title: Role of individual histidines in the pH-dependent global stability of human chloride intracellular channel 1.
abstract: Chloride intracellular channel proteins exist in both a soluble cytosolic form and a membrane-bound form. The mechanism of conversion between the two forms is not properly understood, although one of the contributing factors is believed to be the variation in pH between the cytosol (~7.4) and the membrane (~5.5). We systematically mutated each of the three histidine residues in CLIC1 to an alanine at position 74 and a phenylalanine at positions 185 and 207. We examined the effect of the histidine-mediated pH dependence on the structure and global stability of CLIC1. None of the mutations were found to alter the global structure of the protein. However, the stability of H74A-CLIC1 and H185F-CLIC1, as calculated from the equilibrium unfolding data, is no longer dependent on pH because similar trends are observed at pH 7.0 and 5.5. The crystal structures show that the mutations result in changes in the local hydrogen bond coordination. Because the mutant total free energy change upon unfolding is not different from that of the wild type at pH 7.0, despite the presence of intermediates that are not seen in the wild type, we propose that it may be the stability of the intermediate state rather than the native state that is dependent on pH. On the basis of the lower stability of the intermediate in the H74A and H185F mutants compared to that of the wild type, we conclude that both His74 and His185 are involved in triggering the pH changes to the conformational stability of wild-type CLIC1 via their protonation, which stabilizes the intermediate state.
authors.affiliation: Protein Structure-Function Research Unit, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg 2050, South Africa.
authors.forename: Ikechukwu
authors.initials: I
authors.lastname: Achilonu
authors.forename: Sylvia
authors.initials: S
authors.lastname: Fanucchi
authors.forename: Megan
authors.initials: M
authors.lastname: Cross
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; D018118:Chloride Channels / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D006639:Histidine / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007424:Intracellular Fluid / Q000737:chemistry / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D055550:Protein Stability; D017433:Protein Structure, Secondary / Q000235:genetics; D011522:Protons
chemical_list: C501966:CLIC1 protein, human; D018118:Chloride Channels; D011522:Protons; D006639:Histidine