title: Characterization of the dynamics of an essential helix in the U1A protein by time-resolved fluorescence measurements.
abstract: 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 beta-sheet in the free protein (closed conformation), but is directed away from this beta-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 degrees 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 beta-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.
authors.affiliation: Department of Chemistry and Molecular Biophysics Program, Wesleyan University, Middletown, Connecticut 06459, USA.
authors.forename: Divina
authors.initials: D
authors.lastname: Anunciado
authors.forename: Michael
authors.initials: M
authors.lastname: Agumeh
authors.forename: Bethany L
authors.initials: BL
authors.lastname: Kormos
authors.forename: David L
authors.initials: DL
authors.lastname: Beveridge
authors.forename: Joseph L
authors.initials: JL
authors.lastname: Knee
authors.forename: Anne M
authors.initials: AM
authors.lastname: Baranger
mesh_terms: D003198:Computer Simulation; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012997:Solvents; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D013997:Time Factors
chemical_list: D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; D012997:Solvents; C075339:U1A protein; D012313:RNA