Structure and Function of Modular Elements in Proteins.

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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