Faculty Profiles - TAMA Florence

Papers - TAMA Florence

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NMMD: Efficient cryo-EM flexible fitting based on simultaneous Normal Mode and Molecular Dynamics atomic displacements. Reviewed International journal Open Access

Rémi Vuillemot, Osamu Miyashita, Florence Tama, Isabelle Rouiller, Slavica Jonic

Journal of molecular biology Vol. 434 ( 7 ) page： 167483 - 167483 2022.4

Language：English Publishing type：Research paper (scientific journal)

Atomic models of cryo electron microscopy (cryo-EM) maps of biomolecular conformations are often obtained by flexible fitting of the maps with available atomic structures of other conformations (e.g., obtained by X-ray crystallography). This article presents a new flexible fitting method, NMMD, which combines normal mode analysis (NMA) and molecular dynamics simulation (MD). Given an atomic structure and a cryo-EM map to fit, NMMD simultaneously estimates global atomic displacements based on NMA and local displacements based on MD. NMMD was implemented by modifying EMfit, a flexible fitting method using MD only, in GENESIS 1.4. As EMfit, NMMD can be run with replica exchange umbrella sampling procedure. The new method was tested using a variety of EM maps (synthetic and experimental, with different noise levels and resolutions). The results of the tests show that adding normal modes to MD-based fitting makes the fitting faster (40% in average) and, in the majority of cases, more accurate.

DOI： 10.1016/j.jmb.2022.167483

Structures of human pannexin-1 in nanodiscs reveal gating mediated by dynamic movement of the N terminus and phospholipids. Reviewed International journal

Maki Kuzuya, Hidemi Hirano, Kenichi Hayashida, Masakatsu Watanabe, Kazumi Kobayashi, Tohru Terada, Md Iqbal Mahmood, Florence Tama, Kazutoshi Tani, Yoshinori Fujiyoshi, Atsunori Oshima

Science signaling Vol. 15 ( 720 ) page： eabg6941 2022.2

Language：English Publishing type：Research paper (scientific journal)

Pannexin (PANX) family proteins form large-pore channels that mediate purinergic signaling. We analyzed the cryo-EM structures of human PANX1 in lipid nanodiscs to elucidate the gating mechanism and its regulation by the amino terminus in phospholipids. The wild-type channel has an amino-terminal funnel in the pore, but in the presence of the inhibitor probenecid, a cytoplasmically oriented amino terminus and phospholipids obstruct the pore. Functional analysis using whole-cell patch-clamp and oocyte voltage clamp showed that PANX1 lacking the amino terminus did not open and had a dominant negative effect on channel activity, thus confirming that the amino-terminal domain played an essential role in channel opening. These observations suggest that dynamic conformational changes in the amino terminus of human PANX1 are associated with lipid movement in and out of the pore. Moreover, the data provide insight into the gating mechanism of PANX1 and, more broadly, other large-pore channels.

DOI： 10.1126/scisignal.abg6941

A hybrid approach to study large conformational transitions of biomolecules from single particle XFEL diffraction data Reviewed International journal Open Access

H. Asi, B. Dasgupta, T. Nagai, O. Miyashita, F. Tama

Front. Mol. Biosci Vol. 9 page： 913860 2022

A Methodology to Generate Efficient Neural Networks for Classification of Scientific Datasets Reviewed International journal Open Access

Patel Ria, Rorabaugh Ariel Keller, Olaya Paula, Caino-Lores Silvina, Channing Georgia, Schuman Catherine, Miyashita Osamu, Tama Florence, Taufer Michela

2022 IEEE 18TH INTERNATIONAL CONFERENCE ON E-SCIENCE (ESCIENCE 2022) page： 389 - 390 2022

Identifying Structural Properties of Proteins from X-ray Free Electron Laser Diffraction Patterns Reviewed International journal Open Access

Olaya Paula, Caino-Lores Silvina, Lama Vanessa, Patel Ria, Rorabaugh Ariel Keller, Miyashita Osamu, Tama Florence, Taufer Michela

