Probing the alpha-complementing domain of E. coli beta-galactosidase with use of an insertional pentapeptide mutagenesis strategy based on Mu in vitro DNA transposition.
Protein structure-function relationships can be studied by using linker insertion mutagenesis, which efficiently identifies essential regions in target proteins. Bacteriophage Mu in vitro DNA transposition was used to generate an extensive library of pentapeptide insertion mutants within the alpha-complementing domain 1 of Escherichia coli beta-galactosidase, yielding mutants at 100% efficiency. Each mutant contained an accurate 15-bp insertion that translated to five additional amino acids within the protein, and the insertions were distributed essentially randomly along the target sequence. Individual mutants (alpha-donors) were analyzed for their ability to restore (by alpha-complementation) beta-galactosidase activity of the M15 deletion mutant (alpha-acceptor), and the data were correlated to the structure of the beta-galactosidase tetramer. Most of the insertions were well tolerated, including many of those disrupting secondary structural elements even within the protein's interior. Nevertheless, certain sites were sensitive to mutations, indicating both known and previously unknown regions of functional importance. Inhibitory insertions within the N-terminus and loop regions most likely influenced protein tetramerization via direct local effects on protein-protein interactions. Within the domain 1 core, the insertions probably caused either lateral shifting of the polypeptide chain toward the protein's exterior or produced more pronounced structural distortions. Six percent of the mutant proteins exhibited temperature sensitivity, in general suggesting the method's usefulness for generation of conditional phenotypes. The method should be applicable to any cloned protein-encoding gene.
Program in Cellular Biotechnology, Institute of Biotechnology, Viikki Biocenter, University of Helsinki, Finland.
Eini
E
Poussu
Mauno
M
Vihinen
Lars
L
Paulin
Harri
H
Savilahti
D000595:Amino Acid Sequence; D010583:Bacteriophage mu / Q000235:genetics*; D004251:DNA Transposable Elements / Q000235:genetics*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005816:Genetic Complementation Test; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012995:Solubility; D013329:Structure-Activity Relationship; D013696:Temperature; D001616:beta-Galactosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D004251:DNA Transposable Elements; D001616:beta-Galactosidase