Combinatorial cassette mutagenesis as a probe of the informational content of protein sequences.
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.
Reidhaar-Olson
J F
JF
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Sauer
R T
RT
D000595:Amino Acid Sequence; D001665:Binding Sites; D003062:Codon; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011487:Protein Conformation; D012097:Repressor Proteins / Q000235:genetics*; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000235:genetics*; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D003062:Codon; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D012097:Repressor Proteins; D014157:Transcription Factors; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D004247:DNA