Jain Sandhya, Welch Joel T, Horrocks William D, Franklin Sonya J
Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Inorg Chem. 2003 Dec 1;42(24):8098-104. doi: 10.1021/ic034736j.
A series of Eu(III) metallopeptides, designed on the basis of the structural similarity of the helix-turn-helix and EF-hand motifs, have been studied by Eu(III) (7)F(0) --> (5)D(0) excitation spectroscopy. The impact of EF-hand ligand set differences on the hydration number and Eu(III) coordination environment are compared among the peptides. The conditional binding affinities were determined by Eu titration (P3, log K(a) = 6.0 +/- 0.4; P3W, log K(a) = 5.9 +/- 0.2; P5b, log K(a) = 5.3 +/- 0.1). Two similar coordination environments occur in each case, consistent with structural flexibility about the metal site. The coordination environments are consistent with 8- or 9-coordinate Eu(III), including six peptide-based ligands and two to three water molecules (P3, q = 1.9 +/- 0.2; P3W, q = 2.3 +/- 0.2; P4a, q = 1.9 +/- 0.3; P5b, q = 2.6 +/- 0.2). The Eu(III) (7)F(0) --> (5)D(0) excitation spectra are pH-dependent, as reported for several EF-hand proteins (oncomodulin, parvalbumin). A higher energy transition occurs at pH > 6, and has been assigned to deprotonation of coordinated water. The pK(a) leading to this new transition is dependent on Eu(III) Lewis acidity, which varies with the inner and outer sphere ligand set. The noncoordinating ninth position of the Eu-binding loop, which is poised to make second-sphere contacts to the coordinated water, stabilizes the deprotonated form of the coordinated solvent more effectively when it is Thr (P5b) than Asp (P3W). Upon DNA-binding by the metallopeptides, the pK(a) of the pH-dependent peak increases, but no new DNA-dependent transitions are observed. This indicates no DNA-based Eu(III) ligands are introduced, such as phosphate oxygen atoms of the DNA backbone. The hydration number decreases in the presence of DNA (P3W + DNA, q = 1.9 +/- 0.2; P5b + DNA, q = 1.7 +/- 0.2), indicating that DNA-binding by the metallopeptides organizes rather than compromises the Eu-binding site within the peptide.
基于螺旋-转角-螺旋和EF-手基序的结构相似性设计了一系列铕（III）金属肽，并通过铕（III）的（7）F（0）→（5）D（0）激发光谱对其进行了研究。比较了这些肽中EF-手配体集差异对水合数和铕（III）配位环境的影响。通过铕滴定确定了条件结合亲和力（P3，log K（a）= 6.0±0.4；P3W，log K（a）= 5.9±0.2；P5b，log K（a）= 5.3±0.1）。每种情况下都出现了两种相似的配位环境，这与金属位点周围的结构灵活性一致。配位环境与8或9配位的铕（III）一致，包括六个基于肽的配体和两到三个水分子（P3，q = 1.9±0.2；P3W，q = 2.3±0.2；P4a，q = 1.9±0.3；P5b，q = 2.6±0.2）。铕（III）的（7）F（0）→（5）D（0）激发光谱与pH有关，这与几种EF-手蛋白（癌调蛋白、小清蛋白）的情况相同。在pH > 6时会发生更高能量的跃迁，这被归因于配位水的去质子化。导致这种新跃迁的pK（a）取决于铕（III）的路易斯酸度，其随内球和外球配体集而变化。铕结合环的非配位第九位准备与配位水进行第二球接触，当它是苏氨酸（P5b）时比天冬氨酸（P3W）更有效地稳定配位溶剂的去质子化形式。当金属肽与DNA结合时，pH依赖性峰的pK（a）增加，但未观察到新的DNA依赖性跃迁。这表明没有引入基于DNA的铕（III）配体，如DNA主链的磷酸氧原子。在DNA存在下，水合数降低（P3W + DNA，q = 1.9±0.2；P5b + DNA，q = 1.7±0.2），这表明金属肽与DNA的结合使肽内的铕结合位点有序化而非破坏它。
