Structural studies of a membrane embedded chloride channel from E.Coli, a prokaryotic homologue of ClC channels

Thesis

The ClC family of chloride channels are found throughout the eukaryotes and homologues are found among the prokaryotes. There are nine ClC chloride channels found in mammals that are expressed either ubiquitously or in specific tissues. They have a role in a broad spectrum of cellular functions, ranging from the control of electrical excitability of neurons and the maintenance of the balance of charge required in the acidification of organelles, to the regulation of cell volume. The physiological importance of these channels is often demonstrated by disease causing mutations. Mutations in ClC chloride channels cause myotonias, Becker’s Disease and have been implicated in the spread of cancer. The structural information on members of the ClC family is extremely limited. For this reason ClC prokaryotic homologues, ecClC1 and ecClC2 from E.coli, were cloned and expressed in the hope that this system would produce sufficient amounts of protein to grow crystals in order to obtain three-dimensional information. This information will provide a detailed mechanism of chloride conduction and molecular architecture of the channel necessary for the further physiological and pharmacological characterisation of this important family of chloride channels. The E.coli ClC chloride channel ecClC was cloned from E.coli genomic DNA and overexpressed in E.coli. The recombinant protein was purified from the E.coli membranes using detergents. Metal affinity and size exclusion chromatography were used to purify the protein to near homogeneity. Crystals of ecClC2 have been grown that diffract to 4Å and structural analysis is underway. This thesis describes the development of the purification protocol, the crystallisation of the protein and the preliminary analysis of the X-ray diffraction data.

Authors: Bowler, M; Matthew Bowler

• Publication date: 2001
• Type of award: MSc
• Level of award: Masters
• Awarding institution: University of Oxford
• Language: English
• Keywords: prokaryote; ion channel; CLC; purification; Chloride channel; membrane protein; X-ray crystallography; structure
• Subjects: Molecular biophysics (biochemistry); Biochemistry