Original Paper: Mechanically switching single-molecule fluorescence of GFP by unfolding and refolding
For the most part of biology, it is form that follows function. Proteins are a perfect example of this — they are made of a sequence of amino acids (the protein building units), which are synthesized by the ribosome. Once synthesized, the long strings of amino acids fold up into a particular 3D shape or conformational state. Proteins take less than a thousandth of a second to attain their preferred conformational state (called “native state”) that — if nothing goes wrong — ends up being the same for a given sequence. This process is called protein folding. Explaining how a protein finds its folding preference out of all possible ways in such a short time is a longstanding problem in biology.
But, how do scientists know if – and when – a protein is in its folded state? The most straightforward way to do this is by observing its function — the way that a protein performs some biochemical task within the cell. If the protein is functionally active, then it has achieved its proper structure. However, most proteins are too small to observe directly without damaging the cell. To solve this problem researchers frequently use Green Fluorescent Protein (GFP), a protein that glows when it is hit by light of a specific wavelength. By attaching GFP to other proteins, researchers can see exactly where those proteins are at different timepoints. GFP’s stability, lack of interaction with other proteins, and non-toxicity make it an extremely popular candidate for visualizing protein localization. In other words, one “function” of GFP is to fluoresce. Today’s paper seeks to understand how structure correlates with function in GFP, one of biology’s most important tools.
To control the folding process, the authors used dual optical tweezers to mechanically stretch and relax the protein. Optical tweezers — as the name suggests — manipulate the position of particles (beads) using laser light. These beads are typically in the size range of micrometers. To apply forces on the GFP, the beads are attached to the protein via DNA “handles,” so that a DNA strand attached to the protein will stick to the DNA strand attached to the bead. These strands are then bound together ensuring that the force on the beads is transferred to the GFP. The construct looks as follows:
Bead – DNA – Protein – DNA – Bead
When the beads move apart, the protein is stretched to its maximal possible length (also called its contour length) and is unfolded, but when the beads get closer together, the protein folds back to its preferred structure. This process is illustrated in Figure 1.
The authors observed that during unfolding, the GFP protein has undergone two intermediate states before unfolding completely. After unfolding, the beads were brought closer together and the protein folded itself back through the intermediate stages. The GFP molecule stopped emitting light when it was unfolded, which was expected. However, it started fluorescing only when it was completely in its folded state. This important finding showed that this protein is functionally inactive in any of the intermediate folding stages. The authors also observed that this process is reversible; they could unfold and refold the GFP molecule multiple times (see Figure 2).
These findings contribute towards understanding the functionality of proteins that could be used as in vivo optical sensors in force transduction. This work also opens up new avenues in studying biomolecules at the single-molecule level, such as DNA-protein complexes that can induce changes in conformation. Although the experiment only pulled the protein along one axis, this technique could be extended to pulling in several directions at once. If one could control the applied force in 3D, then it could be possible to gain more information on how exactly the protein folds and/or what happens during that process.
