Time-Resolved X-ray Observation of Intracellular Crystallized Protein in Living Animal.
Masahiro KuramochiIbuki SugawaraYoichi ShinkaiKazuhiro MioYuji C SasakiPublished in: International journal of molecular sciences (2023)
Understanding the cellular environment as molecular crowding that supports the structure-specific functional expression of biomolecules has recently attracted much attention. Time-resolved X-ray observations have the remarkable capability to capture the structural dynamics of biomolecules with subnanometre precision. Nevertheless, the measurement of the intracellular dynamics within live organisms remains a challenge. Here, we explore the potential of utilizing crystallized proteins that spontaneously form intracellular crystals to investigate their intracellular dynamics via time-resolved X-ray observations. We generated transgenic Caenorhabditis elegans specifically expressing the crystallized protein in cells and observed the formation of the protein aggregates within the animal cells. From the toxic-effect observations, the aggregates had minimal toxic effects on living animals. Fluorescence observations showed a significant suppression of the translational diffusion movements in molecules constituting the aggregates. Moreover, X-ray diffraction measurements provided diffraction signals originating from these molecules. We also observed the blinking behaviour of the diffraction spots, indicating the rotational motion of these crystals within the animal cells. A diffracted X-ray blinking (DXB) analysis estimated the rotational motion of the protein crystals on the subnanometre scale. Our results provide a time-resolved X-ray diffraction technique for the monitoring of intracellular dynamics.
Keyphrases
- electron microscopy
- high resolution
- dual energy
- induced apoptosis
- reactive oxygen species
- binding protein
- protein protein
- cell cycle arrest
- room temperature
- magnetic resonance imaging
- small molecule
- endoplasmic reticulum stress
- single molecule
- oxidative stress
- cell death
- magnetic resonance
- working memory
- gram negative
- long non coding rna
- energy transfer