Details

Autor(en) / Beteiligte

• Anunciado, Divina
• Agumeh, Michael
• Kormos, Bethany L
• Beveridge, David L
• Knee, Joseph L
• Baranger, Anne M

Titel

Characterization of the Dynamics of an Essential Helix in the U1A Protein by Time-Resolved Fluorescence Measurements

Ist Teil von

• The journal of physical chemistry. B, 2008-05, Vol.112 (19), p.6122-6130

Ort / Verlag

United States: American Chemical Society

Erscheinungsjahr

2008

Link zum Volltext

Quelle

Alma/SFX Local Collection

Beschreibungen/Notizen

• The RNA recognition motif (RRM), one of the most common RNA-binding domains, recognizes single-stranded RNA. A C-terminal helix that undergoes conformational changes upon binding is often an important contributor to RNA recognition. The N-terminal RRM of the U1A protein contains a C-terminal helix (helix C) that interacts with the RNA-binding surface of a β-sheet in the free protein (closed conformation), but is directed away from this β-sheet in the complex with RNA (open conformation). The dynamics of helix C in the free protein have been proposed to contribute to binding affinity and specificity. We report here a direct investigation of the dynamics of helix C in the free U1A protein on the nanosecond time scale using time-resolved fluorescence anisotropy. The results indicate that helix C is dynamic on a 2−3 ns time scale within a 20° range of motion. Steady-state fluorescence experiments and molecular dynamics simulations suggest that the dynamical motion of helix C occurs within the closed conformation. Mutation of a residue on the β-sheet that contacts helix C in the closed conformation dramatically destabilizes the complex (Phe56Ala) and alters the steady-state fluorescence, but not the time-resolved fluorescence anisotropy, of a Trp in helix C. Mutation of Asp90 in the hinge region between helix C and the remainder of the protein to Ala or Gly subtly alters the dynamics of the U1A protein and destabilizes the complex. Together these results show that helix C maintains a dynamic closed conformation that is stable to these targeted protein modifications and does not equilibrate with the open conformation on the nanosecond time scale.