• Skip to main content
  • Skip to after header navigation
  • Skip to site footer
ERN: Emerging Researchers National Conference in STEM

ERN: Emerging Researchers National Conference in STEM

  • About
    • About AAAS
    • About the NSF
    • About the Conference
    • Partners/Supporters
    • Project Team
  • Conference
  • Abstracts
    • Undergraduate Abstract Locator
    • Graduate Abstract Locator
    • Abstract Submission Process
    • Presentation Schedules
    • Abstract Submission Guidelines
    • Presentation Guidelines
  • Travel Awards
  • Resources
    • Award Winners
    • Code of Conduct-AAAS Meetings
    • Code of Conduct-ERN Conference
    • Conference Agenda
    • Conference Materials
    • Conference Program Books
    • ERN Photo Galleries
    • Events | Opportunities
    • Exhibitor Info
    • HBCU-UP/CREST PI/PD Meeting
    • In the News
    • NSF Harassment Policy
    • Plenary Session Videos
    • Professional Development
    • Science Careers Handbook
    • Additional Resources
    • Archives
  • Engage
    • Webinars
    • ERN 10-Year Anniversary Videos
    • Plenary Session Videos
  • Contact Us
  • Login

Developing a peptide tagging strategy to improve tethering in optical tweezers

Undergraduate #43
Discipline: Biological Sciences
Subcategory: Biochemistry (not Cell and Molecular Biology and Genetics)
Session: 3
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, kaiser@jhu.edu

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.

Sidebar

Abstract Locators

  • Undergraduate Abstract Locator
  • Graduate Abstract Locator

This material is based upon work supported by the National Science Foundation (NSF) under Grant No. DUE-1930047. Any opinions, findings, interpretations, conclusions or recommendations expressed in this material are those of its authors and do not represent the views of the AAAS Board of Directors, the Council of AAAS, AAAS’ membership or the National Science Foundation.

AAAS

1200 New York Ave, NW
Washington,DC 20005
202-326-6400
Contact Us
About Us

  • LinkedIn
  • Facebook
  • Instagram
  • Twitter
  • YouTube

The World’s Largest General Scientific Society

Useful Links

  • Membership
  • Careers at AAAS
  • Privacy Policy
  • Terms of Use

Focus Areas

  • Science Education
  • Science Diplomacy
  • Public Engagement
  • Careers in STEM

Focus Areas

  • Shaping Science Policy
  • Advocacy for Evidence
  • R&D Budget Analysis
  • Human Rights, Ethics & Law

© 2023 American Association for the Advancement of Science