Folding of the multidomain ribosomal protein L9: The two domains fold independently with remarkably different rates

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 (T(m) = 67 °C for the C-terminal domain; 80 °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 °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 °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 °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.