). The orthogonal induction allows for the independent expression optimization of both proteins. In cases when the fluorescence output is not needed, the cellular metabolic load can be reduced by only expressing the protein of interest. The arabinose induction is tightly regulated and further repressed under high glucose concentrations, leading to minimal expression of mNG2(1-10) without addition of arabinose.
In cases when the stability of the fluorescent protein is greater than that of a protein to which it is fused, the stability of the protein of interest can be assayed by a heat challenge followed be centrifugation. The denaturation and aggregation of the protein of interest pulls the soluble fluorescent protein into the pellet.54 This approach was feasible with our fluorescent system as mNG2(1-10) linked to mNG2(11) is stable at 60 °C for over 1 hour. Lysate from cells co- expressing PsEFE VMM-His6-mNG2(11) showed fluorescence, but following incubation in a water bath at 55 °C for one hour and centrifugation, the supernate fluorescence was depleted (Figure 4-28). The fluorescent protein was still folded and stable, as the pellet was brightly fluorescent.
Figure 4-28. Split-fluorescent protein labeling enables measurement of PsEFE folding and aggregation. Fluorescence intensity is proportional to the protein of interest in the solution; as PsEFE VMM is denatured and aggregates below 55 °C, the associated split-fluorescent protein pellets out with the aggregated PsEFE VMM to reduce supernate fluorescence.
This fluorescent stability assay is amenable to laboratory automation. With automated colony picking and 96-tip head liquid handlers, each liquid transfer can be performed robotically, increasing throughput potential. With the addition of single-tip liquid handling, variants with improved stability by this screen can be robotically re-arrayed into 96-well plates and assayed for an activity of interest.