Abbott, Geoffrey Winston; (1997) Structural characterisation of recombinant and synthetic voltage-gated potassium channel domains. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

A number of recombinant and synthetic domains from several voltage-gated potassium channels have been produced and characterised using biochemical and biophysical techniques. A recombinant protein corresponding to the cytoplasmic N-terminal region (amino acids 14-162) of Kv1.l delayed rectifier, voltage-gated potassium (Kv) channel, containing the T1 domain implicated as being important in the assembly of ?-subunits into functional tetrameric channels, has been expressed in E.coli, extracted, purified and its structure examined using Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopy. The N-terminal region is found to adopt a loosely-folded conformation, comprising 20-25 % α-helix, 20-25 % β-sheet, 20 % turns and 35 % aperiodic structures. Using sucrose gradient sedimentation it is demonstrated that the Kv1.l N-terminal region is capable of self-assembling into tetramers, and that this process is disrupted by addition of a non-ionic detergent. The spectroscopic results are compared to the structure predicted using THREADER molecular modelling, and a putative supersecondary structure is proposed. A recombinant protein corresponding to the cytoplasmic N-terminal region (amino acids 1-227) of Ky3.4 A-type Kv channel, containing the inactivation ball domain which facilitates fast-inactivation of potassium channels, has also been expressed in E.coli, extracted, purified and examined spectroscopically. Differences occur in secondary structure content dependent upon mode of extraction and purification; furthermore there are discrepancies between structure content predictions from FTIR and CD spectroscopy. These differences are discussed and compared with the results of THREADER structure prediction. A synthetic peptide corresponding to the Kv3.4 inactivation ball domain (amino acids 1-28) is structurally characterised using FTIR and CD spectroscopy, and THREADER modelling. Two forms of the ball peptide are examined: one is a reduced form and one is an oxidised form in which the cysteine residues at positions 6 and 24 are linked by disulphide bond. The inactivation ball is proposed to bind to a hydrophobic, anionic site in the Kv channel to facilitate fast-inactivation. Spectroscopic results indicate that the reduced form of the ball peptide is non-ordered in aqueous solution and in zwitterionic lipids, but that it adopts to some extent a ?-hairpin structure when challenged by anionic lipid micelles or anionic detergent. The oxidised form of the ball peptide is capable of adopting the ?-hairpin conformation in aqueous solution, but adopts a greater proportion of ordered structure in anionic lipids and detergents. Results are discussed in terms of functional implications, and suggestions that oxidation of the ball peptide may be a mode of regulation in vivo. A synthetic polypeptide corresponding to the S4S5H5S6 domains (voltage sensor and pore region) of the Shaker B A-type Kv channel is used in conjunction with the synthetic Kv3.4 inactivation ball peptide to develop an in vitro model of the events which occur during channel inactivation in vivo. The peptides are solubilised in a mixture containing trifluorocthanol and dimyristoyl phosphatidylcholine (DMPC), and conformational changes monitored using FTIR spectroscopy. Temperature changes which result in a shift of the DMPC below' its fluid-gel transition temperature are used to move the S4 region out of the lipid and into the aqueous medium, mimicking the movement which the S4 region undergoes in response to membrane depolarisation in vivo. A shift in the structure of parts of the S4S5H5S6 polypeptide from a-helix to partly β-sheet is observed following this S4 movement, but only in the presence of the inactivation ball peptide. This shift, which is fully reversible upon a temperature change which results in movement of the S4 region back into the DMPC, is characterised in terms of effect of differing salt concentrations, concentrations of ball peptide and pH, and the effect of oxidation of the ball peptide is also examined. Results are related to existing ideas about channel inactivation and a model of inactivation is proposed. Finally, a recombinant protein corresponding to the S5H5S6CT (pore region and C-terminal region) of Shaker B channel is expressed in two different strains of E.coli. In UT5600 strain E.coli, which are deficient in the outer membrane protease ompT, a full-length protein is expressed. In JM109 strain E.coli which express ompT, however, the recombinant Shaker B pore region is cleaved away from the C-terminal region. The relevance of this ompT processing in terms of improving existing methods of production of recombinant K v channel membrane-spanning regions is discussed. Preliminary purification and structural characterisation of the recombinant pore region are performed.

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