Mikihiro Shibata

High-speed atomic force microscopy shows dynamic molecular processes in photoactivated bacteriorhodopsin

Dynamic changes in protein conformation in response to external stimuli are important in biological processes, but it has proved difficult to directly visualize such structural changes under physiological conditions. Here, we show that high-speed atomic force microscopy can be used to visualize dynamic changes in stimulated proteins. High-resolution movies of a light-driven proton pump, bacteriorhodopsin, reveal that, upon illumination, a cytoplasmic portion of each bacteriorhodopsin monomer is brought into contact with adjacent trimers. The bacteriorhodopsin-bacteriorhodopsin interaction in the transiently formed assembly engenders both positive and negative cooperative effects in the decay kinetics as the initial bacteriorhodopsin recovers and, as a consequence, the turnover rate of the photocycle is maintained constant, on average, irrespective of the light intensity. These results confirm that high-resolution visualization is a powerful approach for studying elaborate biomolecular processes under realistic conditions.

FTIR studies of internal water molecules in the Schiff base region of bacteriorhodopsin.

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.

Long-tip high-speed atomic force microscopy for nanometer-scale imaging in live cells

Visualization of morphological dynamics of live cells with nanometer resolution under physiological conditions is highly desired, but challenging. It has been demonstrated that high-speed atomic force microscopy is a powerful technique for visualizing dynamics of biomolecules under physiological conditions. However, application of high-speed atomic force microscopy for imaging larger objects such as live mammalian cells has been complicated because of the collision between the cantilever and samples. Here, we demonstrate that attaching an extremely long (~3 μm) and thin (~5 nm) tip by amorphous carbon to the cantilever allows us to image the surface structure of live cells with the spatiotemporal resolution of nanometers and seconds. We demonstrate that long-tip high-speed atomic force microscopy is capable of imaging morphogenesis of filopodia, membrane ruffles, pit formation, and endocytosis in COS-7, HeLa cells and hippocampal neurons.

Functional extension of high-speed AFM for wider biological applications.

High-speed atomic force microscopy (HS-AFM) has been established and used, which can visualize biomolecules in dynamic action at high spatiotemporal resolution without disturbing their function. Various studies conducted in the past few years have demonstrated that the dynamic structure and action of biomolecules revealed with HS-AFM can provide greater insights than ever before into how the molecules function. However, this microscopy has still limitations in some regards. Recently, efforts have been carried out to overcome some of the limitations. As a result, it has now become possible to visualize dynamic processes occurring even on live cells and perform simultaneous observations of topographic and fluorescent images at a high rate. In this review, we focus on technical developments for expanding the range of objects and phenomena observable by HS-AFM as well as for granting multiple functionalities to HS-AFM.

Protein-Bound Water as the Determinant of Asymmetric Functional Conversion between Light-Driven Proton and Chloride Pumps

Bacteriorhodopsin (BR) and halorhodopsin (HR) are light-driven outward proton and inward chloride pumps, respectively. They have similar protein architecture, being composed of seven-transmembrane helices that bind an all-trans-retinal. BR can be converted into a chloride pump by a single amino acid replacement at position 85, suggesting that BR and HR share a common transport mechanism, and the ionic specificity is determined by the amino acid at that position. However, HR cannot be converted into a proton pump by the corresponding reverse mutation. Here we mutated 6 and 10 amino acids of HR into BR-like, whereas such multiple HR mutants never pump protons. Light-induced Fourier transform infrared spectroscopy revealed that hydrogen bonds of the retinal Schiff base and water are both strong for BR and both weak for HR. Multiple HR mutants exhibit strong hydrogen bonds of the Schiff base, but the hydrogen bond of water is still weak. We concluded that the cause of nonfunctional conversion of HR is the lack of strongly hydrogen-bonded water, the functional determinant of the proton pump.

Strongly hydrogen-bonded water molecules in the Schiff base region of rhodopsins

In many rhodopsins, a positively charged retinal chromophore is stabilized by a negatively charged carboxylate, and the presence of bound water molecules has been found in the Schiff base region by X-ray crystallography of various rhodopsins. Low-temperature Fourier-transform infrared (FTIR) spectroscopy can directly monitor hydrogen-bonding alterations of internal water molecules of rhodopsins. In particular, we found that a bridged water molecule between the Schiff base and Asp 85 in bacteriorhodopsin (BR), a light-driven proton-pump protein, forms an extremely strong hydrogen bond. It is likely that a hydration switch of the water from Asp 85 to Asp 212 plays an important role in the proton transfer in the Schiff base region of BR. Comprehensive studies of archaeal and visual rhodopsins have revealed that strongly hydrogen-bonded water molecules are only found in the proteins exhibiting proton-pump activities. Strongly hydrogen-bonded water molecules and its transient weakening may be essential for the proton-pump function of rhodopsins.

