An experimental approach to testing modular evolution: directed replacement of alpha-helices in a bacterial protein.
We have used oligonucleotide site-directed mutagenesis to ask whether certain structural motifs in proteins are determined mainly by local interactions among amino acids. Multiple consecutive amino acids in three alpha-helices in the alkaline phosphatase (EC 3.1.3.1) of Escherichia coli have been replaced with helical sequences from four other sources. Altogether, 12 distinct helical replacements were created, 9 of which retain enzymatic activity. Most short stretches of helical sequence can be replaced with unrelated helical sequences without eliminating enzyme activity. Replacements of the carboxyl half of an alpha-helix are less harmful than those of the amino half, and the two together are synergistic rather than additive. These results are consistent with the hypothesis that proteins originally evolved by the assembly of small functional folding units.
DuBose
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Department of Genetics, Washington University School of Medicine, Saint Louis, MO 63110-1095.
Hartl
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D000469:Alkaline Phosphatase / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000648:ultrastructure; D005075:Biological Evolution; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation
D001426:Bacterial Proteins; D000469:Alkaline Phosphatase