Alanine Scanning Mutagenesis Script

This Python script (Alanine_Replacement.py) automates alanine scanning mutagenesis on a given protein sequence. It systematically replaces each amino acid residue in the sequence with alanine (Glycine if naturally occurring Alanine), one at a time, generating a series of mutant sequences from the input. The output is a FASTA file containing all the generated variants, which can be used for downstream analysis or synthesis, such as identifying critical residues for binding or structure.

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🧬 Purpose

Alanine scanning is a common method in structural and functional biology to identify the importance of specific amino acids in a protein. This script facilitates the rapid generation of all possible alanine substitution variants from a given protein input for use with peptide/gene synthesis

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🔧 Usage

positional arguments:
  input_file            Input protein file in FASTA format

options:
  -h, --help            show this help message and exit
  -o OUTPUT, --output OUTPUT
                        Output file name for mutated sequences in FASTA format

After processing, the script will output a FASTA file in the same directory with the format:

<Provided Name>_mutated.fasta

Each entry in the FASTA file will represent one substitution, with 3 duplicate entrys for each of the single, double, and triple Alanine replacements.

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🗂 Example

If your input sequence is:

>Test
MAKTISVVTLLCVLPAVVYST

The output will include entries like:

>Test_mut1
AAKTISVVTLLCVLPAVVYST
>Test_mut1_2
AGKTISVVTLLCVLPAVVYST
>Test_mut1_2_3
AGATISVVTLLCVLPAVVYST
>Test_mut2
MGKTISVVTLLCVLPAVVYST
>Test_mut2_3
MGATISVVTLLCVLPAVVYST
>Test_mut2_3_4
MGAAISVVTLLCVLPAVVYST
...

Naturally occuring Alanine's are replace with Glycine (G) instead.

Note - this does not factor in any other existing amino acid residues, therefore residues habrouring disulfide bonds (e.g., cystines) will be altered.

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✅ Features

• Alanine (and Cystine) amino acid replacements - single, double, and triple replacement options
• Clean FASTA output with informative headers
• Fast and lightweight — no external dependencies

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📄 License

MIT License

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✍️ Author

Ashley Otter
GitHub: @asherichia

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💡 Future Enhancements

• Support batch processing of multiple sequences