March 10 2022

Understanding the molecular forces driving the unfolded polypeptide chain to self-assemble into a functional native structure remains an open question. However, identifying the states visited during protein folding (e.g., the transition state between the unfolded and native states) is tricky due to their transient nature. Here, we introduce calorimetric force spectroscopy in a temperature jump optical trap to determine the enthalpy, entropy, and heat capacity of the transition state of protein barnase. We find that the transition state has the properties of a dry molten globule, that is, high free energy and low configurational entropy, being structurally similar to the native state. This experimental single-molecule study characterizes the thermodynamic properties of the transition state in funneled energy landscapes.

Abstract

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 ^∘C to 37 ^∘C using a temperature-jump optical trap. We derive the folding free energy, entropy and enthalpy, and heat capacity change (ΔCp = 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 ΔCp (∼90%) between the unfolded and the transition states. In contrast, the major change in enthalpy (∼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.

See: https://www.pnas.org/doi/10.1073/pnas.2112382119

Figure: CFS of protein barnase. (A) Three-dimensional view of native barnase obtained with X-ray diffraction with 1.50-Å resolution (44). Four external α-helices (helix 1: Phe7-Tyr17 [red]; helix 2: Lys27-Leu33 [blue]; helix 3: Ala37-Lys39 [cyan]; helix 4: Leu42-Val45 [orange]) contain a total of 25 amino acids surrounding four β-strands (purple) located in the protein core. (B) Schematics of the temperature-jump optical trap setup. The diode lasers (red and blue) form a single optical trap, while the collimated heating laser (green) passes through the microfluidic chamber. CCD, charge-coupled device; PBS, polarizing beam splitter. (C) Illustration of the molecular construct and experimental setup: barnase is flanked by two identical 500-bp dsDNA handles and tethered between two beads. One bead is captured in the optical trap while the other one is kept fixed by air suction on the tip of a glass micropipette.