31P NMR spectra of oligodeoxyribonucleotide duplex lac operator-repressor headpiece complexes: importance of phosphate ester backbone flexibility in protein-DNA recognition.
The 31P NMR spectra of various 14-base-pair lac operators bound to both wild-type and mutant lac repressor headpiece proteins were analyzed to provide information on the backbone conformation in the complexes. The 31P NMR spectrum of a wild-type symmetrical operator, d(TGTGAGCGCTCACA)2, bound to the N-terminal 56-residue headpiece fragment of a Y7I mutant repressor was nearly identical to the spectrum of the same operator bound to the wild-type repressor headpiece. In contrast, the 31P NMR spectrum of the mutant operator, d(TATAGAGCGCTCATA)2, wild-type headpiece complex was significantly perturbed relative to the wild-type repressor-operator complex. The 31P chemical shifts of the phosphates of a second mutant operator, d(TGTGTGCGCACACA)2, showed small but specific changes upon complexation with either the wild-type or mutant headpiece. The 31P chemical shifts of the phosphates of a third mutant operator, d(TCTGAGCGCTCAGA)2, showed no perturbations upon addition of the wild-type headpiece. The 31P NMR results provide further evidence for predominant recognition of the 5'-strand of the 5'-TGTGA/3'-ACACT binding site in a 2:1 protein to headpiece complex. It is proposed that specific, strong-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 strong complexes is entropically favorable and provides a new (and speculative) mechanism for protein discrimination of different operator binding sites. It demonstrates the potential importance of phosphate geometry and flexibility on protein recognition and binding.
Karslake
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C
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907.
Botuyan
M V
MV
Gorenstein
D G
DG
D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D004952:Esters / Q000737:chemistry*; D007763:Lac Operon; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D010710:Phosphates / Q000737:chemistry*; D010957:Plasmids; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*
D004952:Esters; D009838:Oligodeoxyribonucleotides; D010710:Phosphates; D012097:Repressor Proteins; D004247:DNA