Structure-activity relationship of human immunodeficiency virus nucleocapsid protein

1. NCp8-f1/ n11a的结构-活性关系HIV-2的NC蛋白NCp8是一个49 AA的肽，含有两个锌指，由七个称为“碱性氨基酸簇”的氨基酸残基连接。结果表明，以碱性氨基酸簇为侧翼的n端锌指是活性最小的结构域。1998年，测定了包含最小活性结构域的29个氨基酸肽（NCp8-f1）的三维结构。碱性氨基酸簇具有环状构象。团簇的构象可能是由团簇和锌指之间的氢键稳定的：在Arg^<27>和Asn^<11> N_sH之间（Y. Kodera et al., Biochemistry, 37, 1998）。本研究采用紫外交联法分析了Asn^<11>被Ala取代的NCp8-f1衍生物（NCp8-f1/N11A）的rna结合特性，并采用模拟退火计算的^1H NMR谱法确定了该肽的三维结构。NCp8-f1/N11A的rna结合特性与NCp8-f1不同。碱性氨基酸团簇的构象尚未确定。这些结果表明，碱性氨基酸簇的构象对特异性RNA结合很重要。NCp8-f2NCp8-f2的三维结构测定是一个27个氨基酸的肽，包括c端锌指和碱性氨基酸簇。测定了NCp8-f2的三维结构。通过与NCp8-f1、NCp7-f2和NCp10.3的结构比较，讨论了其构效关系。制备均匀富集^<15>N的NCp8NCp8，这是确定NCp8和NCp8- rna复合物三维结构所必需的，在大肠杆菌细胞中表达并使用泛素融合蛋白系统纯化。该样品已制备5mg。

1. Structure-activity relationships of NCp8-f1/N11AThe NC protein, NCp8, of HIV-2 is a 49 AA peptide containing two zinc fingers connected by seven amino acid residues called "basic amino acid cluster". It has been shown that the N-terminal zinc finger flanked by basic amino acid cluster is the minimal active domain. In 1998, three-dimensional structure of a 29 amino acid peptide (NCp8-f1) including the minimal active domain was determined. The basic amino acid cluster was well-defined with a loop-like conformation. The conformation of the cluster is likely to be stabilized by the hydrogen bonds between the cluster and the zinc finger: between Arg^<27> 0 and Asn^<11> N_sH (Y. Kodera et al., Biochemistry, 37, 1998).In the present study, the RNA-binding property of NCp8-f1 derivative with Asn^<11> replaced by Ala (NCp8-f1/N11A) was analyzed using ultraviolet cross-linking assay and the three-dimensional structure of this peptide was determined by ^1H NMR spectroscopy with simulated annealing calculations. The RNA-binding property of NCp8-f1/N11A was different from that of NCp8-f1. The conformation of the basic amino acid cluster was not defined. These results suggest mat the conformation of the basic amino acid cluster is important for specific RNA binding.2. Determination of the three-dimensional structure of NCp8-f2NCp8-f2 is a 27 amino acid peptide, which includes the C-terminal zinc finger and the basic amino acid cluster. The three-dimensional structure of NCp8-f2 was determined. The structure-activity relationships are discussed on the basis of the comparison of this structure with those of NCp8-f1, NCp7-f2 and NCp10.3. Preparation of uniformly ^<15>N-enriched NCp8NCp8 uniformly enriched with ^<15>N, which is necessary to determine the three-dimensional structure of the NCp8 and the NCp8-RNA complex, was expressed in E. coli cells and purified using a ubiquitin fusion protein system. 5mg of this sample has been prepared.