Interactions of protein side chains with RNA defined with REDOR solid state NMR

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biophysics

Background:

• The human immunodeficiency virus type-1 (HIV-1) Tat protein and its TAR RNA binding are crucial for viral transcriptional elongation.
• Determining the precise structure of the Tat-TAR complex has been challenging due to limitations in traditional NMR and crystallization techniques.

Purpose of the Study:

• To establish a method for obtaining high-resolution structural information of the Tat-TAR complex using solid-state NMR.
• To validate the applicability of solid-state NMR REDOR experiments for protein-RNA complex structural determination.

Main Methods:

• Utilized solid-state NMR rotational-echo double-resonance (REDOR) experiments.
• Incorporated (15)N and (13)C labels in the Tat peptide and (19)F and (31)P labels in the TAR RNA.
• Measured distances between specific atoms in the Tat peptide and TAR RNA.

Main Results:

• Successfully obtained multiple distance constraints between arginine residues in the Tat peptide and the U23 base and phosphodiester backbone of TAR RNA.
• REDOR-derived distances closely matched those from solution NMR structural models.
• Demonstrated the consistency of Tat-TAR interactions in both amorphous solid and solution states.

Conclusions:

• Solid-state NMR REDOR is a powerful technique for elucidating the structure of challenging protein-RNA complexes.
• The findings confirm that Tat-TAR interactions are conserved between solution and solid states.
• This methodology offers a viable alternative for structural studies when crystallization or solution NMR is insufficient.