標題: Linker and N-Terminal Domain Engineering of Pyrrolysyl-tRNA Synthetase for Substrate Range Shifting and Activity Enhancement
作者: Jiang, Han-Kai
Lee, Man-Nee
Tsou, Jo-Chu
Chang, Kuan-Wen
Tseng, Hsueh-Wei
Chen, Kuang-Po
Li, Yaw-Kuen
Wang, Yane-Shih
應用化學系
Department of Applied Chemistry
關鍵字: non-canonical amino acids;pyrrolysyl-tRNA synthetase;linker engineering;amber codon suppression;tRNA binding domain
公開日期: 7-四月-2020
摘要: The Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS).tRNA(Pyl) pair can be used to incorporate non-canonical amino acids (ncAAs) into proteins at installed amber stop codons. Although engineering of the PylRS active site generates diverse binding pockets, the substrate ranges are found similar in charging lysine and phenylalanine analogs. To expand the diversity of the ncAA side chains that can be incorporated via the PylRS.tRNA(Pyl) pair, exploring remote interactions beyond the active site is an emerging approach in expanding the genetic code research. In this work, remote interactions between tRNA(Pyl), the tRNA binding domain of PylRS, and/or an introduced non-structured linker between the N- and C-terminus of PylRS were studied. The substrate range of the PylRS.tRNA(Pyl) pair was visualized by producing sfGFP-UAG gene products, which also indicated amber suppression efficiencies and substrate specificity. The unstructured loop linking the N-terminal and C-terminal domains (CTDs) of PylRS has been suggested to regulate the interaction between PylRS and tRNA(Pyl). In exploring the detailed role of the loop region, different lengths of the linker were inserted into the junction between the N-terminal and the C-terminal domains of PylRS to unearth the impact on remote effects. Our findings suggest that the insertion of a moderate-length linker tunes the interface between PylRS and tRNA(Pyl) and subsequently leads to improved suppression efficiencies. The suppression activity and the substrate specificity of PylRS were altered by introducing three mutations at or near the N-terminal domain of PylRS (N-PylRS). Using a N-PylRS.tRNA(Pyl) pair, three ncAA substrates, two S-benzyl cysteine and a histidine analog, were incorporated into the protein site specifically.
URI: http://dx.doi.org/10.3389/fbioe.2020.00235
http://hdl.handle.net/11536/154436
ISSN: 2296-4185
DOI: 10.3389/fbioe.2020.00235
期刊: FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY
Volume: 8
起始頁: 0
結束頁: 0
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