title: Backbone assignment and secondary structure of the PLAT domain of human polycystin-1.
abstract: Polycystin-1 is a large transmembrane protein mutated in the common genetic disorder autosomal dominant polycystic kidney disease. One of the predicted intracellular domains of polycystin-1 is PLAT (Polycystin-1, Lipoxygenase and Alpha Toxin), which consists of 116 amino acids and is anchored to the membrane by linkers at both ends. It is predicted to have a large number of hydrophobic residues on the surface. Assignment of the NMR spectrum was hampered by considerable line broadening, and hence a programme of site-directed mutagenesis and searching for suitable solution conditions was undertaken. The optimum construct required fusion of the GB1 domain at the N-terminus and a His tag at the C-terminus, and proved to have several additional amino acids at both ends beyond the canonical domain boundaries, as well as mutation of W3128 to alanine. Optimum solubility required 500 mM sodium chloride, and usable spectra could only be obtained by perdeuteration. Backbone assignment was made using standard triple resonance spectra and is 88 % complete. The chemical shifts obtained suggest that a loop consisting of residues 3223-3228 is mobile in solution, and that the protein is similar in structure to a prediction produced by Swiss-Model based on the structure of a homologous protein.
authors.affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield, S10 2TN, UK.|Kidney Genetics Group, Academic Unit of Nephrology, Department of Infection and Immunity, University of Sheffield Medical School, Sheffield, S10 2RX, UK.
authors.forename: Yaoxian
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Kidney Genetics Group, Academic Unit of Nephrology, Department of Infection and Immunity, University of Sheffield Medical School, Sheffield, S10 2RX, UK. a.ong@sheffield.ac.uk.
authors.forename: Albert C M
authors.initials: AC
authors.lastname: Ong
authors.affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield, S10 2TN, UK. m.williamson@sheffield.ac.uk.
authors.forename: Mike P
authors.initials: MP
authors.lastname: Williamson
authors.affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield, S10 2TN, UK.
authors.forename: Andrea M
authors.initials: AM
authors.lastname: Hounslow
mesh_terms: D006801:Humans; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D050396:TRPP Cation Channels / Q000737:chemistry*
chemical_list: D050396:TRPP Cation Channels
keywords: ADPKD; Hydrophobic; Kidney; PLAT; Perdeuteration; Polycystin-1