Real-Time Observation of Capsaicin-Induced Intracellular Domain Dynamics of TRPV1 Using the Diffracted X-ray Tracking Method.

The transient receptor potential vanilloid type 1 (TRPV1) is a multimodal receptor which responds to various stimuli, including capsaicin, protons, and heat. Recent advances in cryo-electron microscopy have revealed the structures of TRPV1. However, due to the large size of TRPV1 and its structural complexity, the detailed process of channel gating has not been well documented.

Visualizing Intramolecular Dynamics of Membrane Proteins.

Membrane proteins play important roles in biological functions, with accompanying allosteric structure changes. Understanding intramolecular dynamics helps elucidate catalytic mechanisms and develop new drugs. In contrast to the various technologies for structural analysis, methods for analyzing intramolecular dynamics are limited.

Diffracted X-ray Tracking Method for Measuring Intramolecular Dynamics of Membrane Proteins.

Membrane proteins change their conformations in response to chemical and physical stimuli and transmit extracellular signals inside cells. Several approaches have been developed for solving the structures of proteins. However, few techniques can monitor real-time protein dynamics.

Angle change of the A-domain in a single SERCA1a molecule detected by defocused orientation imaging.

The sarcoendoplasmic reticulum Ca-ATPase (SERCA) transports Ca ions across the membrane coupled with ATP hydrolysis. Crystal structures of ligand-stabilized molecules indicate that the movement of actuator (A) domain plays a crucial role in Ca translocation. However, the actual structural movements during the transitions between intermediates remain uncertain, in particular, the structure of E2PCa has not been solved.

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

Serotonin receptors play important roles in neuronal excitation, emotion, platelet aggregation, and vasoconstriction. The serotonin receptor subtype 2A (5-HTR) is a Gq-coupled GPCR, which activate phospholipase C. Although the structures and functions of 5-HTRs have been well studied, little has been known about their real-time dynamics.

Agonist and Antagonist-Diverted Twisting Motions of a Single TRPV1 Channel.

Transient receptor potential vanilloid type 1 (TRPV1) channels are activated by heat, vanilloids, and extracellular protons. Cryo-EM has revealed various conformations of TRPV1, and these structures suggest an intramolecular twisting motion in response to ligand binding. However, limited experimental data support this observation.

N-terminal β-strand of single-headed kinesin-1 can modulate the off-axis force-generation and resultant rotation pitch.

In in vitro microtubule gliding assays, most kinesins drive the rotation of gliding microtubules around their longitudinal axes in a corkscrew motion. The corkscrewing pitch is smaller than the supertwisted protofilament pitch of microtubules, indicating that the corkscrewing pitch is an inherent property of kinesins. To elucidate the molecular mechanisms through which kinesins corkscrew the microtubule, we performed three-dimensional tracking of a quantum dot bound to a microtubule translocating over a surface coated with single-headed kinesin-1 s under various assay conditions to alter the interactions between the kinesin and microtubule.

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

G protein-coupled receptors (GPCRs) are seven-transmembrane proteins, which transmit extracellular signals inside cells via activating G proteins. GPCRs are involved in a wide variety of physiological functions, such as signal sensing, immune system processes, and neurotransmission. Although the structures and functions of GPCRs have been well studied, little has been known about their real-time dynamics on live cells.

Dissection of the angle of single fluorophore attached to the nucleotide in corkscrewing microtubules.

Direct dissection of the angles of single fluorophores under an optical microscope has been a challenging approach to study the dynamics of proteins in an aqueous solution. For angle quantifications of single substrates, however, there was only one report (Nishizaka et al., 2014) because of difficulties of construction of experimental systems with active proteins working at the single-molecule level.