Protein Folding

One of the most fundamental and challenging problems in molecular biophysics is understanding how proteins fold into their complex three-dimensional structures enabling them to act as channels, enzymes or even molecular motors.

The unique three-dimensional structure of a protein is determined by its amino acid sequence. It is stabilized by non-covalent interactions: hydrogen bonds, electrostatic interactions and hydrophobic interactions, which together weigh up against the loss of entropy that is caused by folding of the polypeptide chain.

It is clear that proteins fold via certain pathways, because it would take a polypeptide chain a time longer than the age of the universe to randomly sample all possible conformations to find its native structure. The folding process of proteins is generally described as diffusion in a high dimensional energy-landscape (Figure 1), in which the polypeptide chain searches for conformations with a lower free energy until the most stable native state has been reached.

Figure 1. Schematics of a folding landscape for a protein. Red trace: hypothetical unfolding pathway. Black trace: hypothetical mechanical unfolding pathway.

 

Why single-molecule force spectroscopy?

To study protein folding and unfolding, single-molecule force spectroscopy is a very suitable technique. It enables monitoring the folding pathway of a single protein molecule, which is not possible with other techniques such as NMR spectroscopy, because this only allows detection of the average state of a population of molecules. During single-molecule force spectroscopy unfolding experiments a known force in the piconewton-range is applied to a protein between two arbitrary attachment points (Figure 2). Different pulling directions usually lead to different unfolding pathways, thus allowing a broader study of the energy-landscape of the protein (M. Bertz, et al., Angewandte Chemie 2008).

Figure 2. During a typical single–molecule force spectroscopy experiment a protein is stretched between a surface and the tip of a cantilever. To provide a quality control the protein of interest is inserted into a series of well characterized proteins. One end of the protein is chemically attached to a functionalized surface via a His-tag while the other end is picked up randomly with the cantilever tip. When the surface is retracted from the tip, the protein is stretched. The acting force can be calculated from the measured cantilever bending.

 

How do knotted proteins fold?

Recently, proteins with a knotted structure have been identified.  It is difficult to reconcile the existence of these knotted folds with energy funnel models for protein folding because the polypeptide chain has to be threaded into forming the knot somehow; simply moving towards conformations with lower free energy is not sufficient. Thus, the discovery of this type of fold challenges the current paradigms in the field of protein folding. Using single-molecule force spectroscopy, we have tightened a protein knot (Bornschlögl et al., Biophysical Journal 2009), shedding light on the mechanical stability and the size of the knot. Current research aims at unraveling the folding pathways of these intriguing protein structures.