Introduction:

In Nature, proteins assemble via multivalent supramolecular interactions that allow them to assemble tenderly and reversibly to form functional nanostructures and retain their functions even in the assembled form. Nucleic acid helicase, tubulin, myosin, kinesin, molecular chaperon, ATP-synthase, channel protein and ribosome are some perfect examples of functional nanostructures of proteins in Nature which are built up from supramolecular assembly of their subunits. Structural and functional excellence of proteins has inspired scientists to synthetically manipulate them and design protein-based nanostructures with a view to achieve novel functional materials. In the present study, I have developed a chemical approach of using multivalent supramolecular interactions to develop nanotubes by assembling cylindrical molecular chaperons one-dimensionally. Innate biological functions of the chaperons, such as, trapping of denatured proteins and their release by adenosine triphosphate (ATP)- fueled machine-like open/close motion were successfully retained in the nanotubes. As a result, the synthetically developed chaperone nanotubes exhibits some novel functions and properties based on the functions and properties of its constituent chemical and biological building blocks. Such functional nanotubes have bright prospects to trigger a paradigm shift in the filed of nanomaterial and nanomedicine.

One-dimensional assembly of chaperonin GroEL:[1]

GroEL is a barrel-shaped tetradecameric protein assembly with an inner diameter of 4.5 nm (Fig. 1a). In biological systems, GroEL traps denatured proteins into its hollow cavity and utilizes its ATP-induced mechanical motion to assist refolding of denatured proteins. GroEL used for the present study (GroEL(SP/MC)) is modified in the entrance parts of its cavity by a number of photochromic (spiropyran/merocyanine) units site-specifically; thereby one may switch certain functions of GroEL by ATP and light. In course of this study, we noticed that GroEL(SP/MC) sometimes displays a higher molecular-mass fraction in size-exclusion chromatography (SEC) and confirmed later that this fraction is caused by one-dimensional (1D) assembly of GroEL(SP/MC) in the presence of divalent metal ions. For obtaining GroEL(SP/MC), we prepared mutaNT(GroEL) (GroELCys: C→A; K311→C, L314→C), which bears 14 cysteine (Cys) residues in each entrance part of the cavity (Fig. 1a). Then, the Cys residues were allowed to react with spirobenzopyran-appended maleimide (SPMI) in 25 mM tris-HCl buffer (pH 7.4; Fig. 1b). During incubation for 12 h at 4 °C, the colorless mixture gradually turned light purple, due to partial isomerization of SP to MC, a known spontaneous process occurring in buffers, to give GroEL(SP/MC). When a 0.6 μM tris-HCl buffer solution of GroEL(SP/MC) was subjected to size exclusion chromatography (SEC), a small shoulder at a shorter retention time was observed along with a major peak due to GroEL(SP/MC) (red, Fig. 2a). To our surprise, addition of MgCl2 to the above solution gave rise to a significant change in SEC. For example, when GroEL(SP/MC) was immersed for 0.5 h with MgCl2 (5 mM) at 37 ℃, a broad elution curve (blue, Fig. 2a) with a peak top elution volume of 2.5 mL emerged at the expense of the peak due to GroEL(SP/MC) (elution volume; 3.2 mL). Dynamic light scattering (DLS) analysis (Fig. 2b) of the resulting mixture (blue) indicated the presence of ca. 7 μm-sized particles (without MgCl2; red). As observed by TEM (Fig. 2d), the mixture contained very long cylindrical nanofibers with a uniform diameter of 15 nm (without MgCl2; Fig. 2c). We found that the supramolecular polymerization of GroEL(SP/MC) takes place via multivalent MC・・metal ion bridging.

Stimuli responsive properties of GroEL nanotubes (NT(GroEL)):[1], [2], [3]

NT(GroEL) consists of three components mainly: (1) divalent metal ions that bridges the MC units via MC・・metal ion complex, (2) photochromic MC units that act as linkers at both ends of cylindrical GroEL and (3) GroEL units that undergoes machine-like open/close motion when fueled by ATP. Due to the stimuli responsiveness of respective components, NT(GroEL) can exhibit unique and novel stimuli responsive properties.

[a] Dissociation of NT(GroEL) in presence of EDTA:[1]

When EDTA, a strong metal ion chelator, was added to the pre-assembled system of GroEL and incubated for 30 min at 37 ℃, the long nanotubes, were cut into short-chain oligomers and, eventually, monomeric GroEL(SP/MC). Dissociation of the nanotubes is confirmed with SEC and TEM.

