| 研究実績の概要 |
The aim of the project is to express, purify, reconstitute and visualzie the operation of a prokaryotic cyclic nucleotide-gated channels by means of high-speed atomic force microscopy and advanced optical microscopy. So far I have gathered all the necessary material and equipment for the protein production, including competent cells, purification columns, lipids etc. The SthK channel gene (UniProtKB G0GA88)was subcloned in the appropriate vector (pCGFP-BC) for overexpression in bacteria (BL21-DE3). Channel expression, purification and reconstitution in artificial bilayer was implemented succesfully. Sample was also sent to collaborators in Italy for complementary force spectroscopy measurements which already produced unfolding patterns of the protein in the presence and absence of ligand. The latter experiment and research axis (ion channel force spectroscopy) was not initially envisioned and rapresent a positive unexpected project development which will complement the atomic force microscopy topographic measurements.
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| 今後の研究の推進方策 |
Despite the slight delay, no change in the proposal or initially envisioned goals seems necessary at this stage, as I believe it shall be possible to easily make up for the lost time. Within the next six months I expect to optimize reconstitution conditions for high-speed atomic force microscopy (HS-AFM) imaging and gather the first HS-AFM movies of SthK channel conformational changes in responce to cyclic nucleotides. In the following semster we will monitor ligand binding via optical microscopy using fluorescent-labelled ligands and correlate this data to structural information obtained by HS-AFM. It will also be attempted to measure the channel physiological state using established ion flux assays and holey MICA substarates.
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