2022 IEEE 18TH INTERNATIONAL CONFERENCE ON E-SCIENCE (ESCIENCE 2022) page： 21 - 31 2022

Protocol for Retrieving Three-Dimensional Biological Shapes for a Few XFEL Single-Particle Diffraction Patterns. Reviewed International journal Open Access

Sandhya P Tiwari, Florence Tama, Osamu Miyashita

Journal of chemical information and modeling Vol. 61 ( 8 ) page： 4108 - 4119 2021.8

Language：English Publishing type：Research paper (scientific journal)

X-ray free-electron laser (XFEL) scattering promises to probe single biomolecular complexes without crystallization, enabling the study of biomolecular structures under near-physiological conditions at room temperature. However, such structural determination of biomolecules is extremely challenging thus far. In addition to the large numbers of diffraction patterns required, the orientation of each diffraction pattern needs to be accurately estimated and the missing phase information needs to be recovered for three-dimensional (3D) structure reconstruction. Given the current limitations to the amount and resolution of the data available from single-particle XFEL scattering experiments, we propose an alternative approach to find plausible 3D biological shapes from a limited number of diffraction patterns to serve as a starting point for further analyses. In our proposed strategy, small sets of input (e.g., five) XFEL diffraction patterns were matched against a library of diffraction patterns simulated from 1628 electron microscopy (EM) models to find potential matching 3D models that are consistent with the input diffraction patterns. This approach was tested for three example cases: EMD-3457 (Thermoplasma acidophilum 20S proteasome), EMD-5141 (Escherichia coli 70S ribosome complex), and EMD-5152 (budding yeast Nup84 complex). We observed that choosing the best strategy to define matching regions on the diffraction patterns is critical for identifying correctly matching diffraction patterns. While increasing the number of input diffraction patterns improved the matches in some cases, we found that the resulting matches are more dependent on the uniqueness or complexity of the shape as captured in the individual input diffraction patterns and the availability of a similar 3D biological shape in the search library. The protocol could be useful for finding candidate models for a limited amount of low-resolution data, even when insufficient for reconstruction, performing a quick exploration of new data upon collection, and the analysis of the conformational heterogeneity of the particle of interest as captured within the diffraction patterns.

DOI： 10.1021/acs.jcim.1c00602

Integrative modeling to characterize structure and dynamics of biomolecules International journal

Tama Florence

ACTA CRYSTALLOGRAPHICA A-FOUNDATION AND ADVANCES Vol. 77 page： C53 - C53 2021.8

Structural differences in the FAD-binding pockets and lid loops of mammalian CRY1 and CRY2 for isoform-selective regulation. Reviewed International journal Open Access

Simon Miller, Ashutosh Srivastava, Yoshiko Nagai, Yoshiki Aikawa, Florence Tama, Tsuyoshi Hirota

Proceedings of the National Academy of Sciences of the United States of America Vol. 118 ( 26 ) 2021.6

Language：English Publishing type：Research paper (scientific journal)

The circadian clock is a biological timekeeper that operates through transcription-translation feedback loops in mammals. Cryptochrome 1 (CRY1) and Cryptochrome 2 (CRY2) are highly conserved core clock components having redundant and distinct functions. We recently identified the CRY1- and CRY2-selective compounds KL101 and TH301, respectively, which provide useful tools for the exploration of isoform-selective CRY regulation. However, intrinsic differences in the compound-binding FAD (flavin adenine dinucleotide) pockets between CRY1 and CRY2 are not well understood, partly because of nonoptimal properties of previously reported apo form structures in this particular region constituted by almost identical sequences. Here, we show unliganded CRY1 and CRY2 crystal structures with well-defined electron densities that are largely free of crystal contacts at the FAD pocket and nearby lid loop. We revealed conformational isomerism in key residues. In particular, CRY1 W399 and corresponding CRY2 W417 in the FAD pocket had distinct conformations ("out" for CRY1 and "in" for CRY2) by interacting with the lid loop residues CRY1 Q407 and CRY2 F424, respectively, resulting in different overall lid loop structures. Molecular dynamics simulations supported that these conformations were energetically favorable to each isoform. Isoform-selective compounds KL101 and TH301 preferred intrinsic "out" and "in" conformations of the tryptophan residue in CRY1 and CRY2, respectively, while the nonselective compound KL001 fit to both conformations. Mutations of lid loop residues designed to perturb their isoform-specific interaction with the tryptophan resulted in reversed responses of CRY1 and CRY2 to KL101 and TH301. We propose that these intrinsic structural differences of CRY1 and CRY2 can be targeted for isoform-selective regulation.

