16-22 October 2021

Iwona Lewandowska

Home Institution
University of Warsaw, Warsaw, Poland

Host Contact
Dr. Katarzyna Tych

Host Institution
University of Groeningen, Groeningen, Netherlands

Aim of the mission
The goal of STSM is to determine the topology of potentially double-knotted proteins and to establish new collaboration and development as a scientist. The main goal of STSM in Groningen is to learn from Dr Tych how to derive mechanical, structural and topological properties using an optical tweezer. During the trip, I was going to learn how to conduct experiments with the use of the C-trap to bind and stretch proteins, as well as gain practical knowledge about the topology of knotted proteins and about ways of manipulating them. During the time I planned to spend in Groningen, I wanted to try to define the topology of the SPOUT superfamily proteins (including double-knotted proteins) and confirm the existence of a knot and a double knot in these proteins. Another goal was to collect data that would enable a better understanding of knotty proteins and their topological properties. The data I have collected will be used in the future to develop computational methods that will help predict the folding process of knotted proteins from a molecular point of view.

Summary of the Results
Before arriving in Groningen and actually starting the STSM, some things had to be prepared earlier. These preparation included: preparation of proteins containing mutated cysteines for optical tweezers experiments (TrmD, Tm1570 and fusion of TrmD-Tm1570 (potentially double knotted protein), examination of proteins activity and getting acquainted with the theoretical foundations of protein dynamics at a single molecule level. During the STSM, first I got acquainted with the information on how to use Lumicks equipment, a device for taking the single molecule experiments by C-trap and optical tweezers. Then, I took part in a training of the device usage, so that I could use it independently and perform proper experiments. The experiments which I carried out during the STSM were composed of several steps, which I will briefly describe.
Step 1 protein labeling with functionalized DNA handles In Step 1 the protein labeling procedure uses maleimide-cysteine chemistry to attach two fragments of DNA handles (modified with 5 -digoxgenin and 5 -biotin) and forming a protein-handle chimera. First, malemide-modified DNA oligos are attached to two cysteins of the protein, then coupled to two oligos (protein-oligo) and purified from the excess of unreacted DNA oligos (by affinity chromatography). At this stage it is important that the proteins have a HisTag. In the final step, the oligos hybrizide to the complementary overhand on the DNA handles (DNA has a known length).
Step 2 protein tethering to beads for optical tweezers experiments (in C-trap) This step involves attaching the protein to beads coated streptavidin and anti-digoxigenin and tethering in C-trap. First, we connect DNA protein-handles with properly prepared anti-digoxigenin coated beads and load into channel 1 of C-trap. Into the channel 2 we load the buffer supplemented with the oxygen scavenger system. To channel 3 we load a streptavidin-coated beads. The most important thing in this step is to attach protein DNA-handles between antidigoxenin-coated bead and a streptavidin-coated bead. This is done by appropriately positioning and calibrating the position of the balls in the traps.
Step 3 Protein stretching In this step, the proteins are stretched at a constant velocity or with a constant force in order to study their topology. The plots obtained from the experiments showed for example how the stretching distance depends on the measured forces. It is sometimes called a mechanical fingerprint of the protein. Based on the analysis of the obtained graphs and models of the structure,
it is predicted which elements of the second-row structure have stretched. During the STSM I prepared DNA handles for each protein, buffers for measurements and I did some of stretching experiments and obtained trajectories for proteins from the SPOUT family, which will be analyzed after returning.

The main result of STSM was to learn how to use C-trap and optical tweezers to carry out single molecule experiments and expand knowledge about the topology of knotted proteins. Additionally,
a collaboration which was established with dr Tych will allow further research on the topology of proteins from the SPOUT family and the double knotted proteins. During the experiment, it was also possible to conduct many other experiments on proteins in parallel and obtain a large number of trajectories, which now will be analyzed and will allow to confirm the presence of knots in proteins. I will be able to share collected data with co-workers from prof Sulkowska lab that are involved in development of computational models. They will be used for improvement models to analyze the energy landscape of protein with non-trivial topology by using numerical methods to prediction the effect of pulling experiments.