title: Glycine insertion in the hinge region of lactose repressor protein alters DNA binding.
abstract: Amino acid alterations were designed at the C terminus of the hinge segment (amino acids approximately 51-59) that links two functional domains within lactose repressor protein (LacI). Gly was introduced between Gly(58) and Lys(59) to generate Gly(58+1); Gln(60) was changed to Gly or Pro, and up to three additional glycines were inserted following Gln(60) --> Gly. All mutant proteins exhibited purification behavior, CD spectra, assembly state, and inducer binding properties similar to wild-type LacI and only small differences in trypsin proteolysis patterns. In contrast, significant differences were observed in DNA binding properties. Gly(58+1) exhibited a decrease of approximately 100-fold in affinity for O(1) operator, and sequential Gly insertion C-terminal to Gln(60) --> Gly resulted in progressively decreased affinity for O(1) operator, approaching nonspecific levels for insertion of >/=2 glycines. Where sufficient affinity for O(1) operator existed, decreased binding to O(1) in the presence of inducer indicated no disruption in the allosteric response for these proteins. Collectively, these results indicate that flexibility and/or spacing between the core and N-terminal domains did not significantly affect folding or assembly, but these alterations in the hinge domain profoundly altered affinity of the lactose repressor protein for its wild-type target sequence.
authors.affiliation: Department of Biochemistry, Rice University, Houston, Texas 77251, USA.
authors.forename: C M
authors.initials: CM
authors.lastname: Falcon
authors.forename: K S
authors.initials: KS
authors.lastname: Matthews
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D005973:Glutamine; D005998:Glycine; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007700:Kinetics; D056968:Lac Repressors; D008239:Lysine; D008958:Models, Molecular; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D011392:Proline; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D012097:Repressor Proteins; D005973:Glutamine; D007544:Isopropyl Thiogalactoside; D011392:Proline; D008239:Lysine; D005998:Glycine