[b] Photoreversible supramolecular polymerization of GroEL:[3]

Photochromic MC units at both ends of GroEL(SP/MC) can be reversibly photoisomerized to SP. Since NT(GroEL) is formed via a 1:2 complexation of divalent metal ions and MC units, photoisomerization of MC to nonionic SP may trigger the dissociation of the NT(GroEL). In fact, when a polymerized mixture containing NT(GroEL) was exposed to visible light (λ > 400 nm) for 15 min at 25 ℃, a fraction corresponding to original NT(GroEL) in the SEC profile was considerably diminished. Instead, an oligomeric fraction of GroEL consisting mainly of 1mer-4mer emerged which indicates the photoreversible cleavage of long NT(GroEL).

[c] ATP-fueled dissociation of NT(GroEL) due to chemomechanical bond scission:[2]

NT(GroEL) depolymerizes readily into monomeric GroEL units when it senses ATP. As shown in the SEC traces, after a 30-min incubation with ATP, original NT(GroEL) almost diminished, affording its oligomeric fraction consisting mainly of 1mer-3mer (TEM). Not only ATP but also other nucleoside triphosphates such as cytidine-5'-triphosphate (CTP) and uridine-5'-triphosphate (UTP) gave rise to the dissociation of NT(GroEL). In contrast, no dissociation resulted when NT(GroEL) was treated with inosine-5'-triphosphate (ITP) and guanosine-5'-triphosphate (GTP). According to the literature, only CTP and UTP, among the four ATP analogues tested, can induce a conformational change of GroEL. Thus, it is likely that the machine-like open/close motion of each GroEL units in the nanotube drives the dissociation of NT(GroEL). In fact, NT(GroEL) displayed an ATPase activity, which is as large as that of non-polymerized GroELCys.

Trapping and release of denatured protein by NT(GroEL):[2]

Similar to GroEL, GroEL(SP/MC) can trap denatured GFP (GroELMC⊃GFPdenat). In presence of Mg2+ GroEL(SP/MC)⊃GFPdenat can polymerize to form nanotubular NT(GroEL)⊃GFPdenat. Importantly, NT(GroEL)⊃GFPdenat can disassemble too in the presence of ATP (100 μM) and release GFPdenat. Release of non-fluorescent GFPdenat can be traced from its spontaneous refolding to GFP in the neutral buffer, which fluoresces strongly at 508 nm. Importantly, dissociation of NT(GroEL)⊃GFPdenat and release of GFP takes place in a sigmoidal manner in response to the concentration of ATP. Such a nonlinear [ATP]-dependency of the guest-release activity is extremely advantageous for aiming at targeted drug delivery with a fail-safe mechanism. Dissociation of NT(GroEL)⊃GFPdenat and release of GFP was also observed in an ATP-rich intracellular environment (HeLa cell lysate; [ATP] = 125 μM), however, no such phenomena took place in an extracellular environment (fetal bovine serum; [ATP] = 2 μM). These interesting results suggest that NT(GroEL) can differentiate environments on the basis of [ATP] and specifically release guests only to an ATP-rich environment. Furthermore, NT(GroEL) bearing a particular type of boronic acid on the surface can smoothly penetrate into HeLa cells. It is observed that upon penetration, a major portion of the penetrated NT(GroEL) is not trapped in lysosome. Further investigation of this interesting property of NT(GroEL) is underway.

Conclusion:

Thus, from chemically and genetically engineered chaperonin protein, a bionanotube with unique properties and novel functions is achieved. These nanotubes can be further modified at their surface for their targeted delivery and [ATP]-responsive functions. In contrast to most nanocarriers so far reported, an ATP-fueled robotic nanocarriers such as NT(GroEL) may provide a universal strategy for on-demand targeting, since activated immune cells such as lymphocytes and macrophages are known to release large amounts of ATP into extracellular matrices.

Publications:

[1] Biswas, S. et al. J. Am. Chem. Soc. 2009, 131, 7556.

[2] Biswas, S. et al. submitted.

[3] Sendai, T.; Biswas, S. et al. submitted.

Figure 1. (a) A molecular model of mutant chaperonin GroEL(Cys). (b) Schematic illustrations for Mg(2+)-induced one-dimensional assembly of GroEL(SP/MC).

