Time-Resolved X-ray Observation of Intracellular Crystallized Protein in Living Animal

Author:

Kuramochi Masahiro1ORCID,Sugawara Ibuki1,Shinkai Yoichi2ORCID,Mio Kazuhiro3ORCID,Sasaki Yuji C.4ORCID

Affiliation:

1. Graduate School of Science and Engineering, Ibaraki University, Hitachi 316-8511, Japan

2. Molecular Neurobiology Research Group, Biomedical Research Institute, National Institute of Advance Industrial Science and Technology (AIST), Tsukuba 305-8566, Japan

3. AIST-UTokyo Advanced Operando-Measurement Technology Open Innovation Laboratory (OPERANDO-OIL), National Institute of Advanced Industrial Science and Technology (AIST), Kashiwa 277-8565, Japan

4. Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa 277-8561, Japan

Abstract

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.

Funder

JSPS KAKENHI

Top Runners in Strategy of Transborder Advanced Researches (TRiSTAR) program

Foundation for Interaction in Science & Technology

Nakatani Foundation for Advancement of Measuring Technologies in Biomedical Engineering

Publisher

MDPI AG

Subject

Inorganic Chemistry,Organic Chemistry,Physical and Theoretical Chemistry,Computer Science Applications,Spectroscopy,Molecular Biology,General Medicine,Catalysis

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