NMR Structure of a Double-Transmembrane Fragment from NHE1

Abstract

The Na+/H+ exchanger isoform 1 (NHE1) is an integral membrane protein important in the regulation of intracellular pH in the heart. NHE1 contains an N-terminal transmembrane domain that exchanges one intracellular H+ for one extracellular Na+ and a C-terminal cytoplasmic tail involved in regulation. Its membrane domain is predicted to contain 12-14 transmembrane helices, however no other detailed structural information is currently available. Our labs have previously used solution-state NMR spectroscopy to determine structures of individual transmembrane helices of NHE1 in either detergent micelles or organic solvents and have found them to contain non-helical regions which correspond to functionally important regions in the protein as determined by mutagenesis studies.To gain additional insights into the structure and function of NHE1 we are investigating the structure of a construct such as a double-transmembrane helix fragment of NHE1 containing TM 6-7 (amino acids 226-274). We have been able to produce unlabelled and 15N labelled peptide using a maltose binding protein fusion construct. We found the peptide has favourable spectral properties for solution-state NMR structure determination in detergent micelles. We will characterize the structure of the peptide in detergent micelles using solution-state NMR and in magnetically aligned bicelles using solid-state NMR experiments.