Structural Changes in Bacteriorhodopsin in Response to Alternate Illumination Observed by High‐Speed Atomic Force Microscopy

Shibata, Mikihiro; Uchihashi, Takayuki; Yamashita, Hayato; Kandori, Hideki; Ando, Toshio

doi:10.1002/anie.201007544

Cited by 54 publications

(63 citation statements)

References 43 publications

(30 reference statements)

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“…We improved the spatial resolution to capture the dynamics of membrane proteins at a high resolution and succeeded in visualizing lateral movement and structural changes of proteins in isolated biologic membranes. [17][18][19] HS-AFM is, at present, capable of imaging soft biologic molecules non-invasively without damage, suggesting that it has the potential to capture the molecular dynamics of the surface of a living cell. The bacterial cell, however, has a substantially higher topography than isolated molecules.…”

Section: Discussionmentioning

confidence: 99%

“…[14][15][16] Moreover, the dynamics of membrane proteins isolated from bacterial cells can be visualized at high resolution with HS-AFM. [17][18][19] HS-AFM will become even more useful in biologic science if it can be used to observe the molecular dynamics of living cell membranes. A large number of membrane proteins play important roles in cell function (e.g., transport, signal transduction for extracellular stimuli, and maintenance of cellular structure).…”

Section: Introductionmentioning

confidence: 99%

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Single-Molecule Imaging on Living Bacterial Cell Surface by High-Speed AFM

Yamashita

Taoka

Uchihashi

et al. 2012

Journal of Molecular Biology

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114

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Advances in microscopy have contributed to many biologic discoveries. Electron microscopic techniques such as cryo-electron tomography are remarkable tools for imaging the interiors of bacterial cells in the near-native state, whereas optical microscopic techniques such as fluorescence imaging are useful for following the dynamics of specific single molecules in living cells. Neither technique, however, can be used to visualize the structural dynamics of a single molecule at high resolution in living cells.In the present study, we used high-speed atomic force microscopy (HS-AFM) to image the molecular dynamics of living bacterial cell surfaces. HS-AFM visualizes the dynamic molecular processes of isolated proteins at sub-molecular resolution without the need for complicated sample preparation. In the present study, magnetotactic bacterial cells were anchored in liquid medium on substrate modified by poly-L-lysine and glutaraldehyde. High-resolution HS-AFM images of live cell surfaces showed that the bacterial outer membrane was covered with a net-like structure comprising holes and the hole rims framing them. Furthermore, HS-AFM captured the dynamic movement of the surface ultrastructure, showing that the holes in the net-like structure slowly diffused in the cell surface. Nano-dissection revealed that porin trimers constitute the net-like structure. Here, we report for the first time the direct observation of dynamic molecular architectures on a live cell surface using HS-AFM.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Single-Molecule Imaging on Living Bacterial Cell Surface by High-Speed AFM

Yamashita

Taoka

Uchihashi

et al. 2012

Journal of Molecular Biology

Self Cite

114

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“…To slow down the photocycle, we then used the D96N bR mutant that has a longer photocycle (~10 s at pH 7) but still retains a proton pumping ability [140]. another study as mentioned later [113].…”

Section: Br In Response To Lightmentioning

confidence: 99%

High-speed atomic force microscopy coming of age

Ando¹

2012

Nanotechnology

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314

246

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High-speed atomic force microscopy (HS-AFM) is now materialized. It allows direct visualization of dynamic structural changes and dynamic processes of functioning biological molecules in physiological solutions, at high spatiotemporal resolution. Dynamic molecular events unselectively appear in detail in an AFM movie, facilitating our understanding of how biological molecules operate to function. This review describes a historical overview of technical development towards HS-AFM, summarizes elementary devices and techniques used in the current HS-AFM, and then highlights recent imaging studies. Finally, future challenges of HS-AFM studies are briefly discussed.

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“…Previously, numerous structural studies of bR in PM were performed under unphotolyzed (Sass et al, 2000) or frozen activated (Luecke et al, 1999;Subramaniam and Henderson, 2000;Vonck, 2000, Lanyi andSchobert, 2003;Hirai and Subramaniam, 2009) states. Recently, by means of high-speed atomic force microscopy (HS-AFM) (Ando et al, 2001;Ando et al, 2008), light-induced conformational changes of bR have been visualized in real-time and real-space (Shibata et al, 2010;Shibata et al, 2011). HS-AFM movies have revealed that the E-F inter-helical loop of each bR monomer moves outwards from its trimer center upon photo-excitation from the ground state to the M N state, which results in contact among three nearest-neighbor bR monomers, each belonging to a different adjacent trimer.…”

Section: Introductionmentioning

confidence: 99%

Role of trimer–trimer interaction of bacteriorhodopsin studied by optical spectroscopy and high-speed atomic force microscopy

Yamashita

Inoue

Shibata

et al. 2013

Journal of Structural Biology

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Bacteriorhodopsin (bR) trimers form a two-dimensional hexagonal lattice in the purple membrane of Halobacterium salinarum. However, the physiological significance of forming the lattice has long been elusive. Here, we study this issue by comparing properties of assembled and non-assembled bR trimers using directed mutagenesis, high-speed atomic force microscopy (HS-AFM), optical spectroscopy, and a proton pumping assay. First, we show that the bonds formed between W12 and F135 amino acid residues are responsible for trimer-trimer association that leads to lattice assembly; the lattice is completely disrupted in both W12I and F135I mutants. HS-AFM imaging reveals that both crystallized D96N and non-crystallized D96N/W12I mutants undergo a large conformational change (i.e., outward E-F loop displacement) upon light-activation. However, lattice disruption significantly reduces the rate of conformational change under continuous light illumination. Nevertheless, the quantum yield of M-state formation, measured by low-temperature UV-visible spectroscopy, and proton pumping efficiency are unaffected by lattice disruption. From these results, we conclude that trimer-trimer association plays essential roles in providing bound retinal with an appropriate environment to maintain its full photo-reactivity and in maintaining the natural photo-reaction pathway.

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Structural Changes in Bacteriorhodopsin in Response to Alternate Illumination Observed by High‐Speed Atomic Force Microscopy

Cited by 54 publications

References 43 publications

Single-Molecule Imaging on Living Bacterial Cell Surface by High-Speed AFM

Single-Molecule Imaging on Living Bacterial Cell Surface by High-Speed AFM

High-speed atomic force microscopy coming of age

Role of trimer–trimer interaction of bacteriorhodopsin studied by optical spectroscopy and high-speed atomic force microscopy

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