Intermolecular protein-RNA interactions revealed by 2D 31P-15N magic angle spinning solid-state NMR spectroscopy

Area of Science:

• Structural Biology
• Biophysics
• Nuclear Magnetic Resonance (NMR) Spectroscopy

Background:

• X-ray crystallography faces challenges in determining the structure of large, flexible ribonucleoprotein (RNP) complexes due to difficulties in crystallization.
• Solution NMR is limited by the size of RNP complexes, restricting its applicability.
• Solid-state NMR spectroscopy offers a size-independent alternative, not requiring large crystals and accommodating flexibility and disorder.

Purpose of the Study:

• To demonstrate the utility of solid-state NMR spectroscopy for investigating intermolecular interactions within large RNP complexes.
• To establish solid-state NMR as a viable method for structural studies of challenging protein-RNA assemblies.

Main Methods:

• Utilizing solid-state NMR spectroscopy, specifically Transferred Echo Double Resonance (TEDOR) experiments.
• Measuring distances between nitrogen-15 ((15)N) in the protein backbone and phosphorus-31 ((31)P) in the RNA backbone.
• Employing measured distances as restraints for computational structure calculations.

Main Results:

• Demonstrated the capability of solid-state NMR to probe protein-RNA interfaces in RNP complexes.
• Accurately measured inter-nuclear distances, validated using the L7Ae-box C/D RNA complex with a known crystal structure.
• Showcased the potential of solid-state NMR for structural analysis of large, flexible, and partially disordered RNP complexes.

Conclusions:

• Solid-state NMR spectroscopy is a powerful and versatile tool for structural investigations of large RNP complexes.
• This technique overcomes limitations of crystallography and solution NMR for studying protein-RNA interactions.
• Opens new avenues for understanding the structure and dynamics of complex biological assemblies.