Alanine scan

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Principle of the alanine scan. Systematic replacement of an amino acid in a native protein sequence for alanine

The alanine scan is a method used in molecular biology and protein biochemistry to identify the molecular components that are essential for the function, form and stability of proteins or peptides . It is based on the systematic sequential exchange of every single amino acid or, alternatively, several amino acids of a peptide, protein or protein part for the amino acid alanine . Replacing a functionally or structurally essential amino acid in a protein with alanine usually results in a loss of protein function.

principle

The alanine scan is based on the systematic, sequential exchange of every single amino acid for alanine. The consequence of this substitution is that all the side chain atoms concerned are removed except for one functionally largely neutral methyl group . Theoretically, a complete removal of the side chains through an exchange with the amino acid glycine would be conceivable, but such a substitution has a strong influence on the secondary structure of the protein thanks to the conformational flexibility of the peptide bond of the glycine . If an amino acid that is essential for function is replaced by alanine, a loss of protein function can usually be observed. A loss of protein function can also occur when an amino acid that is essential for the internal structure or shape is replaced. The method works anyway, as alanine mimics the secondary structure preferences of the majority of proteinogenic amino acids . This fact is fully explained by the alanine world hypothesis .

methodology

Classical methods of the alanine scan of a protein are based on the sequential exchange of each individual amino acid with the help of site-specific mutagenesis . This method is very complex, since each individual modified protein has to be constructed, expressed and analyzed separately. The amount of work can be reduced by combining random mutagenesis with a display method such as phage display . Combinatorial approaches of the alanine scan are also possible and facilitate the investigation of the function of proteins after the simultaneous exchange of several amino acids. An alanine scan of a small peptide is possible using chemical synthesis.

literature

  • Morrison KL, Weiss GA: Combinatorial alanine scanning . In: Curr Opin Chem Biol . 5, No. 3, June 2001, pp. 302-7. PMID 11479122 .

Individual evidence

  1. ↑ Vladimir Kubyshkin, Nediljko Budisa: The Alanine World Model for the Development of the Amino Acid Repertoire in Protein Biosynthesis . In: Int. J. Mol. Sci. . 20, No. 21, September 24, 2019, p. 5507. doi : 10.3390 / ijms20215507 .
  2. ^ Cain SA, Ratcliffe CF, Williams DM, Harris V, Monk PN: Analysis of receptor / ligand interactions using whole-molecule randomly-mutated ligand libraries . In: J. Immunol. Methods . 245, No. 1-2, November 2000, pp. 139-45. PMID 11042291 .
  3. ^ Morrison KL, Weiss GA: Combinatorial alanine scanning . In: Curr Opin Chem Biol . 5, No. 3, June 2001, pp. 302-7. PMID 11479122 .