Structure and Function of Modular Elements in Proteins.

1.我们开展了与蛋白质结构和功能单元相对应的多肽的制备工作。为此，我们构建了一个‘启动子-终止子载体’，其中起始密码子和终止密码子都是由该载体提供的，从而能够直接表达截短的基因。首先将表达载体pKK223-3改造成‘ATG载体’，然后将标签密码子作为Tth111-I重组子的一部分加入。GACTTAGTC，产生表达载体pHT6。用BamHI和Tth111-I消化，然后操纵粘连末端，分别暴露ATG和标签密码子。用同样的方法从pKK233-2中构建了另一个表达载体pHT8。为了裂解5‘-seq.在Lys-1密码子(Aaa)之前的溶菌酶cdna中，我们构建了一个质粒pht20；用ba131酶切pht20，经DARAL酶切和分子内连接后，将产生一个可再生DRAL…的克隆更多站点仅用于所需的克隆。克隆的成功选择将导致模块片段的融合表达。在NBRF数据库中广泛搜索鸡溶菌酶与其他蛋白质的局部同源性，结果表明模块2+3与N，O-二乙酰胞壁酰胺酶以及噬菌体Lambda Endo-lysin有显著的同源性，它们的催化功能与溶菌酶相似。模3与纤溶酶原中kringle之间的这种同源性以及模5与Ikappa链之间的同源性加强了HAT模块具有结构和功能单位的内在本质的概念。通过氢交换-红外光谱分析，发现溶菌酶中的链内交联物不是直接稳定天然结构，而是通过链熵降低去稳定未折叠状态而间接稳定天然结构。此外，对三维晶格模型蛋白质的蒙特卡罗计算机模拟表明，两个或更多致密的局部亚结构的融合作为折叠反应的核心。较少

1. We undertook a work for preparation of polypeptides corresponding to modules which are proposed to be structural and functional units of proteins. To this end, we constructed an 'initiator-terminator vector' in which both the initiation and termination codons are supplied from the vector, enabling a direct expression of a truncated genes. The expression vector pKK223-3 was first modified into an 'ATG vector', and then the TAG codon was incorporated as part of Tth111-I recog. seq., GACTTAGTC, yielding an expression vector pHT6. Digestion with BamHI and Tth111-I, followed by manipulation of the cohesive ends, exposes the ATG and TAG codons, respectively. Also, another expression vector pHT8 was constructed from pKK233-2 in a similar way.2. In order to cleave the 5'-seq. of lysozyme cDNA immediately before the codon for Lys-1 (AAA), we constructed a plasmid, pHT20; Ba131 digestion of Xhol-cut pHT20, Dral digestion, and intramolecular ligation will yield a clone that regenerates a Dral … More site only for the desired clone. Successful selection of the clone will lead to expression of fusions of modular segments.3. Extensive search for local homologies between chicken lysozyme and other proteins in the NBRF data base showed a significant homology between modules 2+3 and N,O-diacetylmuramidase as well as bacteriophage lambda endo- lysin, whose catalytic functions are similar to that of lysozyme. This and other homology between module 3 and the kringle in plasminogen, and that between module 5 and I kappa chain reinforced the notion hat modules have intrinsic nature of structural and functional units.4. By means of hydrogen-exchange-ir spectroscopy, an intrachain cross-linking in lysozyme was found not to stabilize the native structure directly, but to stabilize it indirectly by de stabilizing unfolded state through chain-entropy decrease. Also, Monte Carlo computer simulations for 3-D-lattice-model proteins showed that a fusion of two or more compact, local sub-structures served as nucleus for the folding reaction. Less