Figure 2. Ensemble kinetics of I27 tandem constructs.

(a) Ensemble refolding and unfolding kinetics of I27 single-domain (black), two- (blue), three- (green) and eight-domain (purple) constructs. All multidomain constructs display two refolding phases (circles and diamonds), with a deviation from linearity at low GdmCl concentration (rollover) and two unfolding phases (circles and triangles). Unfolding of all ‘never unfolded' I27 tandem constructs is well described by a single-phase corresponding to native-state unfolding (yellow circles). Rate coefficients for both slow and fast unfolding phases match values for the native (red circles) and the stable misfolded state (red triangles) unfolding measured in single-molecule experiments27, respectively. (b,c) Comparison of the relative amplitudes for the misfolding phase of all constructs observed during refolding (b) and unfolding (c) of previously refolded protein. For both reactions, the proportion of misfolded species increases with the number of domains in a manner we would predict (1:1.3:1.8, equation 1 in Methods).