Ultrafast Pump−Probe Study of the Primary Photoreaction Process in pharaonis Halorhodopsin: Halide Ion Dependence and Isomerization Dynamics

Halorhodopsin is a retinal protein that acts as a light-driven chloride pump in the Haloarchaeal cell membrane. A chloride ion is bound near the retinal chromophore, and light-induced all- trans --> 13- cis isomerization triggers the unidirectional chloride ion pump. We investigated the primary ultrafast dynamics of Natronomonas pharaonis halorhodopsin that contains Cl (-), Br (-), or I (-) ( pHR-Cl (-), pHR-Br (-), or pHR-I (-)) using ultrafast pump-probe spectroscopy with approximately 30 fs time resolution. All of the temporal behaviors of the S n <-- S 1 absorption, ground-state bleaching, K intermediate (13- cis form) absorption, and stimulated emission were observed. In agreement with previous reports, the primary process exhibited three dynamics. The first dynamics corresponds to the population branching process from the Franck-Condon (FC) region to the reactive (S 1 (r)) and nonreactive (S 1 (nr)) S 1 states. With the improved time resolution, it was revealed that the time constant of this branching process (tau 1) is as short as 50 fs. The second dynamics was the isomerization process of the S 1 (r) state to generate the ground-state 13- cis form, and the time constant (tau 2) exhibited significant halide ion dependence (1.4, 1.6, and 2.2 ps for pHR-Cl (-), pHR-Br (-), and pHR-I (-), respectively). The relative quantum yield of the isomerization, which was evaluated from the pump-probe signal after 20 ps, also showed halide ion dependence (1.00, 1.14, and 1.35 for pHR-Cl (-), pHR-Br (-), and pHR-I (-), respectively). It was revealed that the halide ion that accelerates isomerization dynamics provides the lower isomerization yield. This finding suggests that there is an activation barrier along the isomerization coordinate on the S 1 potential energy surface, meaning that the three-state model, which is now accepted for bacteriorhodopsin, is more relevant than the two-state model for the isomerization process of halorhodopsin. We concluded that, with the three-state model, the isomerization rate is controlled by the height of the activation barrier on the S 1 potential energy surface while the overall isomerization yield is determined by the branching ratios at the FC region and the conical intersection. The third dynamics attributable to the internal conversion of the S 1 (nr) state also showed notable halide ion dependence (tau 3 = 4.5, 4.6, and 6.3 ps for pHR-Cl (-), pHR-Br (-), and pHR-I (-)). This suggests that some geometrical change may be involved in the relaxation process of the S 1 (nr) state.

Picosecond time-resolved ultraviolet resonance Raman spectroscopy of bacteriorhodopsin: primary protein response to the photoisomerization of retinal.

Protein dynamics in the primary processes during the bacteriorhodopsin (BR) photocycle under physiological conditions were investigated by measuring picosecond time-resolved ultraviolet resonance Raman (UVRR) spectra of the BR suspended solution at ambient temperature. We used a 565 nm pump pulse to initiate the BR photocycle and two kinds of probe pulses with wavelengths of 225 and 238 nm to detect spectral changes in the tryptophan and tyrosine bands, respectively. The observed spectral changes of the Raman bands are most likely due to tryptophan and tyrosine residues located in the vicinity of the retinal chromophore, that is, Trp86, Trp182, Tyr57, and Tyr185. The 225 nm UVRR spectra exhibited bleaching of intensity for all the tryptophan bands within the instrumental response, followed by recovery with a time constant of 30 ps and no further changes up to 1 ns. This suggests that the stepwise structural changes in the tryptophan residues proceed in response to the retinal photoreaction. It is concluded that the initial intensity bleach arises from the J-intermediate formation and the 30 ps recovery is associated with the K-KL transition. The 30 ps process in the BR photocycle has been detected for the first time. In the 238 nm UVRR spectra, spectral features attributable to the K and KL intermediates were observed. The observed spectral changes showed that the temporal behaviors of the observed spectral changes in each Raman band of both tryptophan and tyrosine were different. This indicates that the spectral changes originated from structural changes of at least two tryptophan and two tyrosine residues.

Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy

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.CRISPR RNA-guided endonuclease Cas9 recognizes and cleaves the double-stranded DNA complementary to the RNA guide. Here the authors use high-speed atomic force micropcopy (HS-AFM) to visualize the conformational dynamics of Cas9 during its DNA targeting and cleavage processes.

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

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.