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 require eukaryotic machinery for native folding and post-translational modifications, so repeatedly expressing and assaying hundreds of mutations in human cells for each experiment is challenging.
Membrane proteins make up some of the largest and most structurally complex proteins, spanning the lipid bilayer up to dozens of times, forming oligomers, and often existing in multiple conformational states. The human proteome consists of >5,000 membrane proteins, including an estimated 901 GPCRs, 488 transporters, and 237 ion channels, which transmit key information to the interior of the cell and guide important cellular processes such as signaling, transport, and chemosensation.
Their roles in regulating diverse biological processes, their involvement in human diseases, and their exposure on the cell surface has made membrane proteins the targets for >40% of pharmaceutical drugs. Recent high-resolution crystal structures for GPCRs, ion channels, and other structurally complex membrane proteins are beginning to offer new insights into how these proteins function.
Mutagenesis studies such as alanine scanning complement such structures by defining mechanisms of activation and coupling, interactions with ligands and monoclonal antibodies (mAbs), and engineered variants with desired properties.