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Developing methods to study conformational changes in RNA crystals using a photocaged ligand.

Hyun Kyung Lee1, Chelsie E Conrad1, Valentin Magidson2

  • 1Protein-Nucleic Acid Interaction Section, Center for Structural Biology, Center for Cancer Research, National Cancer Institute, Frederick, MD, United States.

Frontiers in Molecular Biosciences
|September 2, 2022
PubMed
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This summary is machine-generated.

Time-resolved crystallography using photocaged ligands offers a controlled method to study RNA conformational changes. This study demonstrates a light-inducible system for observing solid-to-solid phase transitions in RNA crystals.

Area of Science:

  • Structural Biology
  • Biophysics
  • Crystallography

Background:

  • Real-time crystallographic studies require uniform spatial and temporal changes.
  • Ligand-mixing experiments face challenges with asynchrony due to diffusion and crystal heterogeneity.
  • Photolabile caged ligands offer a solution for controlled, rapid triggering of reactions.

Purpose of the Study:

  • To investigate solid-to-solid phase transitions (SSPT) in RNA crystals induced by light-triggered release of a photocaged ligand.
  • To characterize the kinetics and influencing factors of light-induced SSPT using polarized video microscopy.
  • To demonstrate the feasibility of using photocaged ligands for time-resolved crystallographic studies.

Main Methods:

  • Time-resolved X-ray crystallography (TRX) with an X-ray free electron laser (XFEL).
Keywords:
RNAcrystal phase transitionphoto-inducedphotocaged ligandriboswitch

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  • Polarized video microscopy (PVM) after photoactivation of a photocaged adenine (pcADE).
  • Kinetic analysis using a four-state model for ligand-induced conformational changes.

    • Main Results:

    • Light-triggered release of pcADE induced SSPT in RNA crystals, maintaining Bragg diffraction.
    • Transition times were less dependent on crystal size compared to direct mixing methods.
    • The photo-induced transition was influenced by the equilibrium between caged and uncaged ligand due to uncaging efficiency.

    Conclusions:

    • A method for characterizing phase transitions in RNA crystals using photocaged ligands was successfully demonstrated.
    • The study highlights the feasibility of triggering phase transitions with light-inducible systems for time-resolved crystallography.
    • This approach provides insights into studying time-resolved ligand-induced conformational changes in crystals.