(13)C NMR detects conformational change in the 100-kD membrane transporter ClC-ec1.
 

(13)C NMR detects conformational change in the 100-kD membrane transporter ClC-ec1.

(13)C NMR detects conformational change in the 100-kD membrane transporter ClC-ec1.

J Biomol NMR. 2015 Jan 29;

Authors: Abraham SJ, Cheng RC, Chew TA, Khantwal CM, Liu CW, Gong S, Nakamoto RK, Maduke M

Abstract
CLC transporters catalyze the exchange of Cl(-) for H(+) across cellular membranes. To do so, they must couple Cl(-) and H(+) binding and unbinding to protein conformational change. However, the sole conformational changes distinguished crystallographically are small movements of a glutamate side chain that locally gates the ion-transport pathways. Therefore, our understanding of whether and how global protein dynamics contribute to the exchange mechanism has been severely limited. To overcome the limitations of crystallography, we used solution-state (13)C-methyl NMR with labels on methionine, lysine, and engineered cysteine residues to investigate substrate (H(+)) dependent conformational change outside the restraints of crystallization. We show that methyl labels in several regions report H(+)-dependent spectral changes. We identify one of these regions as Helix R, a helix that extends from the center of the protein, where it forms the part of the inner gate to the Cl(-)-permeation pathway, to the extracellular solution. The H(+)-dependent spectral change does not occur when a label is positioned just beyond Helix R, on the unstructured C-terminus of the protein. Together, the results suggest that H(+) binding is mechanistically coupled to closing of the intracellular access-pathway for Cl(-).


PMID: 25631353 [PubMed - as supplied by publisher]



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