X-ray-based living-cell motion analysis of individual serotonin receptors

Mio, Kazuhiro; Ishihara, Masaki; Fujimura, Shoko; Sasaki, Daisuke; Nozawa, Shiro; Ichiyanagi, Kouhei; Fukaya, Ryo; Adachi, Shin; Kawamura, Masahiro; Sekiguchi, Hiroshi; Kubo, Tai; Sasaki, Yuji

doi:10.1016/j.bbrc.2020.05.200

Cited by 20 publications

(32 citation statements)

References 17 publications

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“…In this study, the mutant DRY/AAY protein which disturbs the ionic lock lost the clockwise torsion motion observed in the wild type. This is consistent with our previous report that the loss of ionic lock lost the fluctuation of the receptor analyzed in the θ direction [ 13 ].…”

Section: Discussionsupporting

confidence: 94%

“…In order to apply DXT to living cells, radiation damage should be minimized. In the previous study, we found that narrowing the bandwidth of X-ray eliminated cell damage [ 13 ]. So we used X-rays with 0.02 energy bandwidth, 15 keV peak energy and 10 13 photon/s flux for the living-cell DXT, instead of the 15.8 keV white X-ray that is commonly used for single molecular DXT.…”

Section: Resultsmentioning

confidence: 99%

“…As reported in the previous study, the slope in the θ direction was suppressed by the full agonist α-methylserotonin (αMS) and the αMS with inverse agonist ketanserin (αMS/ket) ( Figure 2 a). This was considered due to the shift of conformational equilibrium from multiple structures to active or inactive conformation [ 13 , 15 ].…”

Section: Resultsmentioning

confidence: 99%

“…In our previous study using DXT and DXB techniques, we were able to detect various 5-HT 2A Rs motion components according to the recording frame rate [ 13 ]. In that, the ligand-induced conformational shift from multiple structures to active or inactive conformation at different time scales was observed.…”

Section: Introductionmentioning

confidence: 99%

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Living-Cell Diffracted X-ray Tracking Analysis Confirmed Internal Salt Bridge Is Critical for Ligand-Induced Twisting Motion of Serotonin Receptors

Mio

Fujimura

Ishihara

et al. 2021

IJMS

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Serotonin receptors play important roles in neuronal excitation, emotion, platelet aggregation, and vasoconstriction. The serotonin receptor subtype 2A (5-HT2AR) is a Gq-coupled GPCR, which activate phospholipase C. Although the structures and functions of 5-HT2ARs have been well studied, little has been known about their real-time dynamics. In this study, we analyzed the intramolecular motion of the 5-HT2AR in living cells using the diffracted X-ray tracking (DXT) technique. The DXT is a very precise single-molecular analytical technique, which tracks diffraction spots from the gold nanocrystals labeled on the protein surface. Trajectory analysis provides insight into protein dynamics. The 5-HT2ARs were transiently expressed in HEK 293 cells, and the gold nanocrystals were attached to the N-terminal introduced FLAG-tag via anti-FLAG antibodies. The motions were recorded with a frame rate of 100 μs per frame. A lifetime filtering technique demonstrated that the unliganded receptors contain high mobility population with clockwise twisting. This rotation was, however, abolished by either a full agonist α-methylserotonin or an inverse agonist ketanserin. Mutation analysis revealed that the “ionic lock” between the DRY motif in the third transmembrane segment and a negatively charged residue of the sixth transmembrane segment is essential for the torsional motion at the N-terminus of the receptor.

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Section: Discussionsupporting

confidence: 94%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Living-Cell Diffracted X-ray Tracking Analysis Confirmed Internal Salt Bridge Is Critical for Ligand-Induced Twisting Motion of Serotonin Receptors

Mio

Fujimura

Ishihara

et al. 2021

IJMS

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“…Recently, diffracted x-ray blinking (DXB) using monochromatic x-rays, which can measure the intramolecular movement of proteins as a diffusion coefficient, has been proposed, 7 and its use is currently expanding. 4,8,9 In addition, the technique used for producing very good-quality gold nanocrystals, which is essential for DXT and DXB, utilizes a very special technique using epitaxial growth on a single crystal in ultrahigh vacuum. Thus, it is practically difficult for researchers to utilize high-sensitivity high-speed dynamics measurements using DXT and DXB.…”

Section: Introductionmentioning

confidence: 99%

Laboratory diffracted x-ray blinking to monitor picometer motions of protein molecules and application to crystalline materials

Arai

Inamasu

Yamaguchi

et al. 2021

Structural Dynamics

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In recent years, real-time observations of molecules have been required to understand their behavior and function. To date, we have reported two different time-resolved observation methods: diffracted x-ray tracking and diffracted x-ray blinking (DXB). The former monitors the motion of diffracted spots derived from nanocrystals labeled onto target molecules, and the latter measures the fluctuation of the diffraction intensity that is highly correlated with the target molecular motion. However, these reports use a synchrotron x-ray source because of its high average flux, resulting in a high time resolution. Here, we used a laboratory x-ray source and DXB to measure the internal molecular dynamics of three different systems. The samples studied were bovine serum albumin (BSA) pinned onto a substrate, antifreeze protein (AFP) crystallized as a single crystal, and poly{2-(perfluorooctyl)ethyl acrylate} (PC 8 FA) polymer between polyimide sheets. It was found that not only BSA but also AFP and PC 8 FA molecules move in the systems. In addition, the molecular motion of AFP molecules was observed to increase with decreasing temperature. The rotational diffusion coefficients (D R ) of BSA, AFP, and PC 8 FA were estimated to be 0.73 pm 2 /s, 0.65 pm 2 /s, and 3.29 pm 2 /s, respectively. Surprisingly, the D R of the PC 8 FA polymer was found to be the highest among the three samples. This is the first report that measures the molecular motion of a single protein crystal and polymer by using DXB with a laboratory x-ray source. This technique can be applied to any kind of crystal and crystalline polymer and provides atomic-order molecular information.

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Biophysical Characterizations in the Solution State

Rambo

Inoue

2021

Encyclopedia of Virology

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X-ray-based living-cell motion analysis of individual serotonin receptors

Cited by 20 publications

References 17 publications

Living-Cell Diffracted X-ray Tracking Analysis Confirmed Internal Salt Bridge Is Critical for Ligand-Induced Twisting Motion of Serotonin Receptors

Living-Cell Diffracted X-ray Tracking Analysis Confirmed Internal Salt Bridge Is Critical for Ligand-Induced Twisting Motion of Serotonin Receptors

Laboratory diffracted x-ray blinking to monitor picometer motions of protein molecules and application to crystalline materials

Biophysical Characterizations in the Solution State

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