An NMR study of the N-terminal domain of wild-type hERG and a T65P trafficking deficient hERG mutant |
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Authors: | Gayen Shovanlal Li Qingxin Chen Angela Shuyi Nguyen Thi Hanh Thuy Huang Qiwei Hill Jeffrey Kang CongBao |
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Institution: | Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), Singapore. |
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Abstract: | The human Ether-à-go-go Related Gene (hERG) potassium channel plays an important role in the heart by controlling the rapid delayed rectifier current. The N-terminal 135 residues (NTD) contain a Per-Arnt-Sim (PAS) domain and an N-terminal amphipathic helix. NMR relaxation analysis and H/D exchange experiments on the NTD demonstrated that the amphipathic helix is rigid and solvent accessible. An NTD containing a T65P mutation, which causes a hERG channel trafficking deficiency, was purified from E.coli. The mutant protein did not aggregate in gel filtration analysis and the amide cross peaks of its residues disappeared in an HSQC spectrum indicating the possibility of structural changes. A carbon chemical shift comparison of the residues with cross peaks in the HSQC spectrum showed no clear difference between the purified wild-type protein and the purified mutant. There were multiple conformations observed for the T65P mutant protein at high temperatures from HSQC experiments and a thermal stability assay showed that the T65P mutation reduced the thermal stability of NTD. This instability may affect protein folding or structural dynamics of other regions. |
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Keywords: | potassium channel hERG NMR spectroscopy PAS domain amphipathic helix thermal stability |
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