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 degrees C to 37 degrees C using a temperature-jump optical trap. We derive the folding free energy, entropy and enthalpy, and heat capacity change (Delta C-p = 1,050 +/- 50 cal/mol.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 Delta C-p (similar to 90%) between the unfolded and the transition states. In contrast, the major change in enthalpy (similar to 80%) 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.

Automated summary

This study experimentally verifies the existence of a molten globule intermediate in the protein folding process and reveals some thermodynamic properties of this intermediate.