| 研究実績の概要 |
This year I investigated the DNA-mediated assembly of gold nanoparticles (AuNPs) and the protein GroEL into various structures, including GroEL-covered AuNPs, porous hybrid networks, and GroEL-DNA lamellar structures. The chaperonin protein GroEL was employed for its shape persistent barrel structure, and ATP-induced biomolecular machine functions.
Having previously obtained a series of AuNP-GroEL co-monomers with different numbers of GroEL covering individual AuNPs, I identified agarose gel electrophoresis as the most promising technique for the separation of these distinct species. While full separation has not yet been achieved, I obtained separate fractions with over 50% composition of AuNP-GroEL4 and AuNP-GroEL6, having 4 and 6 GroEL per nanoparticle respectively, as observed by transmission electron microscopy.
I studied the assembly of porous clusters of AuNPs and GroEL and found that the assembly proceeds on the order of hours, with the size of formed clusters tunable by factors including AuNP:GroEL ratio, overall concentration, and buffer conditions. Once formed, these clusters are stable against dilution, mixing, and the addition of an excess of one of the monomer species, however can be repeatedly dissociated and re-assembled through heating and cooling cycles. Further, the constituent GroEL in porous clusters retain their ATPase activity.
I made the serendipitous observation of the ATP-induced assembly of GroEL-DNA and began to study the roles of ATP, GroEL, and DNA in the assembly. The investigation of the mechanism involved and broader implications is ongoing.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
So far, I have expanded the class of GroEL-based assembly architectures to include GroEL-covered AuNPs, GroEL-AuNP porous hybrid networks, and GroEL-DNA lamellar structures.
As for the work on GroEL-functionalized AuNPs I set the goal to achieved my goal of complete separation of distinct species so that they may be assembled into an ordered network structure. So far I have not achieved this goal, however the control over protein-nanoparticle stoichiometry, with greater than 50% composition of individual species, is an achievement which can stimulate related work in the field of materials engineering for biomedicine and biotechnology.
The work on GroEL-AuNP porous hybrid networks has yielded a series of clusters with controllable surface character, size-dependent colloidal stability, stimuli-switchable stability/dynamicity, and ATPase activity. The demonstration of further properties such as guest loading and release, ATP-induced structural transformations, or internal enzymatic activity should lead to this work being submitted for publication to a high-level journal.
The recent serendipitous discovery of ATP induced GroEL-DNA assembly was completely unexpected and as such, the mechanistic studies are still ongoing. After confirming the assembly mechanism I should have a better idea about the generalizability and broader implications of these assemblies. This work may have significant implications in biology, DNA nanotechnology, protein-based materials engineering and/or supramolecular chemistry.
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| 今後の研究の推進方策 |
I will focus on three unique architectures of gold nanoparticle (AuNP)-biomolecular machine GroEL assemblies. These are stoichiometrically-controlled GroEL-functionalized AuNPs, AuNP-GroEL porous networks, and GroEL lamellar assemblies.
For the stoichiometrically-controlled GroEL-functionalized AuNPs, I plan to investigate their biomolecular machine function with ATPase and protein refolding assays and further assembly into higher order assembly architectures.
As for the AuNP-GroEL porous networks, I plan to investigate their ATPase activity, guest loading and release capabilities with fluorescent proteins as well as characterize their internal ordering, using TEM, cryo-TEM, TEM tomography, or SAXS.
Finally, for the GroEL lamellar assemblies, I plan to investigate their assembly mechanism and the effect of ATP on the assembly. I finally intend to investigate the generalizability and broader implications of these assemblies.
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