Inducing Cooperative folding through tightening contacts creates an unfavorable two-state folding behavior.

<p>The free energy profile is represented as Q_CA versus F(Q_CA)/k_BT. The wild-type IL-33 is represented in black and IL-33 with tighter contacts introduced in Trefoil 1 (ε^{<i>i</i>,<i>j</i>} = ε*1.03) is represented in dark blue while (ε^{<i>i</i>,<i>j</i>} = ε*1.06) is represented in light blue. IL-33 with tightened contacts in Trefoil 1 induces two-state cooperative folding as evidenced by the presence of only one transition state. Additionally, this two-state folding is unfavorable with respect to native three-state folding as evidenced by the larger barrier to the native state, slowing the folding process.