Diffracted X-ray Blinking Tracks Single Protein Motions

Abstract

Single molecule dynamics studies have begun to use quantum probes. Single particle analysis using cryo-transmission electron microscopy has dramatically improved the resolution when studying protein structures and is shifting towards molecular motion observations. X-ray free-electron lasers are also being explored as routes for determining single molecule structures of biological entities. Here, we propose a new X-ray single molecule technology that allows observation of molecular internal motion over long time scales, ranging from milliseconds up to 103 seconds. Our method uses both low-dose monochromatic X-rays and nanocrystal labelling technology. During monochromatic X-ray diffraction experiments, the intensity of X-ray diffraction from moving single nanocrystals appears to blink because of Brownian motion in aqueous solutions. X-ray diffraction spots from moving nanocrystals were observed to cycle in and out of the Bragg condition. Consequently, the internal motions of a protein molecule labelled with nanocrystals could be extracted from the time trajectory using this diffracted X-ray blinking (DXB) approach. Finally, we succeeded in distinguishing the degree of fluctuation motions of an individual acetylcholine-binding protein (AChBP) interacting with acetylcholine (ACh) using a laboratory X-ray source.

Tilting and rotational motions of silver halide crystal with diffracted X-ray blinking

“…Our DXB analysis, without the tracking of diffraction spots, analyses the time-resolved fluctuations of the diffraction intensity using the ACF. Our previous study showed that the ACF decay constant of protein molecules with nanocrystals correlates with the angular velocity 12 . The time trajectory of the diffraction intensity of silver halides reflects the grain motions during the photolysis chemical reaction.…”

“…S3 ). The time-resolved intensity fluctuation of each pixel was computed to evaluate rotational motions using the following autocorrelation function (ACF) 12 : where I(t) represents the diffracted photon intensity. The brackets 〈〉 indicate time-averaged values.…”

“…Several ACF curves did not show a single exponential decay because the signal-to-noise ratio was low. Thus, we chose decay constants to satisfy the following conditions: (I) 0 < y, 0 < A and 0 < Г 12 and (II) residual values between the fitted and actual ACF curves of less than 0.5. These calculations were performed for ACF data for all pixels.…”

“…1 b). The movements of diffraction spots appear to behave in an on/off (blinking) fashion at the Debye–Scherrer ring, which we term “Diffracted X-ray Blinking” (DXB) 12 , 13 . The tilting and rotational motions of a protein molecule labelled with nanocrystals can be evaluated as its exponential relaxation by ACF.…”

Overview of “2SEA Frontiers of Synchrotron Radiation Biophysics” session of symposia in the 57th Annual Meeting of Biophysical Society of Japan

“…DXT refers to the technique of labeling specific sites of protein molecules with gold nanocrystals and monitoring their local movements by tracking the diffraction spots from the nanocrystals. Previously, intense X-ray beams were required for these measurements, but recently Sekiguchi has developed a technique with much lower doses of X-ray, by observing the diffracted intensity fluctuation from the nanocrystals (Sekiguchi et al 2018).…”