DOI： 10.1073/pnas.2026191118

Reversible modulation of circadian time with chronophotopharmacology. Reviewed International journal Open Access

Dušan Kolarski, Carla Miró-Vinyals, Akiko Sugiyama, Ashutosh Srivastava, Daisuke Ono, Yoshiko Nagai, Mui Iida, Kenichiro Itami, Florence Tama, Wiktor Szymanski, Tsuyoshi Hirota, Ben L Feringa

Nature communications Vol. 12 ( 1 ) page： 3164 - 3164 2021.5

Language：English Publishing type：Research paper (scientific journal)

The circadian clock controls daily rhythms of physiological processes. The presence of the clock mechanism throughout the body is hampering its local regulation by small molecules. A photoresponsive clock modulator would enable precise and reversible regulation of circadian rhythms using light as a bio-orthogonal external stimulus. Here we show, through judicious molecular design and state-of-the-art photopharmacological tools, the development of a visible light-responsive inhibitor of casein kinase I (CKI) that controls the period and phase of cellular and tissue circadian rhythms in a reversible manner. The dark isomer of photoswitchable inhibitor 9 exhibits almost identical affinity towards the CKIα and CKIδ isoforms, while upon irradiation it becomes more selective towards CKIδ, revealing the higher importance of CKIδ in the period regulation. Our studies enable long-term regulation of CKI activity in cells for multiple days and show the reversible modulation of circadian rhythms with a several hour period and phase change through chronophotopharmacology.

DOI： 10.1038/s41467-021-23301-x

Photopharmacological Manipulation of Mammalian CRY1 for Regulation of the Circadian Clock. Reviewed International coauthorship International journal Open Access

Dušan Kolarski, Simon Miller, Tsuyoshi Oshima, Yoshiko Nagai, Yugo Aoki, Piermichele Kobauri, Ashutosh Srivastava, Akiko Sugiyama, Kazuma Amaike, Ayato Sato, Florence Tama, Wiktor Szymanski, Ben L Feringa, Kenichiro Itami, Tsuyoshi Hirota

Journal of the American Chemical Society Vol. 143 ( 4 ) page： 2078 - 2087 2021.2

Language：English Publishing type：Research paper (scientific journal)

CRY1 and CRY2 proteins are highly conserved components of the circadian clock that controls daily physiological rhythms. Disruption of CRY functions are related to many diseases, including circadian sleep phase disorder. Development of isoform-selective and spatiotemporally controllable tools will facilitate the understanding of shared and distinct functions of CRY1 and CRY2. Here, we developed CRY1-selective compounds that enable light-dependent manipulation of the circadian clock. From phenotypic chemical screening in human cells, we identified benzophenone derivatives that lengthened the circadian period. These compounds selectively interacted with the CRY1 photolyase homology region, resulting in activation of CRY1 but not CRY2. The benzophenone moiety rearranged a CRY1 region called the "lid loop" located outside of the compound-binding pocket and formed a unique interaction with Phe409 in the lid loop. Manipulation of this key interaction was achieved by rationally designed replacement of the benzophenone with a switchable azobenzene moiety whose cis-trans isomerization can be controlled by light. The metastable cis form exhibited sufficiently high half-life in aqueous solutions and structurally mimicked the benzophenone unit, enabling reversible period regulation over days by cellular irradiation with visible light. This study revealed an unprecedented role of the lid loop in CRY-compound interaction and paves the way for spatiotemporal regulation of CRY1 activity by photopharmacology for molecular understanding of CRY1-dependent functions in health and disease.

