The intracellular region of ClC-3 chloride channel is in a partially folded state and a monomer.
In contrast to bacterial ClC chloride channels, all eukaryotic ClC chloride channels have a conserved long intracellular region that makes up of the carboxyl terminus of the protein and is necessary for channel functions as a channel gate. Little is known, however, about the molecular structure of the intracellular region of ClC chloride channels so far. Here, for the first time, we have expressed and purified the intracellular region of the rat ClC-3 chloride channel (C-ClC-3) as a water-soluble protein under physiological conditions, and investigated its structural characteristics and assembly behavior by means of circular dichroism (CD) spectroscopy, differential scanning calorimetry (DSC), size exclusion chromatography and analytical ultracentrifugation. The far-UV CD spectra of C-ClC-3 in the native state and in the presence of urea clearly show that the protein has a significantly folded secondary structure consisting of alpha-helices and beta-sheets, while the near-UV CD spectra and DSC experiments indicate the protein is deficient in well-defined tertiary packing. Its Stokes radius is larger than its expected size as a folded globular protein, as determined on size exclusion chromatography. Furthermore, the DisEMBL program, a useful computational tool for the prediction of disordered/unstructured regions within a protein sequence, predicts that the protein is in a partially folded state. Based on these results, we conclude that C-ClC-3 is partially folded. On the other hand, both size exclusion chromatography and sedimentation equilibrium analysis show that C-ClC-3 exists as a monomer in solution, not a dimer like the whole ClC-3 molecule.
Li
Shu Jie
SJ
Department of Biophysics, College of Physics Science, Nankai University, Tianjin 300071, P. R. China. shujieli@protein.osaka-u.ac.jp
Kawazaki
Masanobu
M
Ogasahara
Kyoko
K
Nakagawa
Atsushi
A
D000595:Amino Acid Sequence; D000818:Animals; D002152:Calorimetry, Differential Scanning; D018118:Chloride Channels / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D002712:Chlorides / Q000737:chemistry*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003593:Cytoplasm / Q000737:chemistry; D015640:Ion Channel Gating / Q000502:physiology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry; D014176:Protein Biosynthesis / Q000235:genetics; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D013056:Spectrophotometry, Ultraviolet; D014461:Ultracentrifugation
D018118:Chloride Channels; D002712:Chlorides; C115376:ClC-3 channel; D010446:Peptide Fragments