PMID: 2499352Mar 21, 1989Paper

Genetic engineering of EcoRI mutants with altered amino acid residues in the DNA binding site: physicochemical investigations give evidence for an altered monomer/dimer equilibrium for the Gln144Lys145 and Gln144Lys145Lys200 mutants

Biochemistry
R GeigerA Düsterhöft

Abstract

We have genetically engineered the Arg200----Lys mutant, the Glu144Arg145----GlnLys double mutant, and the Glu144Arg145Arg200----GlnLysLys triple mutant of the EcoRI endonuclease in extension of previously published work on site-directed mutagenesis of the EcoRI endonuclease in which Glu144 had been exchanged for Gln and Arg145 for Lys [Wolfes et al. (1986) Nucleic Acids Res. 14, 9063]. All these mutants carry modifications in the DNA binding site. Mutant EcoRI proteins were purified to homogeneity and characterized by physicochemical techniques. All mutants have a very similar secondary structure composition. However, whereas the Lys200 mutant is not impaired in its capacity to form a dimer, the Gln144Lys145 and Gln144Lys145Lys200 mutants have a very much decreased propensity to form a dimer or tetramer depending on concentration as shown by gel filtration and analytical ultracentrifugation. This finding may explain the results of isoelectric focusing experiments which show that these two mutants have a considerably more basic pI than expected for a protein in which an acidic amino acid was replaced by a neutral one. Furthermore, while wild-type EcoRI and the Lys200 mutant are denatured in an irreversible manner upon heating t...Continue Reading

References

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Citations

May 9, 2001·Biochimica Et Biophysica Acta·T Deva, S Krishnaswamy
Apr 16, 1998·Proceedings of the National Academy of Sciences of the United States of America·C R Robinson, S G Sligar
Sep 15, 1993·Proceedings of the National Academy of Sciences of the United States of America·A JeltschA Pingoud
Nov 24, 2004·Biological Chemistry·Petra Fritsche, Jürgen Alves
Jun 1, 2014·Nucleic Acids Research·Alfred PingoudWolfgang Wende
May 15, 1997·European Journal of Biochemistry·A Pingoud, A Jeltsch
Feb 15, 1997·European Journal of Biochemistry·S Windolph, J Alves
Feb 7, 1998·Journal of Molecular Biology·R S MuirJ Heitman

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