Dynamic motions of ice-binding proteins in living Caenorhabditis elegans using diffracted X-ray blinking and tracking

Abstract

The dynamic properties of protein molecules are involved in the relationship between their structure and function. Time-resolved X-ray observation enables capturing the structures of biomolecules with picometre-scale precision. However, this technique has yet to be implemented in living animals. Here, we examined diffracted X-ray blinking (DXB) and diffracted X-ray tracking (DXT) to observe the dynamics of a protein located on intestinal cells in adult Caenorhabditis elegans . This in vivo tissue-specific DXB was examined at temperatures from 20 °C to −10 °C for a recombinant ice-binding protein from Antarctomyces psychrotrophicus (AnpIBP) connected with the cells through a transmembrane CD4 protein equipped with a glycine-serine linker. AnpIBP inhibits ice growth at subzero temperatures by binding to ice crystals. We found that the rotational motion of AnpIBP decreases at −10 °C. In contrast, the motion of the AnpIBP mutant, which has a defective ice-binding ability, did not decrease at −10 °C. The twisting and tilting motional speeds of AnpIBPs measured above 5 °C by DXT were always higher than those of the defective AnpIBP mutant. These results suggest that wild-type AnpIBP is highly mobile in solution, and it is halted at subzero temperatures through ice binding. DXB and DXT allow for exploring protein behaviour in live animals with subnano resolution precision.

Adenosine triphosphate induces amorphous aggregation of amyloid β by increasing Aβ dynamics

“…The ACF curves were fitted to an exponential curve by ACF(t) = Aexp(−Гt) + y, where A is the amplitude, y is the conversion factor, and Г is the decay constant. The decay constants were chosen to satisfy the following conditions: (i) 0 < y, 0 < A and 0 < Г, and (ii) residual values between the fitted and actual ACF curves of less than 1.0 23 , 24 . These calculations were performed for all pixels.…”

Diffracted X-ray Tracking for Observing the Internal Motions of Individual Protein Molecules and Its Extended Methodologies

“…Since the motion is not in one direction but in random motion, the observation of the diffraction point in motion using monochromatic X-rays, which have only a narrow energy range, causes the appearance of a blinking diffraction point. This concept is the principle of the diffracted X-ray blinking method DXB, as shown in Figure 18 [45][46][47][48][49][50][51][52][53][54]. Unlike DXT, where the exact direction of motion can be determined, the speed of motion can be monitored; the X-ray blinking phenomenon is similar to the X-ray Photon Correlation Spectroscopy: XPCS analysis method [55,56], which is an X-ray technique.…”

Analysis of the Structural Dynamics of Proteins in the Ligand-Unbound and -Bound States by Diffracted X-ray Tracking

“…Protein structural dynamics can be determined using a brilliant light source and a high-speed detector to detect the time-dependent movement of a gold nanocrystal attached to the target protein in real-time, in the range of μsec to msec. Using DXT, the structural dynamics of various proteins and those in ligand–protein interactions have been analyzed [ 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 ]. We used DXT to analyze the change in structural dynamics of relatively smaller globular proteins, a single-chain variable fragment (scFv) antibody (26 k), a de novo designed protein (8 k), and a DNA-binding protein (12 k), as they interacted with other molecules [ 29 , 30 , 31 , 32 ].…”