Structure-activity relationship of human immunodeficiency virus nucleocapsid protein

1. NCp 8-f1/N11 A的构效关系HIV-2的NC蛋白（NCp 8）是一个由7个氨基酸残基连接的含有两个锌指的49个氨基酸的肽段，称为“碱性氨基酸簇”。研究表明，N-末端锌指结构两侧的碱性氨基酸簇是最小的活性结构域。1998年，确定了包含最小活性结构域的29个氨基酸肽（NCp 8-f1）的三维结构。碱性氨基酸簇具有明确的环状构象。簇合物的构象可能是通过簇合物与锌指之间的氢键来稳定的：在Arg^<27>0和Asn^<11>N_sH（Y. Kodera等人，在本研究中，使用紫外交联测定法分析了具有由Ala取代的Asn^的NCp 8-f1衍生物<11>（NCp 8-f1/N11 A）的RNA结合性质，并通过模拟退火计算的^1H NMR光谱确定了该肽的三维结构。NCp 8-f1/N11 A与RNA的结合特性与NCp 8-f1不同。碱性氨基酸簇的构象尚未确定。这些结果表明，碱性氨基酸簇的构象对于RNA的特异性结合是重要的. NCp 8-f2的三维结构测定结果表明，NCp 8-f2是一个由27个氨基酸组成的多肽，其C端含有锌指和碱性氨基酸簇。确定了NCp 8-f2的三维结构。并与NCp 8-f1、NCp 7-f2和NCp 10.3的结构进行了比较，讨论了其构效关系。制备均匀富集^<15>N的NCp 8将均匀富集^ N的NCp <15>8在E. coli细胞中，并使用泛素融合蛋白系统纯化。已制备5 mg该样品。

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.