Van Duynhoven J P, Nooren I M, Swinkels D W, Folkers P J, Harmsen B J, Konings R N, Tesser G I, Hilbers C W
Nijmegen SON Research Centre for Molecular Design, Structure and Synthesis, University of Nijmegen, The Netherlands.
Eur J Biochem. 1993 Sep 1;216(2):507-17. doi: 10.1111/j.1432-1033.1993.tb18169.x.
Scrutiny of NOE data available for the protein encoded by gene V of the filamentous phage IKe (IKe GVP), resulted in the elucidation of a beta-sheet structure which is partly five stranded. The DNA-binding domain of IKe GVP was investigated using a spin-labeled deoxytrinucleotide. The paramagnetic-relaxation effects observed in the 1H-NMR spectrum of IKe GVP, upon binding of this DNA fragment, could be visualized using two-dimensional difference spectroscopy. In this way, the residues present in the DNA-binding domain of IKe GVP can be located in the structure of the protein. They exhibit a high degree of identity with residues in the gene V protein encoded by the distantly related phage M13 (M13 GVP), for which similar spectral perturbations are induced by such a spin-labeled oligonucleotide. Binding studies with negatively charged lanthanide-1,4,7,10-tetraazacyclodecanetrayl-1,4,7-10- tetrakis(methylene)tetrakisphosphonic acid (DOTP) complexes, showed that these complexes bind to IKe and M13 GVP at two spatially remote sites whose affinities have different pH dependencies. Above pH 7, there is one high-affinity binding site for Gd(DOTP)5-/M13 GVP monomer, which coincides with the single-stranded DNA-binding domain as mapped with the aid of spin-labeled oligonucleotide fragments. The results show that single-stranded DNA binds to conserved (phosphate binding) electropositive clusters at the surface of M13 and IKe GVP. These positive patches are interspersed with conserved or conservatively replaced hydrophobic residues. At pH 5, a second Gd(DOTP)(5-)-binding site becomes apparent. The corresponding pattern of spectral perturbations indicates the accommodation of patches of conserved, or conservatively replaced, hydrophobic residues in the cores of the M13 and IKe dimers.
对丝状噬菌体IKe基因V编码的蛋白质（IKe GVP）的核Overhauser效应（NOE）数据进行仔细研究，结果阐明了一种部分为五链的β-折叠结构。使用自旋标记的脱氧三核苷酸对IKe GVP的DNA结合结构域进行了研究。在该DNA片段结合后，在IKe GVP的1H-NMR谱中观察到的顺磁弛豫效应，可以使用二维差异光谱进行可视化。通过这种方式，IKe GVP的DNA结合结构域中存在的残基可以定位在蛋白质结构中。它们与远亲噬菌体M13编码的基因V蛋白中的残基具有高度同一性，对于M13 GVP，这种自旋标记的寡核苷酸会诱导类似的光谱扰动。与带负电荷的镧系元素-1,4,7,10-四氮杂环十二烷四氮基-1,4,7,10-四（亚甲基）四膦酸（DOTP）配合物的结合研究表明，这些配合物在两个空间上相距较远的位点与IKe和M13 GVP结合，其亲和力具有不同的pH依赖性。在pH 7以上，Gd（DOTP）5-/M13 GVP单体有一个高亲和力结合位点，该位点与借助自旋标记寡核苷酸片段定位的单链DNA结合结构域重合。结果表明，单链DNA与M13和IKe GVP表面保守的（磷酸盐结合）正电簇结合。这些正性区域散布着保守的或保守取代的疏水残基。在pH 5时，第二个Gd（DOTP）（5-）结合位点变得明显。相应的光谱扰动模式表明，保守的或保守取代的疏水残基区域存在于M13和IKe二聚体的核心中。
