Molten globule-like transition state of protein barnase measured with calorimetric force spectroscopy

Understanding how proteins fold into their native structure is a fundamental problem in biophysics, crucial for protein design. It has been hypothesized that the formation of a molten globule intermediate precedes folding to the native conformation of globular proteins; however, its thermodynamic properties are poorly known. We perform single-molecule pulling experiments of protein barnase in the range of 7${}^{\circ }$C to 37${}^{\circ }$C using a temperature-jump optical trap. We derive the folding free energy, entropy and enthalpy, and heat capacity change ($\mathrm{\Delta }{C}_p=1050\pm 50$ cal/mol$\cdot$K) at low ionic strength conditions. From the measured unfolding and folding kinetic rates, we also determine the thermodynamic properties of the transition state, finding a significant change in $\mathrm{\Delta }{C}_p$ ($\sim$ 90$\mathrm{\%}$) between the unfolded and the transition states. In contrast, the major change in enthalpy ($\sim$ 80$\mathrm{\%}$) occurs between the transition and native states. These results highlight a transition state of high energy and low configurational entropy structurally similar to the native state, in agreement with the molten globule hypothesis.