Line-shape simulation for the methyl carbon and proton peaks of L99A, in accordance with the two-state exchange model. (a and b) 13C line-shape spectral simulation for the L121Cδ1 carbon (A) and for the L84Cδ2 carbon (b), as a function of the excited state population. (c and d) 13C line-shape spectral simulation for the L121Cδ1 carbon (c) and the L84Cδ2 carbon (d), as a function of kex. The 13C chemical shift difference ΔωGE between the ground (G) and the transiently populated high-energy state (E), the rate constant kex (1449 s−1) for a chemical exchange between the two states, and a population of the high-energy state pE (3.4%) were all obtained from the literature (31). (e and f) 1H line shape spectral simulation for L121Hδ1 proton, as a function of the high-energy state population (e), and as a function of kex (f). The 1H chemical shift values of the ground and high-energy states of L99A/G113A, measured by ZZ-exchange NMR experiments at 1°C (32), are used in the simulations. The program WINDNMR-Pro was used for the simulations (37).