Characterization of the Dynamics of an Essential Helix in the U1A Protein by Time-Resolved Fluorescence Measurements
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- 作者：Divina Anunciado ; Michael Agumeh ; Bethany L. Kormos ; David L. Beveridge ; Joseph L. Knee ; Anne M. Baranger
- 刊名：Journal of Physical Chemistry B
- 出版年：2008
- 出版时间：May 15, 2008
- 年：2008
- 卷：112
- 期：19
- 页码：6122 - 6130
- 全文大小：451K
- 年卷期：v.112,no.19(May 15, 2008)
- ISSN：1520-5207
文摘
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 importantcontributor to RNA recognition. The N-terminal RRM of the U1A protein contains a C-terminal helix (helixC) that interacts with the RNA-binding surface of a 
le">-sheet in the free protein (closed conformation), but isdirected away from this le">-sheet in the complex with RNA (open conformation). The dynamics of helix C inthe free protein have been proposed to contribute to binding affinity and specificity. We report here a directinvestigation 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 withina 20
range of motion. Steady-state fluorescence experiments and molecular dynamics simulations suggestthat the dynamical motion of helix C occurs within the closed conformation. Mutation of a residue on thele">-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 helixC. Mutation of Asp90 in the hinge region between helix C and the remainder of the protein to Ala or Glysubtly alters the dynamics of the U1A protein and destabilizes the complex. Together these results show thathelix C maintains a dynamic closed conformation that is stable to these targeted protein modifications anddoes not equilibrate with the open conformation on the nanosecond time scale.
le">-sheet in the free protein (closed conformation), but isdirected away from this le">-sheet in the complex with RNA (open conformation). The dynamics of helix C inthe free protein have been proposed to contribute to binding affinity and specificity. We report here a directinvestigation 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 withina 20 range of motion. Steady-state fluorescence experiments and molecular dynamics simulations suggestthat the dynamical motion of helix C occurs within the closed conformation. Mutation of a residue on thele">-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 helixC. Mutation of Asp90 in the hinge region between helix C and the remainder of the protein to Ala or Glysubtly alters the dynamics of the U1A protein and destabilizes the complex. Together these results show thathelix C maintains a dynamic closed conformation that is stable to these targeted protein modifications anddoes not equilibrate with the open conformation on the nanosecond time scale.