Remarkably, the bR-bR interaction in the transiently formed assembly elicits both positive and negative cooperative effects on the decay kinetics as the initial bR recovers. By the bipolar nature of the cooperativity, however, the turnover rate of the phtocycle is maintained constant on average, irrespective of the light intensity.Thus, the direct and high-resolution visualization of dynamically acting molecules is a powerful new approach to gaining insight into elaborate bimolecular processes. 2 The biological function of proteins is closely associated with their ability to undergo structural changes. In many cases, these structural changes are triggered by external stimuli including pH, temperature, ligand binding, mechanical stress, and light.Although their direct real-space and real-time visualization is a straightforward approach to understanding the dynamic molecular processes, the lack of suitable techniques has precluded it. Atomic force microscopy (AFM) is a versatile technique to image proteins in liquids at sub-molecular resolution, but its poor temporal resolution has meant an availability of only static or slow time-lapse images of proteins [1][2][3][4][5] . In the last decade, various efforts have been carried out to increase the scan speed of AFM 6-9 .As a result, single protein molecules exhibiting Brownian motion are captured on video at a highest temporal resolution of ~30 ms 10 . However, dynamic visualization of physiologically relevant conformational changes in proteins has been difficult because tip-sample interaction tends to interfere with the physiological functions. To solve this problem, a new method has recently been developed which allows fast and precise control of the tip-sample distance with a minimum load to the sample 7 . This report presents the first ever exemplification of dynamic imaging of a functioning biological sample.Bacteriorhodopsin (bR) is a well-known example of the association between stimulus-triggered structural dynamics and biological function 11,12 , and its direct visualization has long been a goal. bR contains seven transmembrane α-helices (named A-G) enclosing the chromophore retinal 13,14 . In the photocycle, a series of spectral intermediates, designated J, K, L, M, N, and O, occur in that order 12 . The light-induced conformational changes in bR have been investigated by various methods 15-25 , leading to a consensus that the proton channel at the cytoplasmic surface is opened by the tilting of helix F away from the protein center 21,23,24 . Sass et al. reported helix F displacement of ~0.1 nm in the late M state, based on X-ray diffraction of the three-dimensional crystal of wild type (WT) 21 . However, a larger structural change in bR was reported in 3 the electron crystallography study of the D96G, F171C, F219L triple mutant of bR: displacement of helix F by ~0.35 nm away from the center of the protein 23 . The electron crystallography study of the F219L mutant further reported that helices E and F tilt away from the center of the protein, which is ...

The CRISPR-associated endonuclease Cas9 binds to a guide RNA and cleaves double-stranded DNA with a sequence complementary to the RNA guide. The Cas9–RNA system has been harnessed for numerous applications, such as genome editing. Here we use high-speed atomic force microscopy (HS-AFM) to visualize the real-space and real-time dynamics of CRISPR-Cas9 in action. HS-AFM movies indicate that, whereas apo-Cas9 adopts unexpected flexible conformations, Cas9–RNA forms a stable bilobed structure and interrogates target sites on the DNA by three-dimensional diffusion. These movies also provide real-time visualization of the Cas9-mediated DNA cleavage process. Notably, the Cas9 HNH nuclease domain fluctuates upon DNA binding, and subsequently adopts an active conformation, where the HNH active site is docked at the cleavage site in the target DNA. Collectively, our HS-AFM data extend our understanding of the action mechanism of CRISPR-Cas9.

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In a light-driven proton pump protein, bacteriorhodopsin (BR), three water molecules participate in a pentagonal cluster that stabilizes an electric quadrupole buried inside the protein. Previously, low-temperature Fourier-transform infrared (FTIR) difference spectra between BR and the K photointermediate in D(2)O revealed six O-D stretches of water in BR at 2690, 2636, 2599, 2323, 2292, and 2171 cm(-)(1), while five water bands were observed at 2684, 2675, 2662, 2359, and 2265 cm(-)(1) for the K intermediate. The frequencies are widely distributed over the possible range of stretching vibrations of water, and water molecules at <2400 cm(-)(1) were suggested to hydrate negative charges because of their extremely strong hydrogen bonds. In this paper, we aimed to reveal the origin of these water bands in the K minus BR spectra by use of various mutant proteins. The water bands were not affected by the mutations at the cytoplasmic side, such as T46V, D96N, and D115N, implying that the water molecules in the cytoplasmic domain do not change their hydrogen bonds in the BR to K transition. In contrast, significant modifications of the water bands were observed for the mutations in the Schiff base region and at the extracellular side, such as R82Q, D85N, T89A, Y185F, D212N, R82Q/D212N, and E204Q. From these results, we concluded that the six O-D stretches of BR originate from three water molecules, water401, -402, and -406, involved in the pentagonal cluster. Two stretching modes of each water molecule are highly separate (300-470 cm(-)(1) for O-D stretches and 500-770 cm(-)(1) for O-H stretches), which is consistent with the previous QM/MM calculation. The small amplitudes of vibrational coupling are presumably due to strong association of the waters to negative charges of Asp85 and Asp212. Among various mutant proteins, only D85N and D212N lack strongly hydrogen-bonded water molecules (<2400 cm(-)(1)) and proton pumpimg activity. We thus infer that the presence of a strong hydrogen bond of water is a prerequisite for proton pumping in BR. Internal water molecules in such a specific environment are discussed in terms of functional importance for rhodopsins.