Figure 2. (a) SEC traces (observed at λ = 280 nm) of GroEL(SP/MC) (0.6 μM) without (red) and with MgCl2 (5 mM). (b) DLS profiles of GroELSP/MC without (red) and with MgCl2 (5 mM; blue). TEM images GroEL(SP/MC) (c) without and (d) with MgCl2 (5 mM).

癌をはじめとする難病の治療用ナノロボットの開発は約50年も前にリチャード・P・ファインマンによって予言され、特定の疾患を認知し、その疾患特異的に薬物を投与するための方法として期待を集めてきた。このビジョンを共有した多くの研究者がポリマーやミセル、リポソームなどからなる多様な薬物キャリアの開発に取り組み、光、温度変化、酸化還元、pH変化などの物理的・化学的刺激によって薬物を放出できるシステムの構築に成功してきた。しかし、実用性の観点から見ると、これらは薬物放出の精密さ、生体適合性などの点で課題が残るものであり、完全な『ファインマンロボット』の実現には至っていない。一方、タンパク質の集合体からなる生体分子機械が数多くの重要な生理現象を精密に制御していることが、近年、明らかになってきている。本研究は、アデノシン三リン酸(ATP)などの生体内エネルギー物質に応答して構造末端の開閉運動を繰り返すバレル状の生体分子機械「分子シャペロン」に着目した。本論文では、遺伝子工学的・化学的な手法を用い、分子シャペロンからなるATP応答性型ナノキャリアの実現を目的とした研究について述べている。

第1章では、様々な生体分子機械の構造・機能について概説している。特に、生体エネルギー分子であるATPを駆動力として利用するものについて詳しく紹介している。また、分子シャペロンタンパク質の一種であるGroELを取り上げ、薬物キャリアとしての観点からゲスト分子の取り込み・保護機能について詳細を述べている。

第2章では、遺伝子工学的に作成した変異体のGroELタンパク質を光応答性分子で修飾することによって達成した、GroELタンパク質の一次元超分子重合について述べている。GroELの両端に計28個の化学修飾可能なシステインが配置された変異体にマレイミドを利用してスピロピランを導入し、2価の金属イオンを添加することで、約170個のGroELが一次元に集積化し、2.5 μmにも及ぶナノチューブが得られることを見出している。また、金属イオンに対するキレート剤を加えることでナノチューブの解離が観察されることから、このナノチューブは金属イオンを介する多価的な超分子相互作用によって形成されているものと結論づけている。

第3章では、ATP応答性ロボット型ナノキャリアとしてGroELナノチューブの機能と応用について述べている。一次元的に集積化したチューブ状のGroELにおいてもATPase活性が維持され、さらにATP存在下でナノチューブが解離することを見出している。また、ゲストとなる変性タンパク質を捕捉した状態のGroELも重合可能であり、ゲストを内包したナノチューブを与えることを明らかにしている。さらに、このナノチューブは依然としてATP応答性を有しており、ATPの結合によって誘起されるGroELの機械的構造変化が引き金となってモノマーへと解離し、これと同時にゲストが放出されることを明らかにしている。これは、生体内組織におけるATP濃度の違いを利用した新しいタイプのドラッグデリバリーシステムへの応用につながる結果である。ドラッグデリバリーにおいてキャリアの細胞内移行は重要な課題の一つであるが、本論文ではナノチューブの表面をボロン酸誘導体で修飾することにより、ナノチューブの細胞内取込みを誘導できることを明らかにしている。また、ナノチューブへの薬物の内包に関して、不可逆的に変性させたゲストタンパク質を足場として利用するという汎用性の高い戦略を提示している。生分解性のエステル結合を含むリンカーで薬物とゲストタンパク質を連結することで、細胞内での薬物放出が可能であることを示した。ナノチューブの切断速度がATP濃度にS字形依存性を示すことを明らかにしており、癌組織などのATP濃度の高い部位で選択的に薬物を放出することが期待される。事実、癌を発症させたマウスを用いた実験において、血液中に投与したGroELナノチューブは癌組織に効率よく蓄積することを確認している。

以上のように本論文では、生体分子機械を用いたドラッグデリバリーという新しいコンセプトを打ち出しており、これまでに多くの有機・無機材料由来の薬物キャリアとは全く異なるロボティックナノキャリアとしてナノバイオテクノロジー、ナノ医療分野における今後の発展に大きく寄与することが見込まれる。

よって本論文は博士(工学)の学位請求論文として合格と認められる。