Structural investigations of ClC‐ec1, a large integral membrane protein, using solution‐state NMR and nanodisc technology

Abstract

CLC antiporters mediate Cl−/H+ exchange across cell membranes in organisms ranging from E. coli to humans via coupling of protein conformational changes to ion binding, unbinding and translocation. X‐ray crystallographic structures have provided essential molecular pictures, but details of conformational changes remain elusive. We are using solution state NMR to probe conformational change in ClC‐ec1, a CLC homolog of known structure. 13C labeling of lysine residues is achieved by post translational reductive methylation. 1H‐13C HSQC spectra reveal reversible, substrate‐dependent spectral changes that may reflect conformational changes. To address the concern that the detergent‐solubilized protein is not in a native environment, we are also using nanodiscs, which consist of two amphipathic helices wrapped around a bilayer patch of lipids surrounding the membrane‐embedded protein. The monomeric, 13C‐methylated mutant form of ClC‐ec1 was incorporated into discs and tested for homogeneity and monodispersity. The 1H‐13C HSQC spectra show substrate‐dependent spectral changes in this system, although further experiments will be needed to determine if these represent functionally‐relevant conformational changes. These studies demonstrate the potential for using solution‐state NMR and nanodisc technology to study ClC‐ec1 in a native lipid environment.Supported by: National Science Foundation