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Roberto Chica Lab @chicalab.bsky.social · Sep 30

Thrilled to share that our latest article is now out in final form! A great collaboration with @fraserlab.com and @silviaosuna.bsky.social.

Distal mutations enhance catalysis in designed enzymes by facilitating substrate binding and product release

www.nature.com/articles/s41...

Distal mutations enhance catalysis in designed enzymes by facilitating substrate binding and product release - Nature Communications

Distal mutations, though far from the active site, enhance Kemp eliminase catalysis by tuning conformational dynamics that facilitate substrate binding and product release, thereby promoting the full catalytic cycle.

www.nature.com

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Roberto Chica Lab @chicalab.bsky.social · Aug 26

Our latest article is now published online! In collaboration with @thompson-lab.bsky.social, Marc Garcia-Borràs, and @ferranfeixas.bsky.social.

Distal Mutations in a Designed Retro-Aldolase Alter Loop Dynamics to Shift and Accelerate the Rate-Limiting Step

pubs.acs.org/doi/full/10....

Distal Mutations in a Designed Retro-Aldolase Alter Loop Dynamics to Shift and Accelerate the Rate-Limiting Step

Amino acid residues distant from an enzyme’s active site are known to influence catalysis, but their mechanistic contributions to the catalytic cycle remain poorly understood. Here, we investigate the...

pubs.acs.org

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Roberto Chica Lab @chicalab.bsky.social · Jul 31

Similarly, enzyme function can be designed de novo by creating a new active site within a natural protein scaffold that lacks the target activity, even if that catalytic function exists in nature.

See below for an early example:

www.pnas.org/doi/full/10....

Enzyme-like proteins by computational design | PNAS

We report the development and initial experimental validation of a computational design procedure aimed at generating enzyme-like protein catalys...

www.pnas.org

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Roberto Chica Lab @chicalab.bsky.social · Jul 31

No, I don’t think that’s necessarily implied. For example, a TIM barrel can be designed from scratch without referencing any specific natural sequence or structure, even if this fold exists in nature. I consider this de novo design. See example below:

www.nature.com/articles/nch...

De novo design of a four-fold symmetric TIM-barrel protein with atomic-level accuracy - Nature Chemical Biology

Despite substantial effort, the de novo design of a stable TIM-barrel protein fold has remained elusive. A Rosetta-based computational strategy identifies a unique 184-residue sequence that adopts a T...

www.nature.com

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Roberto Chica Lab @chicalab.bsky.social · Jul 31

The design and creation of a protein sequence, structure or function from scratch, rather than modifying a pre-existing sequence, structure or function.

An early pioneer of this field is Bill DeGrado, see below.

www.science.org/doi/10.1126/...

Characterization of a Helical Protein Designed from First Principles

The question of how the primary amino acid sequence of a protein determines its three-dimensional structure is still unanswered. One approach to this problem involves the de novo design of model pepti...

www.science.org

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Roberto Chica Lab @chicalab.bsky.social · Jul 29

Overall, our study:
✅ Introduces a new strategy to transform minimal protein scaffolds into biocatalysts
✅ Provides mechanistic insights from crystallography & molecular dynamics
✅ Opens the door to designing custom lids for more complex reactions, which we’re now exploring

Thanks for reading! 🧵🧬

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Roberto Chica Lab @chicalab.bsky.social · Jul 29

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!

The crystal structure (blue) aligns closely with the design model (minimal TIM barrel and lid colored white and magenta, respectively).

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Roberto Chica Lab @chicalab.bsky.social · Jul 29

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

Michaelis-Menten plot of KempTIM4 showing saturation kinetics.

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Roberto Chica Lab @chicalab.bsky.social · Jul 29

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.

Building a custom lid onto a minimal, de novo TIM barrel using CANVAS.

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Roberto Chica Lab @chicalab.bsky.social · Jul 29

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.

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Roberto Chica Lab @chicalab.bsky.social · Jul 29

In collaboration with @birtehoecker.bsky.social, we’ve unlocked enzymatic activity in a minimal de novo TIM barrel by designing a custom lid for catalysis! 🧵👇
#ProteinDesign #EnzymeDesign

Customizing the Structure of a Minimal TIM Barrel to Create a De Novo Enzyme
www.biorxiv.org/content/10.1...

Customizing the Structure of a Minimal TIM Barrel to Craft a De Novo Enzyme

The TIM barrel is the most prevalent fold in natural enzymes, supporting efficient catalysis of diverse chemical reactions. While de novo TIM barrels have been successfully designed, their minimalisti...

www.biorxiv.org

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Roberto Chica Lab @chicalab.bsky.social · Jul 25

Congratulations! Looking forward to seeing all the exciting science that will come out of your lab! 🧪

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Reposted by Roberto Chica Lab

Stephan Hammer @stephanhammer.bsky.social · Jun 27

Join us! We are looking for a new team member (PhD student) with strong background in organic chemistry.
🙏 RETWEET (We want to recruit internationally)

Organic chemistry meets #DirectedEvolution
Highly interdisciplinary & passionate research group

uni-bielefeld.hr4you.org/job/view/433...

