Linked Life Data

2499352

Source:http://linkedlifedata.com/resource/pubmed/id/2499352

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
6
pubmed:dateCreated
1989-7-27
pubmed:abstractText
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)].
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Mar
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
21
pubmed:volume
28
pubmed:owner
NLM
pubmed:authorsComplete
N
pubmed:pagination
2667-77
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed-meshheading:2499352-Base Sequence, pubmed-meshheading:2499352-Binding Sites, pubmed-meshheading:2499352-DNA, pubmed-meshheading:2499352-Deoxyribonuclease EcoRI, pubmed-meshheading:2499352-Genes, pubmed-meshheading:2499352-Genes, Bacterial, pubmed-meshheading:2499352-Genetic Engineering, pubmed-meshheading:2499352-Genetic Vectors, pubmed-meshheading:2499352-Glutamine, pubmed-meshheading:2499352-Lysine, pubmed-meshheading:2499352-Macromolecular Substances, pubmed-meshheading:2499352-Molecular Sequence Data, pubmed-meshheading:2499352-Mutation, pubmed-meshheading:2499352-Oligonucleotide Probes, pubmed-meshheading:2499352-Protein Binding, pubmed-meshheading:2499352-Protein Conformation, pubmed-meshheading:2499352-Protein Denaturation, pubmed-meshheading:2499352-Restriction Mapping, pubmed-meshheading:2499352-Thermodynamics
pubmed:year
1989
pubmed:articleTitle
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.
pubmed:affiliation
Zentrum Biochemie, Medizinische Hochschule Hannover, West Germany.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't