DOI： 10.1021/jacs.0c12280

Reconstruction of Three-Dimensional Conformations of Bacterial ClpB from High-Speed Atomic-Force-Microscopy Images. Reviewed International journal Open Access

Bhaskar Dasgupta, Osamu Miyashita, Takayuki Uchihashi, Florence Tama

Frontiers in molecular biosciences Vol. 8 page： 704274 - 704274 2021

Language：English Publishing type：Research paper (scientific journal)

ClpB belongs to the cellular disaggretase machinery involved in rescuing misfolded or aggregated proteins during heat or other cellular shocks. The function of this protein relies on the interconversion between different conformations in its native condition. A recent high-speed-atomic-force-microscopy (HS-AFM) experiment on ClpB from Thermus thermophilus shows four predominant conformational classes, namely, open, closed, spiral, and half-spiral. Analyses of AFM images provide only partial structural information regarding the molecular surface, and thus computational modeling of three-dimensional (3D) structures of these conformations should help interpret dynamical events related to ClpB functions. In this study, we reconstruct 3D models of ClpB from HS-AFM images in different conformational classes. We have applied our recently developed computational method based on a low-resolution representation of 3D structure using a Gaussian mixture model, combined with a Monte-Carlo sampling algorithm to optimize the agreement with target AFM images. After conformational sampling, we obtained models that reflect conformational variety embedded within the AFM images. From these reconstructed 3D models, we described, in terms of relative domain arrangement, the different types of ClpB oligomeric conformations observed by HS-AFM experiments. In particular, we highlighted the slippage of the monomeric components around the seam. This study demonstrates that such details of information, necessary for annotating the different conformational states involved in the ClpB function, can be obtained by combining HS-AFM images, even with limited resolution, and computational modeling.

DOI： 10.3389/fmolb.2021.704274

How CRY Regulates the Clock: Structural Studies of a Dynamic Mammalian Circadian Complex Open Access

Sandate C., Fribourgh J., Michael A., Srivastava A., Hura G., Schneidman-Duhovny D., Tripathi S., Takahashi J., Lander G., Hirota T., Tama F., Partch C.

ACTA CRYSTALLOGRAPHICA A-FOUNDATION AND ADVANCES Vol. 76 page： A122 - A122 2020.8

Isoform-selective regulation of mammalian cryptochromes. Reviewed International journal

Simon Miller, You Lee Son, Yoshiki Aikawa, Eri Makino, Yoshiko Nagai, Ashutosh Srivastava, Tsuyoshi Oshima, Akiko Sugiyama, Aya Hara, Kazuhiro Abe, Kunio Hirata, Shinya Oishi, Shinya Hagihara, Ayato Sato, Florence Tama, Kenichiro Itami, Steve A Kay, Megumi Hatori, Tsuyoshi Hirota

Nature chemical biology Vol. 16 ( 6 ) page： 676 - 685 2020.6

Language：English Publishing type：Research paper (scientific journal)

CRY1 and CRY2 are essential components of the circadian clock controlling daily physiological rhythms. Accumulating evidences indicate distinct roles of these highly homologous proteins, in addition to redundant functions. Therefore, the development of isoform-selective compounds represents an effective approach towards understanding the similarities and differences of CRY1 and CRY2 by controlling each isoform individually. We conducted phenotypic screenings of circadian clock modulators, and identified KL101 and TH301 that selectively stabilize CRY1 and CRY2, respectively. Crystal structures of CRY-compound complexes revealed conservation of compound-binding sites between CRY1 and CRY2. We further discovered a unique mechanism underlying compound selectivity in which the disordered C-terminal region outside the pocket was required for the differential effects of KL101 and TH301 against CRY isoforms. By using these compounds, we found a new role of CRY1 and CRY2 as enhancers of brown adipocyte differentiation, providing the basis of CRY-mediated regulation of energy expenditure.

