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How to design alanine scanning peptide libraries

Combinatorial alanine scanning libraries can be used to determine the functional role of amino acid side chains. From these experiments, researchers can establish the relationship between protein structure and function, improve stability, map key binding sites, or alter a protein’s bioactivity.

This approach to peptide library design consists of systematically substituting residues on a specific protein segment with alanine. Alanine, a non-polar hydrophobic residue, is the smallest of amino acids with a single methyl group as its side chain. Due to the low reactivity of its group, alanine is the ideal choice for studying the contribution of specific side chains to bioactivity while preserving the structure of the native protein.

Applications of alanine scanning peptide libraries

However, screening the activity of alanine scanning libraries can become laborious and time-consuming, particularly if the location of the bioactive segments within a native protein are unknown. For this reason, this type of peptide library is routinely used during advanced stages of research when the hundreds to thousands of amino acids contained in a native protein have been narrowed down to a few bioactive residues (i.e., via screening of random or overlapping peptide libraries).

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Applications of alanine scanning peptide libraries

Interactions between proteins are the driving force behind all cellular processes. Understanding these interactions is necessary to learn how to either enhance or disrupt them, which has significant repercussions for human health and drug development.

To unveil these interactions, researchers routinely carry out co-crystallization experiments of receptor-ligand complexes or in silico prediction studies.

However, crystallization experiments are time-consuming and highly delicate, requiring significant amounts of highly pure and stable protein samples. In contrast, in silico studies typically make use of pre-existing data to train machine learning algorithms and carry out predictions. Thus, the accuracy of these algorithms depends on the diversity of the available data. For this reason, when data about a particular protein family is scarce, the predictions tend to be unreliable.

Despite the wealth of knowledge generated by these experiments, they often fail to elucidate the functional role of each specific amino acid. To probe protein function, alanine scanning libraries are a cost-effective alternative to these conventional techniques.

The screening of alanine libraries is particularly useful to systematically map functional binding, providing a detailed picture of the protein-protein interface and identifying key side chains and residues. These maps serve multiple purposes, including:

  • Epitope mapping, instrumental for precision drug development
  • Mapping of enzyme binding pockets, essential for enzyme engineering
  • Membrane protein-ligand interaction studies, key in the context of health and disease

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