Discipline: Biological Sciences
Subcategory: Biochemistry (not Cell and Molecular Biology and Genetics)
Room: Exhibit Hall
Paola M. Miranda-Castrodad - University of Puerto Rico at Cayey
Co-Author(s): Hannah Haller-Hidalgo, Johns Hopkins University, Baltimore, MD; Christian Kaiser, Johns Hopkins University, Baltimore, MD.
Optical tweezers (OT) is a powerful technique that is used to measure sub-nanometer changes associated with protein folding and unfolding. Applying force to a protein acts like a denaturant that is more directed and better controlled than typical methods, which enables reversible single-molecule measurements. Manipulation with OT requires tethering the protein of interest by its ends to molecular handles for force application. Chemical coupling has been a prevalent tethering method, but it can cause protein modification that can affect measurements. A better alternative is peptide tags that spontaneously form covalent bonds to attach the protein of interest to molecular handles. We aim to develop a method to tether proteins efficiently using orthogonal tags. The Kaiser lab has previously demonstrated that the SpyTag/SpyCatcher (Sp/SpC) system is effective for forming tethers in OT experiments by attaching the protein being studied to DNA handles. Since the SnoopTag/SnoopCatcher (Sn/SnC) system is orthogonal to Sp/SpC, we hypothesize that combining both these systems will facilitate sample preparation for OT experiments. To evaluate our hypothesis, we are developing a modular tethering strategy that involves preparing SnC DNA handles and performing OT experiments using an N-terminally Sn-tagged and C-terminally Sp-tagged protein. Preliminarily, experiments indicate that purified SnC robustly reacts with a Sn-tagged model protein to form a covalent bond. The Sn/SnC system thus seems like a useful addition to the OT sample preparation toolkit. We expect the approach to be highly efficient, making it particularly attractive for multimeric complexes and proteins that cannot be obtained in biochemical quantities.
Funder Acknowledgement(s): NSF REU Award #2150379
Faculty Advisor: Christian Kaiser, firstname.lastname@example.org
Role: I conducted most this research under the guidance of my mentor. When I started in the lab, I was introduced to my project and began working immediately on a bacterial transformation to express and purify the SnoopCatcher (SnC) protein. I then designed and performed titration reactions to determine the concentration of SnC before proceeding with the preparation of DNA handles. On the side, I performed cloning to create plasmids that were used for preparing in vitro translated proteins. Lastly, I was able to carry out multiple optical tweezers experiments to test our proposed tethering system.