1. K50N (Lysine → Asparagine at position 50)

Region: Transmembrane

LLR Score: Not top-ranked, but experimental variant showed lysis level = 1.0

Why this mutation?

• Charge modulation: Lysine is positively charged and long-chained; Asparagine is polar but uncharged. Replacing K with N can soften the local charge, possibly improving oligomer stability or membrane insertion.
• Experimental validation: Among the tested K50 mutants (K→F, I, A, V, S, T), K→N showed the strongest lysis, suggesting it's functionally advantageous.
• Structural positioning: K50 lies at the beginning of the TM domain and may face the lipid head groups or help anchor the TM helix. Swapping it with a less charged but still polar residue may maintain insertion while enhancing flexibility.
• Conservation context: Mostly conserved across pBLAST entries, but some strains have deviations (e.g., K→N), meaning it's not intolerant to change.

Hypothesis:

K50N fine-tunes the balance between membrane anchoring and oligomer formation by modulating electrostatic interactions near the TM entry.

2. N53L (Asparagine → Leucine at position 53)

Region: Transmembrane

LLR Score: 1.86 (strong positive)

Why this mutation?

• Hydrophobicity enhancement: Leucine is a hydrophobic residue known to stabilize transmembrane helices. N53L boosts the hydrophobic character of the TM domain, promoting better membrane partitioning.
• Structural role: N53 sits within the membrane-spanning region. Replacing a polar side chain with a bulky hydrophobic one could stabilize the helical bundle or pore structure.
• Conservation: Your pBLAST output shows variability at position 53 — notably N→D, N→H, suggesting it's not rigidly conserved.
• Function link: Oligomerization of MS2-L is driven by TM helices. Enhancing leucine content could encourage tighter TM packing, leading to more efficient pore formation.

Hypothesis:

N53L strengthens hydrophobic interactions in the TM oligomer core, increasing structural stability and lytic potency.