P-31 NMR-SPECTRA OF OLIGODEOXYRIBONUCLEOTIDE DUPLEX LAC OPERATOR REPRESSOR HEADPIECE COMPLEXES - IMPORTANCE OF PHOSPHATE ESTER BACKBONE FLEXIBILITY IN PROTEIN DNA RECOGNITION
P-31 NMR-SPECTRA OF OLIGODEOXYRIBONUCLEOTIDE DUPLEX LAC OPERATOR REPRESSOR HEADPIECE COMPLEXES - IMPORTANCE OF PHOSPHATE ESTER BACKBONE FLEXIBILITY IN PROTEIN DNA RECOGNITION
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DOI：
10.1021/bi00121a038
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发表时间：
1992-02-18
期刊：
影响因子：
2.9
通讯作者：
GORENSTEIN, DG
中科院分区：
文献类型：
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作者：
KARSLAKE, C;BOTUYAN, MV;GORENSTEIN, DG
分析了与野生型和突变型 lac 阻遏头状蛋白结合的各种 14 碱基对 lac 操纵子的 P-31 NMR 谱，以提供有关复合物中主链构象的信息。与 Y7I 突变阻遏物 N 端 56 残基头片段结合的野生型对称操纵基因 d(TGTGAGCGCTCACA)2 的 P-31 NMR 谱几乎与与野生型阻遏蛋白头结合的相同操纵基因的谱相同。相反，相对于野生型阻遏子-操纵子复合物，突变操纵子d(TATGAGCGCTCATA)2-野生型耳机复合物的P-31 NMR谱受到显着干扰。第二个突变操纵子 d(TGTGTGCGCACACA)2 的磷酸盐的 P-31 化学位移在与野生型或突变型头件络合时显示出微小但特定的变化。第三个突变操纵子 d(TCTGAGCGCTCAGA)2 的磷酸盐的 P-31 化学位移在添加野生型头件后未显示出任何扰动。 P-31 NMR 结果为 2:1 蛋白质与耳机复合物中 5'-TGTGA/3'-ACACT 结合位点的 5' 链的主要识别提供了进一步的证据。有人提出，特异性强结合操纵子-蛋白质复合物保留了操纵子本身固有的磷酸酯构象灵活性，而磷酸酯在弱结合操纵子-蛋白质复合物中受到构象限制。这种在强复合物中保留主链扭转自由度在熵上是有利的，并为不同操纵子结合位点的蛋白质区分提供了一种新的（推测性的）机制。它证明了磷酸盐几何形状和灵活性对蛋白质识别和结合的潜在重要性。
The P-31 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 P-31 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 P-31 NMR spectrum of the mutant operator, d(TATGAGCGCTCATA)2,-wild-type headpiece complex was significantly perturbed relative to the wild-type repressor-operator complex. The P-31 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 P-31 chemical shifts of the phosphates of a third mutant operator, d(TCTGAGCGCTCAGA)2, showed no perturbations upon addition of the wild-type headpiece. The P-31 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.