Week 5

L-Protein Mutants Project Summary:

@2026a-nourelden-rihan

The MS2 bacteriophage L-protein is a small 75-residue lysis protein with two functional regions: a soluble N-terminal domain (residues 1–40) that interacts with the bacterial chaperone DnaJ, and a transmembrane domain (residues 41–75) that disrupts the inner membrane to trigger host cell lysis. The goal of this project was to computationally design five single-point mutants with the potential to retain or enhance lysis activity. This was done by first running a Clustal Omega Multiple Sequence Alignment to map out conserved positions across homologs, all of which turned out to cluster exclusively in the soluble domain, pointing to the DnaJ interface as the most functionally constrained region of the protein. An LLR mutation heatmap was then generated and cross-referenced against an experimental lysis dataset; three mutations that appeared in both were excluded after showing a lysis score of zero experimentally, highlighting that LLR scores reflect structural stability rather than functional activity. The final five mutants, Y39L and S9Q in the soluble domain, and K50L, N53L, and T52L in the transmembrane domain, were selected based on the highest LLR scores while avoiding all conserved and experimentally failed positions.

Each mutant was then modeled as an octameric pore complex using AlphaFold Multimer to simulate the expected membrane assembly. The key findings were:

  • All five mutants formed a cylinder-like pore structure, consistent with the expected transmembrane assembly
  • pLDDT scores were low (< 50) across all models, and PAE plots showed near-zero confidence for inter-chain contacts
  • Running the same pipeline on R30Q, an experimentally validated lytic mutant, produced identical low-confidence results, suggesting this is a systematic limitation of AlphaFold for this class of small membrane-disrupting proteins rather than a reflection of the mutants’ actual viability
  • All five mutants remain candidates for wet lab validation

Full documentation, sequences, and AlphaFold outputs are available in the week 5 homework.