No 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.

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.

Research on polymer surfaces has shown that the mobilities of polymer chains, which affect the aggregation state and thus the physical properties of the material, differ between the surface and bulk. However, the mobilities of the surface polymers have not been fully characterized. Therefore, we propose a time-resolved method for evaluating surface mobility. This measurement scheme is called grazing incidence diffracted X-ray blinking (GI-DXB) and can be used to evaluate the molecular motions occurring at polymer surfaces by continuously measuring X-ray diffraction patterns near the total reflection angle over small time periods. In this study, the crystallized polymer poly{2-(perfluorooctyl)ethyl acrylate}(PC8FA) was measured. The decay constants, which are indexes of molecular motions, were calculated to be 3.98 × 10−3 s−1 for the fluoroalkyl groups in the side chains observed along the in-plane direction and 3.36 × 10−3 s−1 for the lamellar structure observed along the out-of-plane direction when 2000 diffraction profiles of 500 ms were recorded and the incident angle was 0.07°. In contrast, transmission DXB indicated decay constants of 2.63 × 10−3 s−1 for the side chains and 2.87 × 10−3 s−1 for the lamellar structures. These results suggested that the PC8FA surface is mobile, because a larger decay constant indicates a higher mobility. GI-DXB can be used to measure surface dynamics. The authors contend that GI-DXB is a highly versatile tool because it allows the evaluation of local motions with a laboratory X-ray system, and these motions cannot be detected by conventional surface analyses. This measurement scheme may facilitate the development of high-performance polymers and discovery of new physical properties.