title: 31P nuclear magnetic resonance spectra and dissociation constants of lac repressor headpiece.duplex operator complexes: the importance of phosphate backbone flexibility in protein.DNA recognition.
abstract: An alkaline phosphatase assay was used to determine the dissociation constants (KD) of the lac repressor N-terminal 56 amino acid fragment of the wild type and of a Y7I mutant complexed to 22 base pair (bp) wild-type and mutant symmetrical operator sequences. KD's in 0.35 M monovalent salt ranged from 5.4 X 10(-8) M for the wild-type repressor.wild-type operator complex to approximately > 1 X 10(-6) M for the wild-type repressor.nonspecific DNA complex. Mutant operators O2 (G5 --> A5 and G16 --> T16) and O4 (G5 --> C5 and C16 --> G16) bind nearly as tightly as the wild-type headpiece, while mutant O3 (A8 --> T8 and T13 --> A13) binds over 5-fold poorer. Operators O1, O2, and O4 bind ca. 10-fold poorer to the Y7I mutant headpiece. Operator O3 binds 2-fold poorer to the mutant headpiece. The temperature and salt dependence on the dissociation constants of wild-type headpiece binding to 22-bp operator support the conclusion that the headpiece contains the major DNA recognition portion of the protein and that electrostatics plays as important a role in the binding of operator to headpiece as it does in the whole repressor. The 31P NMR spectra of shortened 14-bp wild-type and mutant symmetrical operators bound to the N-terminal 56-residue headpiece of the Y7I mutant repressor were compared to the spectra of the same operator bound to the wild-type repressor headpiece. These results are consistent with a recent proposal [Karslake, C., Botuyan, M. V., & Gorenstein, D. G. (1992) Biochemistry 31, 1849-1858] that specific, tight-binding operator.protein complexes retain the inherent phosphate ester conformational flexibility of the operator itself, whereas the phosphate esters are conformationally restricted in the weak-binding operator-protein complexes. This retention of backbone torsional freedom in tight complexes is entropically favorable and provides a mechanism for protein discrimination of different operator binding sites.
authors.affiliation: Department of Chemistry, Purdue University, West Lafayette, Indiana 47907.
authors.forename: M V
authors.initials: MV
authors.lastname: Botuyan
authors.forename: D A
authors.initials: DA
authors.lastname: Keire
authors.forename: C
authors.initials: C
authors.lastname: Kroen
authors.forename: D G
authors.initials: DG
authors.lastname: Gorenstein
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D004952:Esters / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009154:Mutation; D009875:Operator Regions, Genetic; D010759:Phosphorus Isotopes; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism; D012965:Sodium Chloride / Q000737:chemistry; D013696:Temperature
chemical_list: D004268:DNA-Binding Proteins; D004952:Esters; D010759:Phosphorus Isotopes; D012097:Repressor Proteins; D012965:Sodium Chloride; D004247:DNA; D000469:Alkaline Phosphatase