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

Abstract

Significance 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.

Folding Free Energy Determination of an RNA Three-Way Junction Using Fluctuation Theorems

“…From the work W , we have subtracted the energy contributions coming from the bead displacement, the stretching of the DNA-RNA handles, and the 3WJ single-stranded RNA (ssRNA). To do so, we have followed the methodology described in [ 22 , 35 ]. Briefly, the elastic response of the ssRNA and hybrid DNA-RNA handles is calculated using the inextensible worm-like chain model [ 36 ].…”

“…The value of that best matches the two work distributions in the crossing work region (around ) defines the matching estimate [ 22 ]. Below, we will focus on the previous three estimates (ECFT, EJE, BAR), but will use Equation ( 6 ) to illustrate the matching method.…”

“…Instead, in pulling experiments, the force is ramped at a given rate and the unfolding/folding and binding/unbinding reactions are followed under nonequilibrium conditions. In pulling experiments, the barrier height is modulated with time, rendering the folding energy landscape accessible even for molecules with high kinetic barriers, such as long RNAs or proteins [ 18 , 19 , 20 , 21 , 22 ].…”

Nucleic Acid Thermodynamics Derived from Mechanical Unzipping Experiments

“…Even though the irreversibility in RNA have been observed before in pulling experiments of short duplexes [ 70 ], a characterization of this general phenomenon in the unzipping of long RNA hairpins has been fundamental to measure the RNA NNBP free-energies. We do not exclude the possibility that similar intermediates also occur in DNA folding; however, this would require different experimental conditions, such as lower temperatures [ 5 , 71 ].…”

“…In molecular biophysics, the accurate knowledge of the thermodynamics of nucleic acids (NAs) and proteins is essential to obtain reliable predictions of protein folding [ 1 , 2 , 3 , 4 , 5 ], DNA/RNA hybridization [ 6 ], and their interactions with enzymes and ions [ 7 ]. Over the past decades, measurements of molecular free-energies, entropies, and enthalpies have been obtained by using bulk techniques such as UV absorbance, fluorescence, and calorimetry, among others [ 8 ].…”