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

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 to 60 degrees C, the thermal denaturation process as shown by circular dichroism spectroscopy is fully reversible with the Gln144Lys145 double mutant and the Gln144Lys145Lys200 triple mutant. All EcoRI endonuclease mutants described here have a residual enzymatic activity with wild-type specificity, since Escherichia coli cells overexpressing the mutant proteins can only survive in the presence of EcoRI methylase. The detailed analysis of the enzymatic activity and specificity of the purified mutant proteins is the subject of the accompanying paper [Alves et al. (1989) Biochemistry (following paper in this issue)].

Changes in solvation during DNA binding and cleavage are critical to altered specificity of the Eco RI endonuclease

“…EcoRI purified essentially according to the method of Geiger et al (39) was donated by Richard Gumport (College of Medicine, Univ. of Illinois, Urbana).…”

Multiple binding modes of the single-stranded DNA binding protein from Escherichia coli as detected by tryptophan fluorescence and site-directed mutagenesis

“…All mutations were introduced using mismatch primed DNA synthesis on gapped duplex substrates (Geiger et al, 1989;Fritz et al, 1988). Generally, mutant plasmids contained an additional restriction site to facilitate screening.…”

Asp‐59 is not important for the catalytic activity of the restriction endonuclease EcoRI

“…Site directed mutagenesis was carried out according to the gapped duplex method as described by Geiger et al [23]. We mutated a modified ecoRl gene which codes for an enzyme with a C-terminal His6-tag.…”

“…The resulting preparations were >90% pure on denaturing gel electrophoresis. Protein concentration was determined spectrophotometrically [23]. To eliminate any influence of the Hisr-tag or of the short purification protocol on the results wild type His6-tagged EeoRI was purified in parallel by the same procedure and used as a control in all experiments described here.…”

“…The resulting solutions were directly analyzed using a cuvette with a pathlength of 0.1 cm in a Jobin Yvon Dichrograph III with a bandwidth of 2 nm, a rate of scanning of 0.03 nm and a time constant of 2 s as described [23].…”