Week 5 HW: Protein Design Part II

🔎 Part 1: Generate Binders with PepMLM

Sequence with mutation: MATKVVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGCTS AGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGRTLVV HEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ

![[htgaa-hw5-pt1-binders.png]]

In text, the generated ones (plus the one provided to compare) were:

0	WRYYAAALRHKX	9.957530
1	WRYYAVAARHKK	13.948671
2	WRSYVVVLELGX	18.185468
3	HHYPAVAVALKX	7.987091
4	FLYRWLPSRRGG	20.635231

AlphaFold however was throwing errors with these sequences, so I asked Claude to check: ![[Screenshot 2026-03-08 at 17.05.41.png]]

So I changed them to these:

• WRYYAAALRHKG
• WRYYAVAARHKK
• WRSYVVVLELGG
• HHYPAVAVALKG
• FLYRWLPSRRGG

🔎 PeptiVerse results

🦠 Peptide #1: WRYYAAALRHKG

AlphaFold:

• ipTM = 0.38
• pTM = 0.8 ![[Screenshot 2026-03-08 at 18.17.58.png]]

![[Screenshot 2026-03-08 at 19.08.57.png]]

➡️ Record the ipTM score and briefly describe where the peptide appears to bind. Does it localize near the N-terminus where A4V sits? Does it engage the β-barrel region or approach the dimer interface? Does it appear surface-bound or partially buried?

ipTM = 0.38 is low confidence of binding. It would be surface-bound and far from the beta-barrel region.

PeptiVerse:

🦠 Number #2 WRYYAVAARHKK

Alphafold:

• ipTM = 0.31
• pTM = 0.75 ![[Screenshot 2026-03-08 at 18.19.06.png]]

➡️ Record the ipTM score and briefly describe where the peptide appears to bind. Does it localize near the N-terminus where A4V sits? Does it engage the β-barrel region or approach the dimer interface? Does it appear surface-bound or partially buried? Surface-bound, relatively close to the mutation site. It does not engage the beta barrel site. iPTM = 0.31 which shows low confidence.

PeptiVerse:

🦠 Number #3 WRSYVVVLELGG

Alphafold:

• ipTM = 0.75
• pTM = 0.86

![[Screenshot 2026-03-08 at 18.48.53.png]] ➡️ Record the ipTM score and briefly describe where the peptide appears to bind. Does it localize near the N-terminus where A4V sits? Does it engage the β-barrel region or approach the dimer interface? Does it appear surface-bound or partially buried? This one is bound on the surface. ipTM = 0.75 which is somewhat confident (we treat a score between 70 and 90 as “confident”). It is somewhat close to the beta-barrel area. It isn’t too far from the mutation area, but also not super close.

![[Screenshot 2026-03-08 at 18.54.30.png]] PeptiVerse:

🦠 Number 4: HHYPAVAVALKG

Alphafold:

• ipTM = 0.29
• pTM = 0.86 ![[Screenshot 2026-03-08 at 18.15.35.png]]

➡️ Record the ipTM score and briefly describe where the peptide appears to bind. Does it localize near the N-terminus where A4V sits? Does it engage the β-barrel region or approach the dimer interface? Does it appear surface-bound or partially buried?

ipTM = 0.29 which is very low confidence of binding. No, it’s not close to the mutation site. It’s on the opposite side of there the beta-barrel sits, so no. It is surface-bound and closest to the positions 86-87.

PeptiVerse:

🦠 Number 5: FLYRWLPSRRGG

Alphafold:

• ipTM = 0.35
• pTM = 0.8

![[Screenshot 2026-03-08 at 18.20.14.png]]

➡️ Record the ipTM score and briefly describe where the peptide appears to bind. Does it localize near the N-terminus where A4V sits? Does it engage the β-barrel region or approach the dimer interface? Does it appear surface-bound or partially buried?

ipTM = 0.35, hence confidence of binding is low. It’s far from the N-terminus that has the A4V. It is surface-bound. It is close to the beta-barrel region (the arrows).

PeptiVerse:

🔎 Part 1 analysis

1. ➡️ In a short paragraph, describe the ipTM values you observe and whether any PepMLM-generated peptide matches or exceeds the known binder. Yes, WRSYVVVLELGG has a higher iPTM (0.75 vs 0.35) plus a lower perplexity score (18 vs 20), even though it has the highest perplexity score out of the other generated ones. In general, the iPTM values for all of them except this one are low (~0.30), meaning that AlphaFold is not very confident about their vinding affinity.

2. ➡️ In a short paragraph, describe what you see. Do peptides with higher ipTM also show stronger predicted affinity? Are any strong binders predicted to be hemolytic or poorly soluble? Which peptide best balances predicted binding and therapeutic properties? ![[Screenshot 2026-03-08 at 19.22.45.png]]

   WRSYVVVLELGG scores the highest iPTM from AlphaFold and the highest binding affinity according to PeptiVerse. The scores are correlated in both platforms for all peptides. Solubility is around 1.0 for all of them, so none are poorly soluble.

   All show hemolytic probabilities under 0.1 besides one, which is the strongest binder. The strongest binder is also one of the only two with a positive GRAVY score, showing the strongest hydrophobicity. (I learned that “Hydrophobic peptides often damage membranes”, so this would not be a good candidate). Therefore the binder with the strongest affinity also shows the highest toxicity and aggregation risk.

   Peptide #1 WRYYAAALRHKG shows the lowest hemolysis score, but its binding affinity is not too strong, so it would not make for a strong candidate, but it has a good safety profile.

   Peptide #2 has weak binding affinity and a high charge, so we discard it. (Why: “High overall positive charge is usually bad for a peptide drug candidate because it increases nonspecific binding to negatively charged membranes and biomolecules, which can drive toxicity, hemolysis, and off‑target effects”)

3. ➡️ Choose one peptide you would advance and justify your decision briefly. I would choose peptide #1 because it has the second highest binding affinity (despite still being moderately low) but a good safety profile.