Combinatorial Cassette Mutagenesis as a Probe of the Informational Content of Protein Sequences

• Published in: Science (American Association for the Advancement of Science) 1988-07, Vol.241 (4861), p.53-57
• Main Authors: Reidhaar-Olson, John F., Sauer, Robert T.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Analytical, structural and metabolic biochemistry; Bacteriophages; Binding Sites; Biological and medical sciences; Codon; Codons; Dimerization; Dimers; DNA; DNA - genetics; DNA - metabolism; DNA binding proteins; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; Genetic engineering; Macromolecular Substances; Methods; Molecular Sequence Data; Molecular structure; Monomers; Mutagenesis; Mutation; nucleotide sequence; phage lambda; Plasmids; Protein Conformation; Protein research; Proteins; Repressor Proteins - genetics; repressors; Solvents; Structure-Activity Relationship; Transcription Factors - genetics; Viral Proteins; Viral Regulatory and Accessory Proteins
• ISSN: 0036-8075; 1095-9203
• DOI: 10.1126/science.3388019

A method of combinatorial cassette mutagenesis was designed to readily determine the informational content of individual residues in protein sequences. The technique consists of simultaneously randomizing two or three positions by oligonucleotide cassette mutagenesis, selecting for functional protein, and then sequencing to determine the spectrum of allowable substitutions at each position. Repeated application of this method to the dimer interface of the DNA-binding domain of $\lambda $ repressor reveals that the number and type of substitutions allowed at each position are extremely variable. At some positions only one or two residues are functionally acceptable; at other positions a wide range of residues and residue types are tolerated. The number of substitutions allowed at each position roughly correlates with the solvent accessibility of the wild-type side chain.