Figure 2 : Characterization of the charge-reversed mutant NT*.

From: Efficient protein production inspired by how spiders make silk

Figure 2

(a) Monomer–dimer equilibrium measured with Trp fluorescence. Spectra between 300 and 400 nm were measured and the ratios at 339/351 nm (wavelengths corresponding to monomer/dimer conformations) were plotted as a function of pH for NTwt (blue) and NT* (red). (b) Stability of NTwt (blue) and NT* (red) in the presence of 0–7 M urea, measured with Trp fluorescence and presented as transition points between native and denatured states ([den]50%) as a function of pH. (c) SEC analysis at pH 8 shows the NTwt monomer (blue) with a hydrodynamic size similar to the constitutive monomer mutants NTA72R (orange) and NT* (red). The migration profile for a dimer is represented by the constitutive dimer mutant NTE79QE84QE119Q (cyan). Another mutant, NTD40NE79QE119Q functions as a control and shows the profile expected in the presence of both monomers and dimers in equilibrium (dotted grey). (d) SEC analysis at pH 5.5 shows the NTwt dimer (blue) with a hydrodynamic size identical to the constitutive dimer mutant NTE79QE84QE119Q (cyan). At low pH, the constitutive monomer mutants NTA72R (orange) and NT* (red) have similar migration profiles, comparable to those observed at pH 8, proving their inability to form dimers.