Identification of functional residues in proteins by charged-to-alanine scanning mutagenesis

Abstract

“Charged-to-alanine” scanning mutagenesis, in which the charged amino acids in a protein (Asp, Glu, Arg, Lys, and His) are systematically substituted with alanine, is a rational mutagenesis strategy for identifying the functionally important regions of proteins. The targeting of the charged amino acids and their replacement with alanine are designed to minimize disruptions to the tertiary structure of the protein. Thus, the mutant proteins are more likely to be successfully expressed for analysis, and the probability of incorrectly assigning a functional role to a specific residue is reduced. The functionally important polar and neutral residues can be identified by the subsequent substitution of the residues flanking the functional charged residues identified. In this paper we describe the rationale behind the approach, the design of mutagenic oligonucleotides, and a detailed method for high-efficiency site-directed mutagenesis. The conclusions drawn from the analysis of two proteins by means of this technique are discussed in terms of the crystal structures of these proteins, which were solved after the completion of the mutagenesis analysis.