1P296 Simultaneous imaging of dynamic structural and chemical events in protein by high-speed AFM combined with single-molecule TIRFM(27. Bioimaging,Poster,The 52nd Annual Meeting of the Biophysical Society of Japan(BSJ2014))
1P296 高速AFM/1分子蛍光顕微鏡複合機によるタンパク質の構造動態と化学反応の同時イメージング(27. バイオイメージング,ポスター,第52回日本生物物理学会年会(2014年度))

Shingo Fukuda, Takayuki Uchihashi, Ryota Iino, Toshio Ando
2014 Seibutsu Butsuri  
CLEM is one the efficient techniques to elucidate the localization and the structure of target protein. CLEM needs a label, which functions in both fluorescence microscopy and electron microscopy. The candidate of the labels is metallothionein, which is heavy metal binding protein. Our previous studies showed that metallothionein with cadmium ions functions as a label for EM [Nishino et al., 2007]. The other groups showed that, in the presence of cadmium and selenium ions, metallothionein
more » ... nanoparticles like Q-dot, which generates fluorescence [Park et al., 2010]. In fluorescence microscopy, we also observed fluorescence of metallothionein to form nanoparticle. We are now trying to observe larger electron density from metallothionein with cadmium for CLEM. 1P296 高速 AFM/1分子蛍光顕微鏡複合機によるタンパク質の構造 To elucidate the functional mechanism of protein nanomachines driven by chemical reactions, it is crucial to know how chemical events give rise to mechanical events (and vice versa). To this end, fluorescence microscopy has thus far been employed to study chemomechanical coupling mechanism of protein. However it has been difficult to directly visualize the protein's conformational dynamics. Here we develop simultaneous observation system of HS-AFM and single-molecule fluorescence microscopy which enable us to observe conformational dynamics of a molecule and binding/dissociation events of fluorescent ligands. We apply this system to rotor-less F1-ATPase hydrolyzing Cy3-ATP to observe both chemical and structural events that are synchronously occurring therein. Collagenase G (ColG) is a representative bacterial collagenase which is suggested to be processively moving on collagen microfibril and unwinding collagen chains during collagenolysis. The behavior of ColG on collagen fibrils, however, have been still unclarified. To revealing ColG motion on collagen simultaneous observation of substrate collagen structure and collagenase dynamics is beneficial. Here, we applied the high-speed atomic force microscopy to simultaneously observe collagen structure and collagenase dynamics during collagenolysis. In the conference, we show the movies of ColG behavior on collagen degradation and discuss collagen-collagenase dynamics. 1P298 新規共分散 Number and Brightness 法によるグルココルチコ イド受容体二量体の生細胞内空間分布解析 Spatio-temporal distribution analysis of dimeric glucocorticoid receptor using a new Number and Brightness method based on covariance Glucocorticoid receptor (GR) belongs to the nuclear receptor super family. When GR binds to synthetic steroid hormone such as the dexamethasone (Dex), it forms a homodimer and works as a transcription factor for various genes. Most GR exists in cytoplasm in the absence of ligand and translocates to nuclear in the presence of the ligand. However, where GR forms dimer is unclear yet. In this study, the Number and Brightness (N&B) analysis which is a statistical imaging analysis was adapted to visualize the distribution of dimeric GR. For this purpose, a software for the N&B analysis was developed and a shot noise free N&B method using covariance was established. Finally, we performed N&B analysis of EGFP-GR dimers distribution in living cell in the presence of Dex. 1P299 生細胞蛍光イメージングによる ALS 関連変異体 TDP43 の構 造解析 The amyotrophic lateral sclerosis (ALS)-linked mutations of TAR RNA/ DNA-binding protein 43kDa (TDP43) have been identified. However it remains unclear how ALS-linked mutation in TDP43 affects the intact folding state. To investigate structural difference of ALS-linked mutant of TDP43, we performed time-lapse imaging and FRET analysis of TDP43 tagged with fluorescent proteins. As the results, rapid translocation of ALS-linked mutant TDP43 (Q331K) from the nucleus into the cytoplasm was observed during caspase3 activation, suggesting Q331K may form caspase3-sensitive structure. FRET analysis suggests that distance between N and C terminus of TDP43 is more than 10 nm. Further analysis is needed, ALS-linked mutation in TDP43 may potentially involve in protein stability. 1P300 高速原子間力顕微鏡による AAA シャペロン p97 の主要 ATPase(D2)リングの構造変化の直接観察 Direct observation of the structural changes of the major ATPase domain D2 of the AAA chaperone p97 by high-speed atomic force microscopy p97 is an AAA chaperone, which plays crucial roles in a variety of cellular processes. p97 comprises three domains, N-terminal domain and two ATPase domains (D1 and D2), and forms a homo-hexameric ring. Our previous high-speed AFM observation has revealed that the N-D1 ring rotates relative to the D2 ring upon binding to ATP at the major ATPase domain D2. However, the conformational changes of D2 during the cycle of ATP hydrolysis remains to be elucidated. Here, we have succeeded to observe the p97 D2 ring by high-speed AFM. For this purpose, we used Nterminally his-tagged p97 and NTA-conjugated streptavidin. On this substrate, the hexameric ring structure of p97 D2 ring was clearly observed. We will discuss the structural changes of the D2 ring in the presence of ATP.
doi:10.2142/biophys.54.s190_2 fatcat:ktcx5ocaf5dzziccpqz6ilhz7i