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Paper

Org. Biomol. Chem., 2009, 7, 3102 - 3111, DOI: 10.1039/b901118h

Manipulation of protein-complex function by using an engineered heterotrimeric coiled-coil switch

Toshihisa Mizuno, Kumiko Suzuki, Tatsuya Imai, Yuya Kitade, Yuji Furutani, Motonori Kudou, Masayuki Oda, Hideki Kandori, Kouhei Tsumoto and Toshiki Tanaka

Design methodology of variant proteins, in which original functions can be manipulated by additive ligand binding, is an attractive target of protein engineering. Especially for multi-protein complexes, techniques for constructing variants which allow the switching on or off of original functions by ligands have been limited until now. We examined a method of utilizing a de novo designed protein module, IZ-DS, which has a tertiary structure that can be significantly changed from a random coil to a folded coiled-coil structure following binding with peptide ligand, IZ-3K. By introducing a metamorphosis IZ-DS sequence to one of the components in a target multi-protein complex, the IZ-3K binding and the subsequent structural transition of the IZ-DS moiety would affect the tertiary structure of the introduced protein unit, and the function of the total multi-protein complex may also be altered. In this research, we used the T7 RNA polymerase (T7 RNAP)/T7 lysozyme complex as the target multi-protein complex, in which allosteric binding of the T7 lysozyme to T7 RNAP halts the RNA synthesis of T7 RNAP. The IZ-DS sequence was introduced to the T7 lysozyme. By optimizing the introduction site of the IZ-DS sequence in the T7 lysozyme, we succeeded in constructing the T7 lysozyme variant, DS-Lys23. In the absence of IZ-3K, the mixture of T7 RNAP and DS-Lys23 exhibited RNA synthesis due to the weakening of the interaction between T7 RNAP and DS-Lys23. Whereas, after the addition of IZ-3K, RNA synthesis was significantly suppressed by the binding of DS-Lys23/IZ-3K complex. The present methodology using a designed ligand-dependent metamorphosis protein sequence constitutes another possible method for the de novo manipulation of various functions of natural protein complexes.

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