Figure 2.

p8 is a dimer in solution. a, fluorescence signal (a.u., arbitrary units) as a function of temperature giving unfolding temperature of p8 in 50 mm Tris-HCl, 150 mm NaCl, 0.25 mm TCEP at pH 7.5, indicating high thermal stability (T_m ∼88 °C). The presence of 1 and 5% DMSO was found to leave this plot unaffected. b, elution profile obtained by SEC-MALS. The traces of the light scattering (blue), differential refractive index (red), and MALS-derived molar masses (black) are shown. The experimental SEC-MALS experiments gave a mean molar mass of 15,530 ± 120 g/mol⁻¹, confirming the presence of homodimer species in solution (the calculated molecular mass of p8 is 8053.4 Da). c, NMR determination of the global rotational correlation times, at T = 298 K, for p8 and p8_Phe44A (TRACT experiments (17)). The normalized intensity decay of the two ¹⁵N doublet coherences is plotted as a function of the relaxation delay. Fitting the data to mono-exponential functions returned the relaxation rate constants for α and β spin states. The difference between the two relaxation rates gave a global rotational correlation time τ_c of 10.9 ns (R_2α = 23.3 Hz; R_2β = 45.6 Hz) for p8, consistent with a dimer in solution. The τ_c value was significantly reduced in the case of p8_Phe44A (6.1 ns; R_2α = 20.3 Hz; R_2β = 32.8 Hz), which is consistent with the expected value (5.7 ns) for a monomeric protein at this temperature (27).