Gabriel Rocklin
@grocklin.bsky.social
Digests proteins. High-throughput protein biophysics and design, Northwestern University. www.rocklinlab.org
Existing aggregation predictors show only modest correlations with our results, but fine tuning SaProt does well on our (carefully split) test set!
November 19, 2025 at 9:16 PM
Existing aggregation predictors show only modest correlations with our results, but fine tuning SaProt does well on our (carefully split) test set!
Arginine is strongly aggregation promoting! This was known in literature but not to me.
November 19, 2025 at 9:16 PM
Arginine is strongly aggregation promoting! This was known in literature but not to me.
Hydrophobicity and isoelectric point (pI) are correlated with temp- and pH-propensity, but don’t tell the whole story. Other factors also influenced aggregation.
November 19, 2025 at 9:16 PM
Hydrophobicity and isoelectric point (pI) are correlated with temp- and pH-propensity, but don’t tell the whole story. Other factors also influenced aggregation.
Really cool result: changes in soluble abundance from having all protein domains mixed together are nicely correlated with similar experiments on single, purified domains (also at 10 mg/mL).
November 19, 2025 at 9:16 PM
Really cool result: changes in soluble abundance from having all protein domains mixed together are nicely correlated with similar experiments on single, purified domains (also at 10 mg/mL).
Overall, 25-50% of the total protein became insoluble, but the proteomics revealed which proteins aggregated (became insoluble) more and which aggregated less.
November 19, 2025 at 9:16 PM
Overall, 25-50% of the total protein became insoluble, but the proteomics revealed which proteins aggregated (became insoluble) more and which aggregated less.
We expressed the protein domains together as a mixture in one E. coli culture, purified our mixture of domains and concentrated to 10mg/mL, then used TMT proteomics to monitor the change in soluble protein abundance after high temp/low pH.
November 19, 2025 at 9:16 PM
We expressed the protein domains together as a mixture in one E. coli culture, purified our mixture of domains and concentrated to 10mg/mL, then used TMT proteomics to monitor the change in soluble protein abundance after high temp/low pH.
New preprint! We measured temperature- and pH-induced aggregation for over 18,000 natural and de novo designed protein domains!
November 19, 2025 at 9:16 PM
New preprint! We measured temperature- and pH-induced aggregation for over 18,000 natural and de novo designed protein domains!
Even with this much data, we found it difficult to predict cooperativity in our domains. Maybe you can do better!! Still, we tried designing mutations that could increase the cooperativity of two of our low cooperativity domains. We found a few! (tho the effects were fairly small)
March 26, 2025 at 4:21 PM
Even with this much data, we found it difficult to predict cooperativity in our domains. Maybe you can do better!! Still, we tried designing mutations that could increase the cooperativity of two of our low cooperativity domains. We found a few! (tho the effects were fairly small)
But, the trend with stability was the opposite- the more compact domains actually had slightly lower global stability! We think this is because of a tradeoff: higher compactness promotes cooperativity, but forces greater alanine content and fewer large hydrophobics, decreasing stability.
March 26, 2025 at 4:21 PM
But, the trend with stability was the opposite- the more compact domains actually had slightly lower global stability! We think this is because of a tradeoff: higher compactness promotes cooperativity, but forces greater alanine content and fewer large hydrophobics, decreasing stability.
Different features of the domains influenced stability and cooperativity in different ways-and sometimes oppositely. For example, more compact helical bundles were more cooperative (more uniform stability across the full domain). Less compact domains had more fluctuations affecting some residues.
March 26, 2025 at 4:21 PM
Different features of the domains influenced stability and cooperativity in different ways-and sometimes oppositely. For example, more compact helical bundles were more cooperative (more uniform stability across the full domain). Less compact domains had more fluctuations affecting some residues.
We wanted to simplify these multidimensional measurements, so we defined a "cooperativity scale" quantifying the conformational fluctuations in each domain, compared to a "typical" domain with the same stability. Some domains have few fluctuations, some have many- we can see the whole landscape!
March 26, 2025 at 4:21 PM
We wanted to simplify these multidimensional measurements, so we defined a "cooperativity scale" quantifying the conformational fluctuations in each domain, compared to a "typical" domain with the same stability. Some domains have few fluctuations, some have many- we can see the whole landscape!
These differences in dynamics occur in natural domains as well! In these LysM domains, the dark blue domain has low stability helix 2, compared to the green domain which has ~uniform stability across all secondary structures.
March 26, 2025 at 4:21 PM
These differences in dynamics occur in natural domains as well! In these LysM domains, the dark blue domain has low stability helix 2, compared to the green domain which has ~uniform stability across all secondary structures.
By analyzing these proteins with HDX NMR, we found that the third helix in the blue domain is highly dynamic - it can "open up" (exchange H for D) even while the other two helices stay folded.
March 26, 2025 at 4:21 PM
By analyzing these proteins with HDX NMR, we found that the third helix in the blue domain is highly dynamic - it can "open up" (exchange H for D) even while the other two helices stay folded.
For example, these designed helical bundle domains have almost identical (predicted) structures, but they have very different distributions of stability in our assay. The blue protein has the highest stability residues overall, but the orange protein has more residues with stabiliy > 4 kcal/mol.
March 26, 2025 at 4:21 PM
For example, these designed helical bundle domains have almost identical (predicted) structures, but they have very different distributions of stability in our assay. The blue protein has the highest stability residues overall, but the orange protein has more residues with stabiliy > 4 kcal/mol.
These experiments reveal the distribution of stability (or "opening energy") for all the different residues in each domain. Some domains have relatively uniform stability for all residues. But other domains look very different- they have a mix of high and low stability residues.
March 26, 2025 at 4:21 PM
These experiments reveal the distribution of stability (or "opening energy") for all the different residues in each domain. Some domains have relatively uniform stability for all residues. But other domains look very different- they have a mix of high and low stability residues.
It's never been possible to analyze conformational dynamics in high-throughput, so we developed a new approach. We create customized "synthetic proteomes" of domains, then use hydrogen-deuterium exchange mass spec to measure the energies of fluctuations in all the domains at the same time!
March 26, 2025 at 4:21 PM
It's never been possible to analyze conformational dynamics in high-throughput, so we developed a new approach. We create customized "synthetic proteomes" of domains, then use hydrogen-deuterium exchange mass spec to measure the energies of fluctuations in all the domains at the same time!
Small proteins can be more complex than they look!
We know proteins fluctuate between different conformations- but by how much? How does it vary from protein to protein? Can highly stable domains have low stability segments? @ajrferrari.bsky.social experimentally tested >5,000 domains to find out!
We know proteins fluctuate between different conformations- but by how much? How does it vary from protein to protein? Can highly stable domains have low stability segments? @ajrferrari.bsky.social experimentally tested >5,000 domains to find out!
March 26, 2025 at 4:21 PM
Small proteins can be more complex than they look!
We know proteins fluctuate between different conformations- but by how much? How does it vary from protein to protein? Can highly stable domains have low stability segments? @ajrferrari.bsky.social experimentally tested >5,000 domains to find out!
We know proteins fluctuate between different conformations- but by how much? How does it vary from protein to protein? Can highly stable domains have low stability segments? @ajrferrari.bsky.social experimentally tested >5,000 domains to find out!
@standupforscience.bsky.social Science saves lives
March 7, 2025 at 7:47 PM
@standupforscience.bsky.social Science saves lives
Four planets over Lincoln Park 6pm
January 29, 2025 at 2:37 PM
Four planets over Lincoln Park 6pm
Then in comes Pauling…
December 31, 2024 at 3:54 AM
Then in comes Pauling…