Week 5 Lab: Protein Design Part II

Lab Option 1: L-Protein Mutational Analysis

Experimental Validation & DMS

This lab provides a Deep Mutational Scanning (DMS) style validation of the L-Protein. By cross-referencing experimental lysis results—where a score of 1 indicates functional lysis and 0 indicates non-functional—with the Log-Likelihood Ratio (LLR) Heatmap generated via ESM2, we can assess the predictive power of protein language models.

Key Findings

• Correlation: The agreement between the experimental data and the LLR Heatmap is remarkably high, approximately 90-95%.
• Predictive Accuracy: The heatmap serves as a highly reliable predictor of whether a specific mutation will allow the protein to retain its ability to lyse bacterial cells.
• Challenges: Designing mutants with high computational confidence remains difficult, highlighting the current limitations of some structure-based models.

Targeted Mutation Strategy

To identify promising variants, I cross-referenced the ESM2 scores with experimental lab data, specifically looking for residues that are not strictly conserved (via pBLAST) and show positive mutational effects.

Selection Criteria:

1. Soluble Region (N-tail): Targeted to assess how surface-exposed changes affect function.
2. Transmembrane Region: Targeted to test the hypothesis that the L-protein assembles to perforate the bacterial membrane.
3. Combined Effects: Testing synergistic effects of multiple “positive-score” mutations.

Proposed Mutants

Structural Hypothesis: Multimeric Assembly

A running hypothesis for L-protein function is that it assembles into a multimeric complex to create perforations in the bacterial membrane. To investigate this, I utilized AF2_Multimer to generate a predicted multimeric assembly.

By modeling these specific mutations (particularly those in the transmembrane region like Mutant 3 and 4) in a multimeric context, we can observe if the substitutions stabilize the pore-forming structure or increase the efficiency of membrane lysis.

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