DOI： 10.1038/s41589-020-0505-1

Computational Protocol for Assessing the Optimal Pixel Size to Improve the Accuracy of Single-particle Cryo-electron Microscopy Maps. Reviewed International coauthorship International journal

Sandhya P Tiwari, Sahil Chhabra, Florence Tama, Osamu Miyashita

Journal of chemical information and modeling Vol. 60 ( 5 ) page： 2570 - 2580 2020.5

Language：English Publishing type：Research paper (scientific journal)

Cryo-electron microscopy (cryo-EM) single-particle analysis has come a long way in achieving atomic-level resolution when imaging biomolecules. To obtain the best possible three-dimensional (3D) structure in cryo-EM, many parameters have to be carefully considered. Here we address the often-overlooked parameter of the pixel size, which describes the magnification of the image produced by the experiment. While efforts are made to refine and validate this parameter in the analysis of cryo-EM experimental data, there is no systematic protocol in place. Since the pixel size parameter can have an impact on the resolution and accuracy of a cryo-EM map, and the atomic resolution 3D structure models derived from it, we propose a computational protocol to estimate the appropriate pixel size parameter. In our protocol, we fit and refine atomic structures against cryo-EM maps at multiple pixel sizes. The resulting fitted and refined structures are evaluated using the GOAP (generalized orientation-dependent, all-atom statistical potential) score, which we found to perform better than other commonly used functions, such as Molprobity and the correlation coefficient from refinement. Finally, we describe the efficacy of this protocol in retrieving appropriate pixel sizes for several examples; simulated data based on yeast elongation factor 2 and experimental data from Gro-EL chaperone, beta-galactosidase, and the TRPV1 ion channel.

DOI： 10.1021/acs.jcim.9b01107

Integrative/Hybrid Modeling Approaches for Studying Biomolecules. Reviewed International journal Open Access

Ashutosh Srivastava, Sandhya Premnath Tiwari, Osamu Miyashita, Florence Tama

Journal of molecular biology Vol. 432 ( 9 ) page： 2846 - 2860 2020.4

Language：English Publishing type：Research paper (scientific journal)

The structural and dynamical characterization of biomolecules holds central importance in the endeavor to understand the molecular mechanisms regulating living systems. However, owing to the inherent heterogeneity of biomolecular interactions within cells, it is often difficult to understand the overall structure and dynamics of biomolecules using any experimental method in isolation. In this regard, hybrid methods that combine data from multiple experiments to generate a comprehensive model of biomolecular complexes have gained prominence in the last few years. In this article, we discuss the advancements in hybrid methods, with a particular focus on the role of computation in their development and application. We further outline the future directions that hybrid methods are likely to take, regarding the advancements in techniques such as X-ray free-electron laser single- particle imaging, and electron cryo-tomography. Finally, we conclude the review by highlighting the future goals of broader consensus and collaboration within the integrative/hybrid structural biology community and for disseminating the data generated by hybrid modeling efforts.

DOI： 10.1016/j.jmb.2020.01.039

Dynamics at the serine loop underlie differential affinity of cryptochromes for CLOCK:BMAL1 to control circadian timing. Reviewed International journal Open Access

Jennifer L Fribourgh, Ashutosh Srivastava, Colby R Sandate, Alicia K Michael, Peter L Hsu, Christin Rakers, Leslee T Nguyen, Megan R Torgrimson, Gian Carlo G Parico, Sarvind Tripathi, Ning Zheng, Gabriel C Lander, Tsuyoshi Hirota, Florence Tama, Carrie L Partch

eLife Vol. 9 2020.2

Language：English Publishing type：Research paper (scientific journal)

Mammalian circadian rhythms are generated by a transcription-based feedback loop in which CLOCK:BMAL1 drives transcription of its repressors (PER1/2, CRY1/2), which ultimately interact with CLOCK:BMAL1 to close the feedback loop with ~24 hr periodicity. Here we pinpoint a key difference between CRY1 and CRY2 that underlies their differential strengths as transcriptional repressors. Both cryptochromes bind the BMAL1 transactivation domain similarly to sequester it from coactivators and repress CLOCK:BMAL1 activity. However, we find that CRY1 is recruited with much higher affinity to the PAS domain core of CLOCK:BMAL1, allowing it to serve as a stronger repressor that lengthens circadian period. We discovered a dynamic serine-rich loop adjacent to the secondary pocket in the photolyase homology region (PHR) domain that regulates differential binding of cryptochromes to the PAS domain core of CLOCK:BMAL1. Notably, binding of the co-repressor PER2 remodels the serine loop of CRY2, making it more CRY1-like and enhancing its affinity for CLOCK:BMAL1.

