Week 5 HW: Protein Design Part 2

Part A: SOD1 Binder Peptide Design (From Pranam)

Part 1: Generate Binders with PepMLM

Begin by retrieving the human SOD1 sequence from UniProt (P00441) and introducing the A4V mutation.
Using the PepMLM Colab linked from the HuggingFace PepMLM-650M model card:
Generate four peptides of length 12 amino acids conditioned on the mutant SOD1 sequence.
To your generated list, add the known SOD1-binding peptide FLYRWLPSRRGG for comparison.
Record the perplexity scores that indicate PepMLM’s confidence in the binders
Generated Peptides Generated Peptides

Part 2: Evaluate Binders with AlphaFold3

Navigate to the AlphaFold Server: alphafoldserver.com
For each peptide, submit the mutant SOD1 sequence followed by the peptide sequence as separate chains to model the protein-peptide complex.
known peptide known peptide
unknown peptide unknown peptide
unknown peptide unknown peptide
unknown peptide unknown peptide
unknown peptide unknown peptide

Part 3: Evaluate Properties of Generated Peptides in the PeptiVerse

  1. Paste the peptide sequence.
  2. Paste the A4V mutant SOD1 sequence in the target field.
  3. Check the boxes
  4. Predicted binding affinity
  5. Solubility
  6. Hemolysis probability
  7. Net charge (pH 7)
  8. Molecular weight

propertied of peptides propertied of peptides
propertied of peptides propertied of peptides
propertied of peptides propertied of peptides
propertied of peptides propertied of peptides
propertied of peptides propertied of peptides

Part 4: Generate Optimized Peptides with moPPIt

generating generating

Sample 1: FAPYWPCCNPCR
Hemolysis: 0.0384 | Solubility: 1.0000 | Affinity: 7.6799 | Motif: 0.6357

Sample 2: YCTDCVDGVVWE
Hemolysis: 0.0898 | Solubility: 0.9530 | Affinity: 7.3664 | Motif: 0.5257

Sample 3: TRKPHYAAFFIY
Hemolysis: 0.0115 | Solubility: 1.0000 | Affinity: 6.8142 | Motif: 0.6964

Sample 4: PCKYVPHVHVCF
Hemolysis: 0.0348 | Solubility: 1.0000 | Affinity: 6.7769 | Motif: 0.6278

Sample 5: GFFVKTFEIVMF
Hemolysis: 0.0313 | Solubility: 1.0000 | Affinity: 6.5842 | Motif: 0.6023

Sample 6: AFVTRELVVQIW
Hemolysis: 0.0775 | Solubility: 0.9980 | Affinity: 6.4754 | Motif: 0.7743

Sample 7: HELTFARFEIQL
Hemolysis: 0.0169 | Solubility: 1.0000 | Affinity: 6.3272 | Motif: 0.7435

Sample 8: QEPCEELQFNHF
Hemolysis: 0.0245 | Solubility: 1.0000 | Affinity: 6.2640 | Motif: 0.6353

Sample 9: CTKVLIVKFEFK
Hemolysis: 0.0224 | Solubility: 1.0000 | Affinity: 6.0939 | Motif: 0.7347

Sample 10: PSEKQCVKFHTT
Hemolysis: 0.0481 | Solubility: 1.0000 | Affinity: 5.8624 | Motif: 0.7204

Sample 11: ANAPWFPPSSPH
Hemolysis: 0.0167 | Solubility: 1.0000 | Affinity: 5.6936 | Motif: 0.6189

Sample 12: AFAKISNKQQQT
Hemolysis: 0.1067 | Solubility: 1.0000 | Affinity: 5.5742 | Motif: 0.7846

Part C: Final Project: L-Protein Mutants

L-Protein Engineering | Option 3: Random Mutagenesis

  1. Create a python function to generate random mutation combinations with at least 2 residues by using the information found in mutational analysis experiments here.

Variant 1: S35K, Q71L
Sequence: METRFPQQSQQTPASTNRRRPFKHEDYPCRRQQRKSTLYVLIFLAIFLSKFTNQLLLSLLEAVIRTVTTLLQLLT

Variant 2: F47I, L44D
Sequence: METRFPQQSQQTPASTNRRRPFKHEDYPCRRQQRSSTLYVLIFDAIILSKFTNQLLLSLLEAVIRTVTTLQQLLT

Variant 3: V63I, V67I
Sequence: METRFPQQSQQTPASTNRRRPFKHEDYPCRRQQRSSTLYVLIFLAIFLSKFTNQLLLSLLEAIIRTITTLQQLLT

Variant 4: R31K, F43P
Sequence: METRFPQQSQQTPASTNRRRPFKHEDYPCRKQQRSSTLYVLIPLAIFLSKFTNQLLLSLLEAVIRTVTTLQQLLT

Variant 5: F5N, L60C
Sequence: METRNPQQSQQTPASTNRRRPFKHEDYPCRRQQRSSTLYVLIFLAIFLSKFTNQLLLSLCEAVIRTVTTLQQLLT

  1. Co-fold the random mutation with DnaJ using Af2_Multimer.

    Variant 2 pAE variant 2 pAE variant 2 pAE

    Structure figure figure