Structural‐functional studies of purified transmembrane segment VI‐VII of the human isoform 1 of the Na+/H+ exchanger

Abstract

The Na+/H+ exchanger isoform 1 (NHE1) is a ubiquitous pH membrane protein in mammalian cells. NHE1 regulates intracellular pH exchanging one intracellular H+ for one extracellular Na+. Though the structure of membrane proteins is highly desirable, expression remains problematic. In this study we expressed and purified transmembrane (TM) VI‐VII of the NHE1 protein. A 49 kDa Maltose‐Binding‐Protein‐TMVI‐VII fusion protein was expressed in E. coli XL1 Blue cells and purified with an Amylose resin. After cleavage with Tobacco Etch Virus, free TM VI‐VII, 6.2 kDa protein was isolated and purified by reverse phase HPLC. The identity was confirmed by Mass Spectroscopy. The structure of 15N isotopically labeled TM VI‐VII peptide was determined in dodecylphosphocholine micelles using solution‐state 2D and 3D NMR. TM VI‐VII contains a helix‐kink‐helix structure for TM VI with helices between amino acids 229–236 and 239–247. TM VII was a continuous helix from 252–274. The data suggests that the C‐terminal half of TM VI interacts with the N‐terminal half of TM VII. Future studies are further defining the helix‐helix interface between residues of TM VI and TM VII.