DOI： 10.7554/eLife.55275

Conformational ensemble of an intrinsically flexible loop in mitochondrial import protein Tim21 studied by modeling and molecular dynamics simulations. Reviewed International journal

Arpita Srivastava, Siqin Bala, Hajime Motomura, Daisuke Kohda, Florence Tama, Osamu Miyashita

Biochimica et biophysica acta. General subjects Vol. 1864 ( 2 ) page： 129417 - 129417 2020.2

Language：English Publishing type：Research paper (scientific journal)

BACKGROUND: Tim21, a subunit of a highly dynamic translocase of the inner mitochondrial membrane (TIM23) complex, translocates proteins by interacting with subunits in the translocase of the outer membrane (TOM) complex and Tim23 channel in the TIM23 complex. A loop segment in Tim21, which is in close proximity of the binding site of Tim23, has different conformations in X-ray, NMR and new crystal contact-free space (CCFS) structures. MD simulations can provide information on the structure and dynamics of the loop in solution. METHODS: The conformational ensemble of the loop was characterized using loop modeling and molecular dynamics (MD) simulations. RESULTS: MD simulations confirmed mobility of the loop. Multidimensional scaling and clustering were used to characterize the dynamic conformational ensemble of the loop. Free energy landscape showed that the CCFS crystal structure occupied a low energy region as compared to the conventional X-ray crystal structure. Analysis of crystal packing indicates that the CCFS provides larger conformational space for the motions of the loop. CONCLUSIONS: Our work reported the conformational ensemble of the loop in solution, which is in agreement with the structure obtained from CCFS approach. The combination of the experimental techniques and computational methods is beneficial for studying highly flexible regions of proteins. GENERAL SIGNIFICANCE: Computational methods, such as loop modeling and MD simulations, have proved to be useful for studying conformational flexibility of proteins. These methods in integration with experimental techniques such as CCFS has the potential to transform the studies on flexible regions of proteins.

DOI： 10.1016/j.bbagen.2019.129417

Reconstruction of low-resolution molecular structures from simulated atomic force microscopy images. Reviewed International journal

Bhaskar Dasgupta, Osamu Miyashita, Florence Tama

Biochimica et biophysica acta. General subjects Vol. 1864 ( 2 ) page： 129420 - 129420 2020.2

Language：English Publishing type：Research paper (scientific journal)

BACKGROUND: Atomic Force Microscopy (AFM) is an experimental technique to study structure-function relationship of biomolecules. AFM provides images of biomolecules at nanometer resolution. High-speed AFM experiments produce a series of images following dynamics of biomolecules. To further understand biomolecular functions, information on three-dimensional (3D) structures is beneficial. METHOD: We aim to recover 3D information from an AFM image by computational modeling. The AFM image includes only low-resolution representation of a molecule; therefore we represent the structures by a coarse grained model (Gaussian mixture model). Using Monte-Carlo sampling, candidate models are generated to increase similarity between AFM images simulated from the models and target AFM image. RESULTS: The algorithm was tested on two proteins to model their conformational transitions. Using a simulated AFM image as reference, the algorithm can produce a low-resolution 3D model of the target molecule. Effect of molecular orientations captured in AFM images on the 3D modeling performance was also examined and it is shown that similar accuracy can be obtained for many orientations. CONCLUSIONS: The proposed algorithm can generate 3D low-resolution protein models, from which conformational transitions observed in AFM images can be interpreted in more detail. GENERAL SIGNIFICANCE: High-speed AFM experiments allow us to directly observe biomolecules in action, which provides insights on biomolecular function through dynamics. However, as only partial structural information can be obtained from AFM data, this new AFM based hybrid modeling method would be useful to retrieve 3D information of the entire biomolecule.

