Important model protein found to fold in unexpected way by single-molecule FRET spectroscopy

Proteins fold into unique shapes that are both thermodynamically stable and fully active. For single-domain proteins, folding is thought to be a simple two-step process, moving the protein directly from an unfolded to a folded state without intermediate steps. But new single-molecule measurements reveal otherwise.

Until now, measurements from a wide range of experiments suggested the B1-IgG binding domain of protein L (PL) was a two-state folder, and was even considered a model for this type of folding. By exploiting the power of single molecule fluorescence, investigators at the Weizmann Institute of Science in Israel report in The Journal of Chemical Physics that this, in fact, is not true for PL. They found a third, poorly populated state for this protein, hidden beneath its folded and unfolded states, that had so far gone undetected.

The third state was found with single-molecule FRET spectroscopy (sm-FRET) of single-protein molecules, immobilized in a lipid vesicle. The encapsulated PL molecules were labeled with fluorescent dyes, which were then probed with a laser while applying guanidinium chloride to cause the PL to denature, or unfold. Analysis of the FRET efficiency plots from the single molecule measurements revealed the existence of the new, third state, located off the main folding pathway, but connected to both the folded and unfolded states.

The investigators tested and ruled out possible artifacts that might have affected the observations. They also used computational modeling to verify their hypothesis that the third state corresponds to a partially unfolded beta hairpin section of the PL molecule. Future work will focus on more complex proteins and will study the dynamics of proteins as they carry out their functions in living systems.

Source: “Two states or not two states: Single-molecule folding studies of protein L,” by Haim Yuval Aviram, Menahem Pirchi, Yoav Barak, Inbal Riven, and Gilad Haran, The Journal of Chemical Physics (2017). The article can be accessed at https://doi.org/10.1063/1.4997584 .