title: Role of arginine 29 and glutamic acid 81 interactions in the conformational stability of human chloride intracellular channel 1.
abstract: The ion channel protein CLIC1 exists in both a soluble conformation in the cytoplasm and a membrane-bound conformation. The conformational stability of soluble CLIC1 demonstrates pH sensitivity which may be attributable to very specific residues that function as pH sensors. These sensors could be histidine or glutamate residues with pK(a) values that fall within the physiological pH range. The role of Glu81, a member of a topologically conserved buried salt bridge in CLIC1, as a pH sensor was investigated here. The mutants E81M, R29M, and E81M/R29M were designed to break the salt bridge between Glu81 and Arg29 and examine the effect of each member on the stability of the protein. Spectroscopic studies and the solved crystal structures indicated that the global structure of CLIC1 was not affected by the mutations. Urea-induced equilibrium unfolding unexpectedly showed E81M to stabilize CLIC1 at pH 7. This was due to stabilizing hydrophobic interactions with Met81 and a water-mediated compensatory H-bond between Met81 and Arg29. R29M and E81M/R29M destabilized CLIC1 at pH 7, and the unfolding transition changed from two-state to three-state, mimicking the wild type at pH 5.5. This observation points out the significance of the salt bridge in stabilizing the native state. The total unfolding free energy change of E81M CLIC1 does not change with pH, implying that Glu81 forms one of a network of pH-sensor residues in CLIC1 responsible for destabilization of the native state. This allows detachment of the N-domain from the C-domain at low pH.
authors.affiliation: Protein Structure-Function Research Unit, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg 2050, South Africa.
authors.forename: Derryn
authors.initials: D
authors.lastname: Legg-E'silva
authors.forename: Ikechukwu
authors.initials: I
authors.lastname: Achilonu
authors.forename: Sylvia
authors.initials: S
authors.lastname: Fanucchi
authors.forename: Stoyan
authors.initials: S
authors.lastname: Stoychev
authors.forename: Manuel
authors.initials: M
authors.lastname: Fernandes
authors.forename: Heini W
authors.initials: HW
authors.lastname: Dirr
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry*; D018118:Chloride Channels / Q000737:chemistry*; D003460:Crystallization; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D018698:Glutamic Acid / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D017510:Protein Folding; D055550:Protein Stability; D013050:Spectrometry, Fluorescence; D014961:X-Ray Diffraction
chemical_list: C546620:CLIC1 protein, rat; D018118:Chloride Channels; D018698:Glutamic Acid; D001120:Arginine