The study was led by Professor Fèlix Ritort, from the Faculty of Physics and the Institute of Nanosciences and Nanotechnology of the UB (IN2UB). Its first author is the researcher Marc Rico-Pasto (UB) and it counts with the collaboration of teams from the University of Padova (Italy), the Institute of Bioengineering in Lausanne (Switzerland) and the company SpliceBio, whose headquarters are in the Barcelona Science Park (PCB).
Optical tweezers to unravel the complexity of living matter
The emergence of innovative techniques such as optical and magnetic tweezers has revolutionized research in biophysics, and specifically, the study of thermodynamic properties in macromolecules: proteins, nucleic acids, etc. This type of technology enables the manipulation of individual molecules with nanometre precision (10-9 meters) applying forces in the piconewton range (10-12 newtons). Therefore, researchers can characterize the thermodynamic properties of complex biomolecules with unprecedented resolution. The application of such techniques provides with new scenarios for the experimental studies in the field of thermodynamics from a statistical approach, an interpretation of thermodynamics that was only possible from a theoretical perspective to date.
However, these techniques have limitations that prevent researchers from differentiating the origins of the measured forces. At the moment, combining different techniques to expand the number of control parameters is a challenge in biophysics. This is precisely what the team in charge of this study has done: introducing a temperature monitor in the optical tweezers to determine, for the first time, the entropy and enthalpy of protein folding.
Energy landscapes in protein folding
During the folding process of proteins and other macromolecules, different kinetic states take place between the native state and the denatured state. Examples are transition states, molecular intermediates and misfolded structures, which have a transient nature that makes thermodynamical characterization more difficult in experiments with a high number of molecules —from the 1023 molecule order, the value known as the Avogadro's number— which are analysed simultaneously. Particularly relevant to protein folding are transition states due to their extremely short lifetime.
“Our results reveal that, during the transition state, the protein skeletal structure is already built. However, most of the van der Waals interactions —weak forces— among the residues are not stabilized”, notes Professor Fèlix Ritort, member of the Department of Condensed Matter Physics of the UB.
“Conclusions show that protein folding can be understood as a process defined by two steps. In the first one, the protein reaches the transition state in which the native skeletal structure is built, and water is expelled from the inside of the polypeptide chain”, continues Ritort. “In the second step, the protein collapses, the interactions between protein residues are stabilized, and the protein reaches the native state”, concludes the researcher.
A first reading of the results reveals that there is a change of enthalpy and entropy during the transition state corresponding to 20% approximately of the total measured in the folding. “This phenomenon shows that the protein skeletal structure requires a 20% of the interactions between residues. The reading we make from the protein folding goes in line with the most recent hypotheses in the field of protein folding”, notes Marc Rico-Pasto, also member of the Department of Condensed Matter Physics.
Despite having stated that the protein skeletal structure is built during the transition state, authors say that they cannot conclude the amount of native interactions that exist in this state. “We can make a first estimation —they say—, but quantifying this result requires some experimental variable that allows us to measure or identify the number of bonds built during the molecular folding in real time”.
The team led by Professor Fèlix Ritort, head of the Small Biosystems Lab of the Faculty of Physics, made significant contributions to the study of the thermodynamic properties of complex systems in biomolecules. In previous studies, the team used the model of the barnase protein —a globular biomolecule secreted by Bacillus amyloliquefaciens— separated by a transition state. The barnase, which does not show intermediate states with a lifetime of more than a millisecond during the folding, is also the reference model for the characterization method of transition states during the protein folding process (phi-value analysis).
Rico-Pasto, M.; Zaltron, A.; Davis, S. J.; Frutos, S.; Ritort, F. “Molten globule-like transition state of protein barnase measured with calorimetric force spectroscopy”. PNAS, March 2022. Doi: 10.1073/pnas.2112382119