DOI： 10.1016/j.bbagen.2019.129420

Crystal contact-free conformation of an intrinsically flexible loop in protein crystal: Tim21 as the case study. Reviewed International journal Open Access

Siqin Bala, Shoko Shinya, Arpita Srivastava, Marie Ishikawa, Atsushi Shimada, Naohiro Kobayashi, Chojiro Kojima, Florence Tama, Osamu Miyashita, Daisuke Kohda

Biochimica et biophysica acta. General subjects Vol. 1864 ( 2 ) page： 129418 - 129418 2020.2

Language：English Publishing type：Research paper (scientific journal)

BACKGROUND: In protein crystals, flexible loops are frequently deformed by crystal contacts, whereas in solution, the large motions result in the poor convergence of such flexible loops in NMR structure determinations. We need an experimental technique to characterize the structural and dynamic properties of intrinsically flexible loops of protein molecules. METHODS: We designed an intended crystal contact-free space (CCFS) in protein crystals, and arranged the flexible loop of interest in the CCFS. The yeast Tim 21 protein was chosen as the model protein, because one of the loops (loop 2) is distorted by crystal contacts in the conventional crystal. RESULTS: Yeast Tim21 was fused to the MBP protein by a rigid α-helical linker. The space created between the two proteins was used as the CCFS. The linker length provides adjustable freedom to arrange loop 2 in the CCFS. We re-determined the NMR structure of yeast Tim21, and conducted MD simulations for comparison. Multidimensional scaling was used to visualize the conformational similarity of loop 2. We found that the crystal contact-free conformation of loop 2 is located close to the center of the ensembles of the loop 2 conformations in the NMR and MD structures. CONCLUSIONS: Loop 2 of yeast Tim21 in the CCFS adopts a representative, dominant conformation in solution. GENERAL SIGNIFICANCE: No single powerful technique is available for the characterization of flexible structures in protein molecules. NMR analyses and MD simulations provide useful, but incomplete information. CCFS crystallography offers a third route to this goal.

DOI： 10.1016/j.bbagen.2019.129418

Single-Cell Fluorescence Analysis of Lipid Droplet Compositional Dynamics during Triacylglycerol Catabolism. Reviewed International journal

Junwei Wang, Keiji Kajiwara, Manish Kesherwani, Florence Tama, Yuki Ohsaki, Shigehiro Yamaguchi, Masayasu Taki

Journal of the American Chemical Society Vol. 147 ( 45 ) page： 41514 - 41523 2025.11

Language：English Publishing type：Research paper (scientific journal)

Lipid droplets (LDs) are dynamic organelles essential to lipid metabolism and energy homeostasis, yet their compositional heterogeneity in living cells remains poorly understood. Here, we present LipiPB Red, a red-emissive fluorescent probe with exceptional photostability, high polarity sensitivity, and long-term retention within LDs, even under serum-containing conditions. When applied to fluorescence lifetime imaging microscopy (FLIM), LipiPB Red enables discrimination between triacylglycerol (TAG) and diacylglycerol (DAG) content within individual LDs. FLIM analysis revealed pronounced compositional heterogeneity among LDs in hepatoma cells. This heterogeneity was abolished, with a concomitant increase in average fluorescence lifetimes, upon genetic knockdown or pharmacological inhibition of adipose triglyceride lipase (ATGL), implicating ATGL-mediated lipolysis as a key regulator of LD diversity. Moreover, coimaging with autophagy markers revealed reduced fluorescence lifetimes in LDs localized within both autophagosomes and autolysosomes. These findings indicate a sequential lipid degradation cascade during lipophagy, where lipid hydrolysis is initiated prior to lysosomal fusion.

DOI： 10.1021/jacs.5c11742

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