Global fitting of relaxation dispersion data measured for twelve residues in nDsbDox at 10°C, 15°C, 20°C, 25°C and 35°C at 500 MHz and at 15°C and 25°C at 750 MHz was performed using CATIA (Cpmg, Anti-trosy, and Trosy Intelligent Analysis)(Alderson et al., 2019; Baldwin et al., 2011a; Vallurupalli et al., 2008), where the specific pulse sequence was simulated incorporating differential relaxation rates between the coherences generated, and off-resonance effects of the 180o pulses. Error bars were determined as described in Figure 6—figure supplement 1—source data 1. Relaxation dispersion data measured for W65 and the curves from the global fitting are shown in (A) (Figure 6—figure supplement 1—source data 1). The global fits at five different temperatures yield a description of the free energy landscape of the exchange process shown in (B). The minor state of nDsbDox is enthalpically more favorable than the ground state. This is reflected in the decrease in the population of the minor state from 7.1% to 1.6% when the temperature is increased from 10°C to 35°C (Figure 6—figure supplement 1—source data 1). The minor state is entropically less favorable than the ground state. Overall, the reaction coordinate in (B) shows that the minor state is less favorable by 8.8 kJ mol−1 at 25°C. The fitting of the experimental curves at the five temperatures also reports on the barrier between the two states. The transition state is enthalphically more favorable than the ground state. There is, however, a sizeable free-energy barrier as the transition state is lower in entropy than the ground state, and so the rate for the forward reaction, while approximately temperature independent, decreases slightly with increasing temperature. Note that and hence depend on the transmission coefficient κ that is used in Equation 1.
and are the activation enthalpy and entropy for the reaction. The transmission coefficient κ was set to 1.4 × 10−7 s−1 K−1 (Fersht, 1984).