title: An NMR study of the N-terminal domain of wild-type hERG and a T65P trafficking deficient hERG mutant.
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.
authors.affiliation: Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), Singapore.
authors.forename: Shovanlal
authors.initials: S
authors.lastname: Gayen
authors.forename: Qingxin
authors.initials: Q
authors.lastname: Li
authors.forename: Angela Shuyi
authors.initials: AS
authors.lastname: Chen
authors.forename: Thi Hanh Thuy
authors.initials: TH
authors.lastname: Nguyen
authors.forename: Qiwei
authors.initials: Q
authors.lastname: Huang
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Hill
authors.forename: CongBao
authors.initials: C
authors.lastname: Kang
mesh_terms: D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D051638:Ether-A-Go-Go Potassium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008969:Molecular Sequence Data; D017510:Protein Folding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D013696:Temperature
chemical_list: D051638:Ether-A-Go-Go Potassium Channels