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Alanine Scanning  Epitope Mapping and Engineering Complex
Membrane Proteins by Comprehensive Mutagenesis    • Alanine
(Ala) scanning is a widely used mutagenesis approach in which
residues in a target protein are systematically substituted for
alanine at selected positions by site-directed mutagenesis,
expressed, and assayed for function. Substitution with alanine
residues eliminates side-chain interactions without altering
main-chain conformation or introducing steric or electrostatic
effects, so is often the preferred choice for testing the
contribution of specific side-chains while preserving native
protein structure.      However, because many target proteins
are hundreds to thousands of amino acids long, individual
alanine replacements across their entire length can be laborious
and time-consuming, typically limiting functional analysis to a
fraction of the protein’s complete sequence. Furthermore,
structurally complex proteins often requir