Native exchange NMR None no exchange for >220 hours MPVDFNGYWKMLSNENFEEYLRALDVNVALRKIANLLKPDKEIVQDGDHMIIRTLSTFRNYIMDFQVGKEFEEDLTGIDDRKCMTTVSWDGDKLQCVQKGEKEGRGWTQWIEGDELHLEMRAEGVTCKQVFKKVH

For the identification of slow exchanging amide protons, the protein sample buffer was replaced with a perdeuterated solution consisting of 20 mM KD2PO4 and 0.05% NaN3 in D2O, as previously described (36). This perdeuterated solution was prepared from the protonated buffer (90% H2O/10% D2O) at pH 6.0 by lyophilizing and redissolving in D2O twice. The buffer exchange was performed at 4°C with Vivaspin centrifugal concentrators (molecular weight cutoff of 10 kDa) in several rounds of filtration over 6–10 h. Following equilibration to 25°C inside the NMR magnet, a series of homonuclear TOCSY (alternating beween 30 and 80 ms spin lock time) and NOESY (alternating between 80 and 150 ms mixing time) experiments were collected over a period of more than 9 days (the first 3.5 days at 600.13 MHz and the remaining time at 499.87 MHz) in order to monitor the amide proton exchange.

Native exchange NMR None no exchange for >20 hours MPVDFNGYWKMLSNENFEEYLRALDVNVALRKIANLLKPDKEIVQDGDHMIIRTLSTFRNYIMDFQVGKEFEEDLTGIDDRKCMTTVSWDGDKLQCVQKGEKEGRGWTQWIEGDELHLEMRAEGVTCKQVFKKVH

For the identification of slow exchanging amide protons, the protein sample buffer was replaced with a perdeuterated solution consisting of 20 mM KD2PO4 and 0.05% NaN3 in D2O, as previously described (36). This perdeuterated solution was prepared from the protonated buffer (90% H2O/10% D2O) at pH 6.0 by lyophilizing and redissolving in D2O twice. The buffer exchange was performed at 4°C with Vivaspin centrifugal concentrators (molecular weight cutoff of 10 kDa) in several rounds of filtration over 6–10 h. Following equilibration to 25°C inside the NMR magnet, a series of homonuclear TOCSY (alternating beween 30 and 80 ms spin lock time) and NOESY (alternating between 80 and 150 ms mixing time) experiments were collected over a period of more than 9 days (the first 3.5 days at 600.13 MHz and the remaining time at 499.87 MHz) in order to monitor the amide proton exchange.