Fold and Unfold

Animation of GFP unfolding

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:

BeadDNAProteinDNABead

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.

Animation of GFP unfolding
Figure 1: The beads (circles) at each end are manipulated by laser beams and move back and forth. The DNA handles (purple) are attached to the GFP protein (green) that folds and unfolds turning to a functionally active and inactive state, respectively.

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).

Correlation between Fluorescence and Contour Length of the protein
Figure 2: Fluorescence signals of the GFP protein as it cycles through the unfolding and folding states. (A) The unfolded protein (light gray line) emits very little light (green signal) and its length fluctuates (purple line). Once the protein refolds (*) it emits more light and its length becomes shorter and consistent (dark gray line). † is the point where the force and state conformation are correlated(B) Cycled transition from dark (unfolded) to bright (folded). The purple circles represent the average contour length of each time. (Image adapted from Ganim’s and Rief’s paper).

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.

Knotty DNA

Original paper: Direct observation of DNA knots using a solid-state nanopore


Try taking out your earphones from your pocket and, in all probability, you’ll find knots and entanglements between the ends. As it turns out, this knotting effect is not limited to macroscopic objects, but occurs on the nanoscale as well. A DNA molecule that carries the genetic information of a living organism is actually a long string-like polymer, so you can imagine that it would also get tangled up just like the cords of your earphones. In fact, scientists know that DNA does form knots when it is in the nucleus of a cell, and these knots need to be removed by specialized bio-molecules, called enzymes, so that a cell can ‘read’ the genetic information encoded in the DNA. [1] In today’s paper, Calin Plesa and his colleagues at TU Delft are able to observe and measure these knots in DNA strands. In the process, they also observe interesting knotting behaviour which has not been observed before.

Knots on DNA

DNA translocation through a solid-state nanopore
Figure 1: This animation shows the DNA moving  through the nanopore. The associated dip in current is mapped onto the graph below. (Animation created by Calin Plesa, available under CC BY-SA license)

The researchers use a nanopore sensor to infer the structural properties of a DNA molecule. The sensor is made up of two reservoirs filled with electrolyte (a solution which separates into cations and anions, which can be used to conduct electricity, e.g. a salt solution), and they are separated by a membrane, or thin sheet, with a tiny hole in it. An electric field applied across the membrane generates an ionic current in the electrolyte and also pulls a negatively charged DNA strand through the tiny opening. The passage of a DNA strand through the nanopore causes a dip in the ionic current that is proportional to the volume of ions displaced—in other words, it’s proportional to the size of the molecule (a typical scenario is shown in Figure 1). Therefore, a knot in the DNA can generate a bigger drop in the current than an untangled strand. From this difference it is possible to infer the characteristics of the knot itself, since a bigger drop indicates a bigger knot.

The typical time for a DNA to pass through the pore is in the order of a few milliseconds, when the DNA is in a solution of potassium chloride (which is the typical salt solution used to carry out nanopore experiments). This makes it difficult technically, to see any features present on the DNA. Previous work has shown that it is possible to slow down the DNA passage by at least 10 times by using lithium chloride as their salt solution. [3] This increase in the translocation time (time it takes for the DNA to pass through the pore) is necessary to clearly see the additional dip in the current as the knot traverses the pore, as illustrated in Figure 2.

Translocation of a DNA molecule containing a trefoil knot through a solid-state nanopore
Figure 2: This animation shows a DNA with a knot moving through the pore. An additional dip in the current can be seen in the current trace as the knot (purple line) passes through the pore. (Animation created by Calin Plesa, available under CC BY-SA license)

The dip in the current signal caused by the knot passing through the pore can then be used to infer characteristics about the knot. In particular, it can be used to calculate the size of the knot, which has not been experimentally determined before. This has both physical and biological significance. Physically, it helps us understand the types of knots being formed on polymers as it can tell us whether the knot is loose or tightly formed. Biologically, it can help us understand how naturally occurring enzymes are able to disentangle knots in DNA strands, a function which is still poorly understood. The size of the knot is estimated by using

$latex d= v t$

where d is the length of the knot along the DNA strand, v is the average speed of the DNA translocation, and t is the time the knot takes to traverse the pore. Using this technique, the researchers estimate that the majority of the knots are less than 100 nm long. Previous research has shown that the DNA strand is rigid over lengths shorter than 50 nm, so considering this, the estimated knot size suggests that the knot is very tight. [2] However, this result needs further analysis, as the process of pulling the DNA through the nanopore might cause the knot to tighten, so this might not be the knot’s size in its natural state.

Slipping and sliding knots

When considering a linear (think: a thread with loose ends) DNA molecule, there is a possibility of the knot ‘slipping’ off the end of the strand before it gets pulled into the nanopore. For the knot to traverse the pore, it needs to be pulled fast enough to get squeezed to the size of the pore. If this process doesn’t happen fast enough the knot ‘halts’ at the pore entrance while the unknotted region translocates through. This allows the knot to disentangle, in case of a linear DNA molecule.

To determine if this slipping process occurs in knotted DNA strands, the researchers repeat their experiment using a circular (think: a thread joined end-to-end) DNA molecule. By using a closed loop they avoid possibility of the knot disentangling, but the knot can still slip towards the trailing end of the DNA during the translocation. The position of the knot is determined by the position of the dip in the current signal (purple line in Figure 2). They measure the probability of finding the knot at each position along the strand using two voltages, 100 mV and 200 mV. As shown in Figure 3, the knots show a preference for sliding toward the trailing end of the molecule at higher voltages, indicating that pulling too hard on the leading end of the DNA strand can indeed cause knots to slip along the strand instead of being pulled through the pore. The researchers also observe a 55% higher knotting occurrence in the circular molecules compared to linear ones. This suggests that knots may have slipped off the end of the linear molecules, thereby not detecting them at all.

Figure 4
Figure 3: The graph shows the probability of detecting the position of the knots along the length of DNA. At 200mV, the knots are observed to be at the trailing end of the DNA motion indicating the slipping phenomenon (adapted from Plesa et al.)

The researchers in this study have shown that naturally induced knots occur in DNA strands and they measured the sizes of those knots, which were previously unknown. This measurement showed that the knots detected are actually quite tight, which was not expected, although this result still needs to be investigated further. Additionally, these knots were seen to slide along the DNA molecules as they traversed the nanopore due to the strong pull at the end of the DNA strand. This was seen clearly by repeating the knotting experiments using circular DNA where there were no ends for the knots to slide off.

This new information about the structure of knots in DNA strands will help inform future studies of the complex topological structures formed in biomolecules such as DNA and proteins. It will also contribute to understanding the effects of topological features on the biological functions of these long, string-like biomolecules. In effect, it can help us explain the consequence of knotted DNA on the cell’s function as well as how the cell is equipped to handle these defects.

[1] http://www.tiem.utk.edu/~gross/bioed/webmodules/DNAknot.html

[2] Baumann, Christoph G., et al. “Ionic effects on the elasticity of single DNA molecules.” Proceedings of the National Academy of Sciences 94.12 (1997): 6185-6190.

[3] Kowalczyk, Stefan W., et al. “Slowing down DNA translocation through a nanopore in lithium chloride.” Nano letters 12.2 (2012): 1038-1044.