Week 05 HW: Protein Design Part II

Part 1: Generate Binders with PepMLM The target is human SOD1 protein (UniProt P00441), focusing on A4V mutation, which is ALS.

Mutant SOD1 Sequence (A4V):

ATKVVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ

Using PepMLM-650M, four peptides of 12 amino acids were generated and compared against known SOD1-binding peptide FLYRWLPSRRGG.

PepMLM Confidence Scores Sequence Description Perplexity FLYRWLPSRRGG Real Binder — WHSPVVAVAHWE Sim 1 10.949699 WSVGWAAIAWWX Sim 2 16.027645 WRSYATAIALWK Sim 3 11.729657 WRYYATGAEWKE Sim 4 13.769973

Part 2: Evaluate Binders with AlphaFold3 Each peptide was modeled against the mutant SOD1 sequence using AlphaFold3 to assess structural docking and interface confidence (ipTM).

Structural Observations Localization: none of the peptides localized specifically to the A4V mutation site at the N-terminus. All peptides were primarily surface-bound. Binding Sites: Sim 3 (WRSYATAIALWK) and Sim 4 (WRYYATGAEWKE) both bound to a region that appears to be a potential polymerization site. Sim 2 (WSVGWAAIAWWX) engaged the β-barrel region. The wild-type binder also localized to a suspected polymerization site.

Eg. of AlphaFold3 Visualization

Part 3: Evaluate Properties in the PeptiVerse therapeutic potential (solubility, hemolysis, and affinity) of the generated sequences.

Peptide Property Comparison Peptide Sequence Solubility Hemolysis Affinity (pKd) MW (Da) Net Charge pI GRAVY ipTM pTM FLYRWLPSRRGG 1.000 0.047 5.96 (Weak) 1507.7 +2.76 11.71 -0.71 0.33 0.78 WHSPVVAVAHWE 1.000 0.048 5.07 (Weak) 1417.6 -1.06 6.02 0.18 0.28 0.78 WSVGWAAIAWWX 1.000 0.174 7.71 (Med) 1314.6 -0.24 5.53 0.78 0.37 0.76 WRSYATAIALWK 1.000 0.056 6.83 (Weak) 1465.7 +1.76 9.99 0.06 0.41 0.72 WRYYATGAEWKE 1.000 0.063 5.71 (Weak) 1559.7 -0.23 6.28 -1.44 0.31 0.86

Analysis of Results The observed ipTM values across the PepMLM-generated candidates range from 0.28 to 0.41, reflecting a low-to-moderate confidence in specific interface orientation. However, two PepMLM peptides outperformed the known binder (ipTM 0.33): WRSYATAIALWK (0.41) and WSVGWAAIAWWX (0.37).

There is a general correlation between structural confidence and affinity; the sequence with the highest affinity (WSVGWAAIAWWX, pKd: 7.71) also showed a strong ipTM. Interestingly, the highest ipTM - WRSYATAIALWK.

Selection Candidate: WRSYATAIALWK While WSVGWAAIAWWX has the highest raw affinity, WRSYATAIALWK offers a superior balance of properties. It provides the highest structural confidence (ipTM 0.41) while maintaining a much safer hemolysis profile (0.056) .

Part 4: Generate Optimized Peptides with moPPIt

moPPIt Generated Peptides Sequence Solubility Hemolysis Affinity MW (Da) Net Charge pI GRAVY GGTTTDDTKAES 1.000 0.054 4.20 1182.1 -2.24 4.05 -1.42 ATTGYCGCTMQN 1.000 0.018 5.39 1249.4 -0.22 5.55 -0.21 DEGYKKQKGQIQ 1.000 0.041 4.75 1421.6 +0.76 8.43 -2.23

Comparison and Evaluation The PepMLM peptides generally focus on high-confidence structural docking and balanced biophysical properties, leaning toward a safe profile while improving affinity over the baseline. In contrast, the moPPIt peptides prioritize diverse chemical spaces and targeted binding.

The moPPIt set introduces extreme charge variations, such as the highly acidic GGTTTDDTKAES (pI 4.05) and the polar-rich DEGYKKQKGQIQ, which differ significantly from the more hydrophobic PepMLM designs. To evaluate these before clinical studies, we would need to perform experimental circular dichroism (CD) to confirm peptide stability and surface plasmon resonance (SPR) to validate the targeted binding affinity at the specific A4V or dimer interface sites chosen during the moPPIt steering process.