Research Position (PhD) in organic chemistry and b...

<div style="text-align: justify;">The  research  group  „Organic  Chemistry  and&nb...

uni-bielefeld.hr4you.org

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Reposted by Roberto Chica Lab

Robert E. Campbell @campbell-lab.bsky.social · May 15

Protein Engineering, Design & Selection (PEDS) invites contributions to a Special Collection titled, “Non-Canonical Amino Acids", with guest editors Prof. Huiwang Ai (Virginia) and Prof. Peng Chen (Peking). Send us your best work!
academic.oup.com/peds/pages/c...

Non-canonical amino acid from PDB ID 8W3Z shown chelating to a magnesium ion. Image made with PyMol.
https://www.rcsb.org/structure/8W3Z
https://www.pymol.org

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Reposted by Roberto Chica Lab

Nick Polizzi @nickpolizzi.bsky.social · Apr 28

Super excited to share a new preprint from our lab on design of small-molecule binding proteins using neural networks! The paper has a bit of everything. A new graph neural network, new design algorithms, and experimental validation. www.biorxiv.org/content/10.1...
🧵🧪

Zero-shot design of drug-binding proteins via neural selection-expansion

Computational design of molecular recognition remains challenging despite advances in deep learning. The design of proteins that bind to small molecules has been particularly difficult because it requ...

www.biorxiv.org

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Reposted by Roberto Chica Lab

Protein Society @proteinsociety.bsky.social · Mar 6

Guess what? TPS has extended the deadline to March 19 to submit abstracts for poster presentations and speaking opportunities at our 39th Annual Symposium. Join us in San Francisco June 26 - 29 for 3.5 days of scientific talks.
hashtag#proteinscience hashtag#annualsymposium
lnkd.in/g7VKqX7C

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Roberto Chica Lab @chicalab.bsky.social · Feb 28

The take-home message? Distal residues actively shape enzyme catalysis. Optimizing them can remove bottlenecks in substrate binding & product release—boosting activity. Want to dive deeper? Read our full study here: www.biorxiv.org/content/10.1...
(6/6)

Distal mutations enhance catalysis in designed enzymes by facilitating substrate binding and product release

The role of amino-acid residues distant from an enzyme's active site in facilitating the complete catalytic cycle—including substrate binding, chemical transformation, and product release—remains poor...

www.biorxiv.org

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Roberto Chica Lab @chicalab.bsky.social · Feb 28

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)

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Roberto Chica Lab @chicalab.bsky.social · Feb 28

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

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Roberto Chica Lab @chicalab.bsky.social · Feb 28

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)

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Roberto Chica Lab @chicalab.bsky.social · Feb 28

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)

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Roberto Chica Lab @chicalab.bsky.social · Feb 28

How do mutations far from an enzyme's active site influence catalysis? 🤔

Part 2: In collaboration with @fraserlab.bsky.social and @silviaosuna.bsky.social, we investigated this question using de novo Kemp eliminases, revealing effects of distal mutations on the catalytic cycle. 🧵 (1/6)

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Roberto Chica Lab @chicalab.bsky.social · Feb 28

Thank you for the feedback!

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Reposted by Roberto Chica Lab

Diego del Alamo @delalamo.xyz · Feb 28

Whereas beneficial active site mutations to enzymes often improve the chemical transformation itself by preorganizing the active site, mutations to second-shell residues instead tune steps like product release by modifying the broader conformational ensemble www.biorxiv.org/content/10.1...

Figure from https://www.biorxiv.org/content/10.1101/2025.02.21.639315v1.full.pdf showing how mutations to the active site (labeled "core" relative to the parental, which is labeled "designed"), to second-shell residues (labeled "shell"), or both (labeled "evolved") affect the conformational dynamics of a de novo designed kemp eliminase.

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Roberto Chica Lab @chicalab.bsky.social · Feb 27

Together, our findings reveal how distal mutations sculpt enzyme function by reshaping the catalytic cycle for more efficient catalysis.

What does this mean for enzyme design? Find out in our preprint: www.biorxiv.org/content/10.1...
#Enzymology #Biophysics #Catalysis #EnzymeDesign

Distal mutations in a designed retro-aldolase alter loop dynamics to shift and accelerate the rate-limiting step

Amino-acid residues distant from an enzyme’s active site are known to influence catalysis, but their mechanistic contributions to the catalytic cycle remain poorly understood. Here, we investigate the...

www.biorxiv.org

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