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 bacteria to mammals. This exchange is accomplished through elegant coupling of protein conformational changes to ion binding, unbinding and translocation events. To understand this mechanism, molecular details of the different conformations and the dynamics of their interchange must be known. While X-ray crystallographic structures have provided essential molecular pictures, the details of protein conformational change have remained elusive. To address this issue, we are using solution-state NMR to probe conformational change in ClC-ec1, a well-characterized CLC homolog of known structure. Selective 13C labeling of lysine residues and the N-terminus is achieved by post-translational reductive methylation. 1H-13C HSQC spectra of these samples reveal reversible, substrate-dependent spectral changes that may reflect protein conformational changes (studies in progress). A chronic concern with the use of detergent-solubilized protein is the possible effects of the non-native detergent environment. To address this concern we are also implementing nanodisc technology. Nanodiscs present a promising alternative for studying membrane proteins such as CLCs, offering both a native bilayer environment and a size small enough for solution-state NMR studies. Each disc consists of two amphipathic helices wrapped around a bilayer patch of lipids surrounding the membrane-embedded protein. The protocol for the incorporation of the monomeric, 13C-methylated mutant form of ClC-ec1 was optimized and the samples tested for homogeneity and monodispersity. The 1H-13C HSQC spectra of methylated, monomeric ClC-ec1 in nanodiscs are being investigated, with preliminary results indicating substrate-dependent spectral changes in this system. While further experiments will be needed to determine if these spectral changes represent functionally-relevant conformational changes, these studies demonstrate the potential for using solution-state NMR and nanodisc technology to study ClC-ec1 structure in a native lipid environment.