Our crystal structure validated the designed fold, confirming that the lid was correctly folded.

However, a subtle 1.8 Å lid shift disrupted a key catalytic contact, likely contributing to the modest activity. But structural analysis reveals paths to improve activity in the next round of design!

One of our designs, KempTIM4, showed catalytic efficiency comparable to many first-round de novo Kemp eliminases generated by traditional methods.

Using CANVAS, we designed a structural lid onto a minimal, de novo TIM barrel to anchor catalytic residues and create an active site for the Kemp elimination reaction.

TIM barrels are among nature’s most powerful enzyme scaffolds but making them from scratch with catalytic function has been a challenge.

Enter CANVAS: a computational pipeline combining Triad, RFdiffusion & ProteinMPNN to customize minimal TIM barrels into functional enzymes.

Molecular dynamics simulations showed that distal mutations enhance active-site accessibility—either by loosening loops covering the active site or widening bottlenecks for substrate entry & product exit. The enzyme breathes more efficiently! 🌬️ (5/6)

Kinetic solvent viscosity effects & stopped-flow experiments showed that distal mutations don’t just tweak structure—they accelerate substrate binding & product release. (4/6)

Crystal structures showed that active-site mutations pre-organize the catalytic machinery. But distal mutations? They subtly tune conformational dynamics—enhancing productive substates & reshaping the energy landscape of the catalytic cycle. (3/6)

We engineered "Core" and "Shell" variants of three evolved Kemp eliminases to dissect the effects of active-site vs. distal mutations. Core mutations dramatically boosted catalysis. Shell mutations alone? Not much—until they worked together in evolved enzymes. 🔍 (2/6)

Crystal structures revealed that distal mutations trigger large-scale conformational shifts in an active-site loop, making the active site more open. Interestingly, these mutations aren't located on the loop—making their effects hard to predict!

We studied a computationally designed & evolved retro-aldolase, creating two variants:
🔹 RA95-Core – Active-site mutations only
🔹 RA95-Shell – Distal mutations only
Comparing them to the evolved RA95.5-8F revealed surprising insights!

Protein Engineering Canada Conference.
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Inspiration for an excellent beer!

Save the date!
The 5th Protein Engineering Canada Conference will take place at the University of Toronto on June 26-28, 2024.

event.fourwaves.com/pec2024

Save the date!
The 5th Protein Engineering Canada Conference will take place at the University of Toronto on June 26-28, 2024.

For more info:
event.fourwaves.com/pec2024

We show that our enzyme design approach outperforms other protocols because the crystallographic data underpinning our backbone ensembles provide the highest-fidelity insight into the true conformational ensemble of the enzyme, including accurate representation of catalytically-competent substates.

Here, we use conformational ensembles generated from dynamics-based refinement against X-ray diffraction data to design artificial enzymes with efficiencies comparable to those obtained after several rounds of directed evolution.