Matzapetakis Manolis, Farrer Brian T, Weng Tsu-Chien, Hemmingsen Lars, Penner-Hahn James E, Pecoraro Vincent L
Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055, USA.
J Am Chem Soc. 2002 Jul 10;124(27):8042-54. doi: 10.1021/ja017520u.
Designed alpha-helical peptides of the TRI family with a general sequence Ac-G(LKALEEK)(4)G-CONH(2) were used as model systems for the study of metal-protein interactions. Variants containing cysteine residues in positions 12 (TRI L12C) and 16 (TRI L16C) were used for the metal binding studies. Cd(II) binding was investigated, and the results were compared with previous and current work on Hg(II) and As(III) binding. The metal peptide assemblies were studied with the use of UV, CD, EXAFS, (113)Cd NMR, and (111m)Cd perturbed angular correlation spectroscopy. The metalated peptide aggregates exhibited pH-dependent behavior. At high pH values, Cd(II) was bound to the three sulfurs of the three-stranded alpha-helical coiled coils. A mixture of two species was observed, including Cd(II) in a trigonal planar geometry. The complexes have UV bands at 231 nm (20 600 M(-1) cm(-1)) for TRI L12C and 232 nm (22 600 M(-1) cm(-1)) for TRI L16C, an average Cd-S bond length of 2.49 A for both cases, and a (113)Cd NMR chemical shift at 619 ppm (Cd(II)(TRI L12C)(3)(-)) or 625 ppm (Cd(II)(TRI-L16C)(3)(-)). Nuclear quadrupole interactions show that two different Cd species are present for both peptides. One species with omega(0) = 0.45 rad/ns and low eta is attributed to a trigonal planar Cd-(Cys)(3) site. The other, with a smaller omega(0), is attributed to a four-coordinate Cd(Cys)(3)(H(2)O) species. At low pH, no metal binding was observed. Hg(II) binding to TRI L12C was also found to be pH dependent, and a 3:1 sulfur-to-mercury(II) species was observed at pH 9.4. These metal peptide complexes provide insight into heavy metal binding and metalloregulatory proteins such as MerR or CadC.
具有通用序列Ac-G(LKALEEK)(4)G-CONH(2)的TRI家族设计α-螺旋肽被用作研究金属-蛋白质相互作用的模型系统。在第12位（TRI L12C）和第16位（TRI L16C）含有半胱氨酸残基的变体用于金属结合研究。研究了Cd(II)的结合情况，并将结果与先前和当前关于Hg(II)和As(III)结合的工作进行了比较。使用紫外光谱（UV）、圆二色光谱（CD）、扩展X射线吸收精细结构谱（EXAFS）、(113)Cd核磁共振（NMR）和(111m)Cd扰动角关联光谱对金属肽组装体进行了研究。金属化肽聚集体表现出pH依赖性行为。在高pH值下，Cd(II)与三股α-螺旋卷曲螺旋的三个硫原子结合。观察到两种物种的混合物，包括呈三角平面几何构型的Cd(II)。对于TRI L12C，配合物在231 nm处有紫外吸收带（20 600 M(-1) cm(-1)），对于TRI L16C，在232 nm处有紫外吸收带（22 600 M(-1) cm(-1)），两种情况下平均Cd-S键长均为2.49 Å，(113)Cd NMR化学位移在619 ppm（Cd(II)(TRI L12C)(3)(-)）或625 ppm（Cd(II)(TRI-L16C)(3)(-)）。核四极相互作用表明两种肽都存在两种不同的Cd物种。一种ω(0) = 0.45 rad/ns且η值较低的物种归因于三角平面Cd-(Cys)(3)位点。另一种ω(0)较小的物种归因于四配位的Cd(Cys)(3)(H(2)O)物种。在低pH值下，未观察到金属结合。还发现Hg(II)与TRI L12C的结合也依赖于pH，在pH 9.4时观察到硫与汞(II)比例为3:1的物种。这些金属肽配合物为深入了解重金属结合以及诸如MerR或CadC等金属调节蛋白提供了依据。
