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A two-step probing method to compare lysine accessibility across macromolecular complex conformations
Andrew J MacRae1,2, Patricia Coltri1,3, Eva Hrabeta-Robinson1
1Department of Molecular, Cell and Developmental Biology, University California , Santa Cruz , USA.
RNA Biology
|June 20, 2019
Summary
This study introduces a chemical probing method to map lysine residue reactivity in large molecular complexes. This technique provides crucial biochemical insights into structural rearrangements, complementing cryo-electron microscopy models.
Area of Science:
- Biochemistry
- Structural Biology
- Molecular Biology
Background:
- Advances in cryo-electron microscopy (cryo-EM) enable structural determination of large, dynamic molecular complexes.
- High-resolution models of complex biological assemblies are often limited, hindering understanding of functional transitions.
- Orthogonal biochemical data is essential for elucidating the molecular interactions driving conformational changes.
Purpose of the Study:
- To develop and validate a novel two-step chemical probing method for assessing lysine residue reactivity in macromolecular complexes.
- To demonstrate the utility of this method in understanding protein-RNA interactions and conformational dynamics.
- To provide a biochemical approach for contextualizing structural models obtained by cryo-EM and X-ray crystallography.
Main Methods:
- A two-step chemical probing strategy targeting lysine residues in purified macromolecular complexes.
- Detection of modified lysine residues using tandem mass spectrometry (MS/MS).
- Application of the method to the *E. coli* 30S ribosomal subunit and human spliceosomes.
Main Results:
- Lysine reactivity patterns quantitatively correlate with existing X-ray crystallography-derived structural models of the *E. coli* 30S ribosome.
- The method successfully identified distinct lysine reactivity profiles across three different human spliceosome conformations.
- Demonstrated the method's effectiveness in revealing structural rearrangements and protein-RNA interaction dynamics.
Conclusions:
- The chemical probing method offers a powerful biochemical approach to complement structural studies of macromolecular complexes.
- This technique is particularly valuable for investigating ribonucleoprotein complexes and changes in protein-RNA interactions.
- The method aids in interpreting architectural rearrangements observed in intermediate-resolution cryo-EM structures solved in multiple conformations.
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