2019 Fiscal Year Research-status Report
Direct visualization of conformational dynamics of hemagglutinin and interaction between hemagglutinin and exosomes using high-speed atomic force microscopy.
| Project/Area Number |
19K23841
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| Research Institution | Kanazawa University |
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Principal Investigator |
LIM KEE・SIANG 金沢大学, ナノ生命科学研究所, 特任助教 (60842987)
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| Project Period (FY) |
2019-08-30 – 2021-03-31
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| Keywords | HS-AFM / Influenz A / Hemagglutinin / Fusogenic transition / Virulence factor |
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| Outline of Annual Research Achievements |
In summary, this project is running smoothly, and has completed two aims. First, native conformation of HA in neutral buffer has been revealed using HS-AFM. Results show that HA conformation in neutral buffer was ellipsoid. Furthermore, HS-AFM images of HA are used for spatial dimension analysis of HA. Second, conformational change of HA triggered by acidic environment has been captured using HS-AFM. Acidification of HA triggered its conformational change from ellipsoid to Y-shape. In addition, the real-time conformational transition of single HA molecule has also been recorded using HS-AFM. In future, experiments will be performed to investigate the interaction between HA and exosome in neutral or acidic condition. After three aims are achieved, manuscript will be submitted for publication.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
Two aims of this study have been done. The first aim is characterization of native conformation of HA in neutral buffer. Results show that HA appeared to be ellipsoid in neutral buffer. Analysis of spatial dimension parameters of HA has completed. The second aim is to visualize real-time conformational changes of HA in acidic environment. Results indicate that HA underwent conformational change, from ellipsoid to Y-shape, after exposed to acidic environment. These findings support results reported in previous studies that used other imaging techniques. Nevertheless, direct real-time visualization of native conformation and acid-induced conformational change of HA can only be achieved using HS-AFM.
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| Strategy for Future Research Activity |
After achieving the aforementioned aims, this project continues to complete the last aim: interaction between HA and exosome. Experiments will be conducted to observe the interaction between HA and exosome in different pH (neutral and acidic) because the interaction could rely on different mechanism. In acidic environment, conformational change allows fusion peptide to be released from HA and it can directly insert into lipid layer (exosomal layer). In contrast, the interaction between HA and exosome in neutral condition only rely on the interaction between receptor binding domain of HA and its receptor in exosome. It will be interesting to observe these phenomena under HS-AFM at real-time fashion. Once the third aim is completed, manuscript will be prepared for publication.
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| Causes of Carryover |
The requested amount will be used to replenish research materials including cantilevers, mica, influenza A hemagglutinin recombinant proteins, buffers, and to buy new materials for exosome preparation (cell culture reagents and isolation kit). These materials will first use for achieving the third aim. After that, additional experiments will be conducted to solidify the findings for three main aims in this study. Furthermore, remaining funding will be kept for experiments that requested by reviewers.
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Research Products
(2 results)
