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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.

Poussu, Eini; Vihinen, Mauno LU ; Paulin, Lars and Savilahti, Harri (2004) In Proteins 54(4). p.681-692
Abstract
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... (More)
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. (Less)
Please use this url to cite or link to this publication:
author
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Mutation: genetics, Insertional: genetics, Mutagenesis, Escherichia coli: genetics, Escherichia coli: enzymology, Bacteriophage mu: genetics, DNA Transposable Elements: genetics, beta-Galactosidase: chemistry, beta-Galactosidase: genetics, beta-Galactosidase: metabolism
in
Proteins
volume
54
issue
4
pages
681 - 692
publisher
John Wiley & Sons
external identifiers
  • PMID:14997564
  • Scopus:1542346468
ISSN
0887-3585
DOI
10.1002/prot.10467
language
English
LU publication?
no
id
f1ead395-e710-4491-ae26-cc68634bde1d (old id 3635483)
alternative location
http://www.ncbi.nlm.nih.gov/pubmed/14997564?dopt=Abstract
date added to LUP
2013-06-12 16:12:28
date last changed
2016-10-23 04:32:23
@misc{f1ead395-e710-4491-ae26-cc68634bde1d,
  abstract     = {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.},
  author       = {Poussu, Eini and Vihinen, Mauno and Paulin, Lars and Savilahti, Harri},
  issn         = {0887-3585},
  keyword      = {Mutation: genetics,Insertional: genetics,Mutagenesis,Escherichia coli: genetics,Escherichia coli: enzymology,Bacteriophage mu: genetics,DNA Transposable Elements: genetics,beta-Galactosidase: chemistry,beta-Galactosidase: genetics,beta-Galactosidase: metabolism},
  language     = {eng},
  number       = {4},
  pages        = {681--692},
  publisher    = {ARRAY(0xa210918)},
  series       = {Proteins},
  title        = {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.},
  url          = {http://dx.doi.org/10.1002/prot.10467},
  volume       = {54},
  year         = {2004},
}