Laplaza Catalina E, Holm R H
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.
J Biol Inorg Chem. 2002 Apr;7(4-5):451-60. doi: 10.1007/s00775-001-0320-4. Epub 2002 Jan 8.
Helix-loop-helix peptides containing 63 residues (HC(4)H(2), HC(4)HC, HC(5)H), designated by their sequence and content of histidyl (H) and cysteinyl (C) residues, have been previously synthesized for the purpose of stabilizing certain bridged metal sites in proteins. These peptides bind one Fe(4)S(4) cluster by means of a ferredoxin tricysteinyl consensus sequence and an additional Cys residue, and one Ni(II) atom (HC(4)H(2), HC(5)H) in predesigned binding sites. In this investigation, the apopeptides and their Fe(4)S(4) derivatives are shown to be relatively stable to unfolding by guanidine hydrochloride, indicating stability of secondary structure. With this property demonstrated, Ni(II) binding equilibria have been evaluated in the terms of site-specific (Scatchard model) and stepwise (stoichiometric) binding constants. Two peptides were designed to have preformed CysHis(3) (HC(4)H(2)) and Cys(2)His(2) (HC(5)H) binding sites. The data indicate one strong binding site in each peptide with preferred binding constants k(1)=4.4x10(5) M(-1)(HC(4)H(2)) and 2.7x10(5) M(-1)(HC(5)H). Based on X-ray absorption spectroscopic data, these binding steps are associated with the formation of the desired coordination units Ni(II)CysHis(3) and Ni(II)Cys(2)His(2). For peptide HC(4)HC, k(1)=2.5x10(5) M(-1), but the binding site could not be fully identified. Collective evidence from this and prior investigations supports the presence of the bridged assemblies Ni(II)-(mu(2)-S x Cys)-[Fe(4)S(4)], stabilized by a scaffolding effect in peptides HC(4)H(2) and HC(5)H. The assembly Ni(II)-X-[Fe(4)S(4)] is the minimal structure of the A-Cluster of carbon monoxide dehydrogenase adduced from spectroscopic evidence; bridge X is currently unidentified. These results suggest that de novo designed peptides may serve as scaffolds for the construction of native bridged sites in proteins. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00775-001-0320-4.
含有63个残基的螺旋-环-螺旋肽（HC(4)H(2)、HC(4)HC、HC(5)H），根据其组氨酸（H）和半胱氨酸（C）残基的序列和含量命名，此前已被合成，目的是稳定蛋白质中某些桥连金属位点。这些肽通过铁氧化还原蛋白三半胱氨酸共有序列和一个额外的半胱氨酸残基结合一个Fe(4)S(4)簇，并在预先设计的结合位点结合一个Ni(II)原子（HC(4)H(2)、HC(5)H）。在本研究中，脱辅基肽及其Fe(4)S(4)衍生物对盐酸胍展开相对稳定，表明二级结构稳定。证明了这一性质后，已根据位点特异性（Scatchard模型）和逐步（化学计量）结合常数评估了Ni(II)的结合平衡。设计了两种肽，使其具有预先形成的CysHis(3)（HC(4)H(2)）和Cys(2)His(2)（HC(5)H）结合位点。数据表明每种肽中有一个强结合位点，其优先结合常数k(1)=4.4×10(5) M(-1)（HC(4)H(2)）和2.7×10(5) M(-1)（HC(5)H）。根据X射线吸收光谱数据，这些结合步骤与所需配位单元Ni(II)CysHis(3)和Ni(II)Cys(2)His(2)的形成有关。对于肽HC(4)HC，k(1)=2.5×10(5) M(-1)，但结合位点无法完全确定。来自本研究和先前研究的综合证据支持在肽HC(4)H(2)和HC(5)H中存在通过支架效应稳定的桥连组装体Ni(II)-(μ(2)-S×Cys)-[Fe(4)S(4)]。组装体Ni(II)-X-[Fe(4)S(4)]是根据光谱证据推断出的一氧化碳脱氢酶A簇的最小结构；桥连X目前尚未确定。这些结果表明，从头设计的肽可作为构建蛋白质中天然桥连位点的支架。本文的电子补充材料可通过位于http://dx.doi.org/10.1007/s00775-001-0320-4的Springer Link服务器获取。
