Mechanical Stretching of Proteins Using Novel Hydrodynamic Single-Molecule Force Spectroscopy System
Ek, Frans (2023)
Ek, Frans
2023
Bioteknologian ja biolääketieteen tekniikan maisteriohjelma - Master's Programme in Biotechnology and Biomedical Engineering
Lääketieteen ja terveysteknologian tiedekunta - Faculty of Medicine and Health Technology
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Hyväksymispäivämäärä
2023-05-17
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tuni-202304244199
https://urn.fi/URN:NBN:fi:tuni-202304244199
Tiivistelmä
Mechanical signals, like biochemical signals, have been shown to guide key cellular processes. The role of mechanobiology has been demonstrated for example in stem cell differentiation, development, physiological processes like bone formation, vascularization, and in diseases like cancer, muscular dystrophies, and cardiovascular diseases. Advances in mechanobiology are still quite recent, and there is a great need for more research in the field.
Mechanical signals act on tissue, cellular and molecular level. On molecular level, mechanobiological processes are mostly associated to the mechanical properties of proteins. Usually, when subjected to mechanical forces, proteins undergo conformational changes that might unveil cryptic binding sites for other proteins, or then in some other way change the protein’s function or relay the force forward through the structure.
To study mechanosensitive proteins, three conventional methods are usually used to perform single-molecule force spectroscopy: atomic force microscopy, magnetic tweezers, and optical tweezers. Since they have their limitations, new methods need to be invented to enhance single-molecule force spectroscopy.
In this thesis, a new approach to protein force spectroscopy is evaluated: hydrodynamic single-molecule force spectroscopy (HDSMFS) system. The system is based on a microfluidic channel, to which the target proteins can be C-terminally conjugated via covalent SpyTag/SpyCatcher bond. The target proteins are conjugated N-terminally to fluorescent microbeads which can be tracked via TIRF microscopy, and the flow rates, which subject the beads and therefore the fragments to shear forces from the buffer flow, can be very accurately controlled via syringe pumps attached to the microfluidic setup. To test the SpyTag/SpyCatcher conjugation technique, 7 protein fragments were selected to be expressed, purified, characterized and reactivity tested, and then ran through the measurements in the system. The goal was also to familiarize with the system setup, present in Freie Universität Berlin, Bionanointerfaces group lab, and to learn how the system is set up and how the measurements are conducted, to bring the setup one day also to our faculty.
For the tests, human talin-1 R1-R3, R1-R3 IVVI mutant, R7-R8 and R9-R10 fragments, as well as human dystrophin SR6-SR10 and SR18-SR24 fragments were selected. In addition, there is a control fragment, which is insensitive to forces of the studied force range. Fragments were chosen based on their existing previous characterization, and because of their importance in key mechanobiological processes like focal adhesion mechanosensing and muscular function.
Protein expression, purification, characterization, and reactivity tests were all successful, and the fragments were confirmed to be suitable for the HDSMFS system. In addition, the ability to control the SpyCatcher surface density was tested, which also proved successful. As for the system itself, the process of setting up the system and conducting measurements with it were learned in good detail, and some preliminary results were obtained for 3 of the fragments. A small qualitative analysis was done, which revealed already that we can see specific and non-specific interactions tracked via the beads, as expected. However, data analysis based on cluster algorithm developed in FU Berlin was out of scope for the thesis and will be conducted outside it.
Overall, the primary goals of the thesis were achieved, and the HDSMFS system was proven to be a great tool for future studies of these adhesion proteins.
Mechanical signals act on tissue, cellular and molecular level. On molecular level, mechanobiological processes are mostly associated to the mechanical properties of proteins. Usually, when subjected to mechanical forces, proteins undergo conformational changes that might unveil cryptic binding sites for other proteins, or then in some other way change the protein’s function or relay the force forward through the structure.
To study mechanosensitive proteins, three conventional methods are usually used to perform single-molecule force spectroscopy: atomic force microscopy, magnetic tweezers, and optical tweezers. Since they have their limitations, new methods need to be invented to enhance single-molecule force spectroscopy.
In this thesis, a new approach to protein force spectroscopy is evaluated: hydrodynamic single-molecule force spectroscopy (HDSMFS) system. The system is based on a microfluidic channel, to which the target proteins can be C-terminally conjugated via covalent SpyTag/SpyCatcher bond. The target proteins are conjugated N-terminally to fluorescent microbeads which can be tracked via TIRF microscopy, and the flow rates, which subject the beads and therefore the fragments to shear forces from the buffer flow, can be very accurately controlled via syringe pumps attached to the microfluidic setup. To test the SpyTag/SpyCatcher conjugation technique, 7 protein fragments were selected to be expressed, purified, characterized and reactivity tested, and then ran through the measurements in the system. The goal was also to familiarize with the system setup, present in Freie Universität Berlin, Bionanointerfaces group lab, and to learn how the system is set up and how the measurements are conducted, to bring the setup one day also to our faculty.
For the tests, human talin-1 R1-R3, R1-R3 IVVI mutant, R7-R8 and R9-R10 fragments, as well as human dystrophin SR6-SR10 and SR18-SR24 fragments were selected. In addition, there is a control fragment, which is insensitive to forces of the studied force range. Fragments were chosen based on their existing previous characterization, and because of their importance in key mechanobiological processes like focal adhesion mechanosensing and muscular function.
Protein expression, purification, characterization, and reactivity tests were all successful, and the fragments were confirmed to be suitable for the HDSMFS system. In addition, the ability to control the SpyCatcher surface density was tested, which also proved successful. As for the system itself, the process of setting up the system and conducting measurements with it were learned in good detail, and some preliminary results were obtained for 3 of the fragments. A small qualitative analysis was done, which revealed already that we can see specific and non-specific interactions tracked via the beads, as expected. However, data analysis based on cluster algorithm developed in FU Berlin was out of scope for the thesis and will be conducted outside it.
Overall, the primary goals of the thesis were achieved, and the HDSMFS system was proven to be a great tool for future studies of these adhesion proteins.