2020 Fiscal Year Research-status Report
Nanoscale probing and visualization of cyclic nucleotide-gated ion channels structural dynamics and function
| Project/Area Number |
20K22626
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| Research Institution | Kanazawa University |
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Principal Investigator |
Marchesi Arin 金沢大学, ナノ生命科学研究所, 特任助教 (80882240)
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| Project Period (FY) |
2020-09-11 – 2022-03-31
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| Keywords | Atomic Force Microscopy / Ion channels / Purification / Gating |
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| Outline of Annual Research Achievements |
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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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
At this stage I would already expected to gather some high-speed atomic force microscopy imaging of the ion channel. There is a slight delay of around 2 months from my original plans. This is attributed to two main factors: (1) gathering of all the necessary consumables took longer than was originally estimated (2) I could not catch up with the lost time becouse of an urgent impending manuscript revision (in this paper I am first and corresponding author) which overlapped with the project in the last two months.
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| Strategy for Future Research Activity |
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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Research Products
(1 results)
