Visualizing Intramolecular Dynamics of Membrane Proteins

Abstract

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. Single-molecule measurements using optical microscopy have been widely used for kinetic analysis. Recently, improvements in detectors and image analysis technology have made it possible to use single-molecule determination methods using X-rays and electron beams, such as diffracted X-ray tracking (DXT), X-ray free electron laser (XFEL) imaging, and cryo-electron microscopy (cryo-EM). High-speed atomic force microscopy (HS-AFM) is a scanning probe microscope that can capture the structural dynamics of biomolecules in real time at the single-molecule level. Time-resolved techniques also facilitate an understanding of real-time intramolecular processes during chemical reactions. In this review, recent advances in membrane protein dynamics visualization techniques were presented.

Keywords: conformation dynamics; diffracted X-ray tracking technique; membrane proteins; single-molecule analysis.

Figure 1

Membrane protein dynamics visualization techniques. (A) Visualizing single molecular dynamics using an optical microscope. The γ subunit of the F₁ stator ring was labeled with a fluorescent actin filament. The anticlockwise rotation of the γ subunit was observed under an epifluorescence microscope [15]. (B) OT use a highly focused laser beam to generate force to trap or move a single molecule. This technique can be applied to understand biological mechanisms, such as conformational change, folding mechanisms, and protein-membrane interactions. (C) Cryo-EM and the classification technique. Two-dimensional-class averaged images of V-ATPase elucidated the rotational model of V-ATPase. ATP hydrolysis causes a 120° rotation step and conformation change in each subunit [17]. (D) HS-AFM scans the sample surface using the tip attached to the cantilever. The conformational changes of the protein are captured by the movement of a photodiode depending on the height of the protein. (E) XFEL imaging and SFX sample loading. Time-resolved X-ray diffraction and conformational changes of a protein are obtained by XFEL irradiation and photoactivation with various intervals (the image of the protein was generated from PDB:1FBK).

Figure 2

Diffracted X-ray tracking (DXT) and diffracted X-ray blinking (DXB) methods. (A) In DXT, the specific domain of a target protein is labeled with gold nanocrystals, with a diameter of 40 nm to 80 nm. The diffraction spots represent the tilting (θ) and twisting (χ) motions. DXT using white X-rays can analyze the trajectories of Laue spots appearing over a wide detector area. (B) DXB uses monochromatic X-rays, and the diffraction spots appear on the diffraction rings. The intramolecular motions are analyzed from the intensity changes of the Au (111) diffraction rings.

Figure 3

Diffracted X-ray tracking (DXT) measurement of ligand-induced rotation of TRPV1 channel. (A) DXT measurement system in SPring-8 BL40XU beamline. Diffraction images were recorded using an X-ray image intensifier (V5445P, Hamamatsu Photonics) and a CMOS camera (FASTCAM SA1.1, Photron, Japan). White X-rays are irradiated to the sample (inset), and the time-resolved movement diffractions are recorded. (B) Trajectory map of X-ray diffraction. Diffractions are obtained from Au(111) according to Bragg’s law. Trajectories move with a two-dimensional axis by tilting (θ) and twisting (χ). (C) Lifetime filtering enabled extracting motion components at different timescales. A CW rotational bias on TRPV1 was sustained by capsaicin (supposed to be channel opening bias) even in the longest lifetime group (8.0 ms ≦ LT < 10.0 ms). The top bars show the selected lifetime. Adapted with permission from Ref. [53]. 2020, American Chemical Society.