title: Folding of the multidomain ribosomal protein L9: the two domains fold independently with remarkably different rates.
abstract: The folding and unfolding behavior of the multidomain ribosomal protein L9 from Bacillus stearothermophilus was studied by a novel combination of stopped-flow fluorescence and nuclear magnetic resonance (NMR) spectroscopy. One-dimensional 1H spectra acquired at various temperatures show that the C-terminal domain unfolds at a lower temperature than the N-terminal domain (Tm = 67 degrees C for the C-terminal domain, 80 degrees C for the N-terminal domain). NMR line-shape analysis was used to determine the folding and unfolding rates for the N-terminal domain. At 72 degrees C, the folding rate constant equals 2980 s-1 and the unfolding rate constant equals 640 s-1. For the C-terminal domain, saturation transfer experiments performed at 69 degrees C were used to determine the folding rate constant, 3.3 s-1, and the unfolding rate constant, 9.0 s-1. Stopped-flow fluorescence experiments detected two resolved phases: a fast phase for the N-terminal domain and a slow phase for the C-terminal domain. The folding and unfolding rate constants determined by stopped-flow fluorescence are 760 s-1 and 0.36 s-1, respectively, for the N-terminal domain at 25 degrees C and 3.0 s-1 and 0.0025 s-1 for the C-terminal domain. The Chevron plots for both domains show a V-shaped curve that is indicative of two-state folding. The measured folding rate constants for the N-terminal domain in the intact protein are very similar to the values determined for the isolated N-terminal domain, demonstrating that the folding kinetics of this domain is not affected by the rest of the protein. The remarkably different rate constants between the N- and C-terminal domains suggest that the two domains can fold and unfold independently. The folding behavior of L9 argues that extremely rapid folding is not necessarily functionally important.
authors.affiliation: Department of Chemistry, Graduate Program in Biophysics, State University of New York at Stony Brook 11794, USA.
authors.forename: S
authors.initials: S
authors.lastname: Sato
authors.forename: B
authors.initials: B
authors.lastname: Kuhlman
authors.forename: W J
authors.initials: WJ
authors.lastname: Wu
authors.forename: D P
authors.initials: DP
authors.lastname: Raleigh
mesh_terms: D001411:Geobacillus stearothermophilus / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D010446:Peptide Fragments; D012269:Ribosomal Proteins; C049650:ribosomal protein L9