title: 13C NMR detects conformational change in the 100-kD membrane transporter ClC-ec1.
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(-).
authors: Department of Molecular & Cellular Physiology, Stanford University School of Medicine, 279 Campus Drive West, Stanford, CA, 94305, USA.
authors: Sherwin J
authors: SJ
authors: Abraham
authors: Ricky C
authors: RC
authors: Cheng
authors: Thomas A
authors: TA
authors: Chew
authors: Chandra M
authors: CM
authors: Khantwal
authors: Corey W
authors: CW
authors: Liu
authors: Shimei
authors: S
authors: Gong
authors: Robert K
authors: RK
authors: Nakamoto
authors: Merritt
authors: M
authors: Maduke
mesh_terms: D017920:Antiporters / Q000648:ultrastructure*; D002250:Carbon Radioisotopes; D066241:Carbon-13 Magnetic Resonance Spectroscopy / Q000379:methods*; D027963:Chloride-Bicarbonate Antiporters / Q000648:ultrastructure*; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000648:ultrastructure*; D008239:Lysine / Q000737:chemistry; D008715:Methionine / Q000737:chemistry; D008745:Methylation; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
chemical_list: D017920:Antiporters; C570787:CLC-ec1 protein, E coli; D002250:Carbon Radioisotopes; D027963:Chloride-Bicarbonate Antiporters; D029968:Escherichia coli Proteins; D008715:Methionine; D008239:Lysine; D003545:Cysteine
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