The goal of most protein folding experiments is detecting and characterization various transient states the protein visits while folding into its native conformation. This process can be visualized as a diffusional conformational search along funnel-like energy landscape with a multitude of unfolded conformation at the rim and a single global minimum representing the native folded conformation.
In a nutshell, such a folding model assumes that there are many paths between unfolded and native state and a protein molecule may sample any structure from the ensemble at each step of the reaction. Analysis of such a complex and heterogeneous sequence of events requires spectroscopic tools that can overcome the inherent averaging in standard techniques. Single-molecule measurements prove to be powerful in dissecting complex protein dynamics.
The study of protein folding reactions at the single-molecule level requires extended observation periods that normally require their tethering to the surface. Special care is needed during immobilization of proteins for folding studies since surface interactions can significantly alter the folding energy landscape. The method which was developed in our lab overcomes this problem by encapsulating protein molecules within surface-tethered lipid vesicles. The diameter of the encapsulating vesicle is much larger than the diameter of trapped protein molecule. Therefore the molecule can freely diffuse inside vesicle interior. This minimizes a perturbation of the energy landscape due to confinement and nonproductive interactions with the vesicle membrane.
One serious disadvantage of vesicle encapsulation is the remarkable impermeability of a lipid membrane for most ions and solutes, which make the exchange of intra-vesicular buffer composition practically impossible and limits studies to the equilibrium experiment with the conditions close to the folding transition midpoint. We are developing a method to induce pores of a precise size into the vesicle membrane, to extend protein-folding study to non-equilibrium experiments. Our strategy is based on the self-assembling properties of channel forming toxin α-hemolysin, secreted from Staphylococus aureus as a water soluble monomer which interacts with the target membrane to form heptameric transmembrane pores. Overcoming the permeability barrier will allow the study of real-time folding dynamics of individual molecules over a broad range of initial and final conditions.