Arranging Small Molecules with Subnanometer Precision on DNA Origami Substrates for the Single‐Molecule Investigation of Protein–Ligand Interactions
Abstract
DNA origami nanostructures are versatile substrates for the single‐molecule investigation of biomolecular interactions as they enable the display of molecular species in complex arrangements. Herein, the fundamental limitations of this approach are explored by displaying pairs of small‐molecule ligands of the protein trypsin on DNA origami substrates and adjusting their ligand–ligand spacing with subnanometer precision. Bidentate binding of trypsin to the ligand pairs is investigated by atomic force microscopy (AFM), microscale thermophoresis (MST), and molecular dynamics simulations. Bidentate trypsin binding is strongly affected by the distance of the ligand pairs and the accessibility of the protein's binding pockets. MST cannot resolve the differences in bidentate trypsin binding because of the nonspecific binding of trypsin to the DNA origami substrates, rendering the AFM‐based single‐molecule detection of binding events superior to ensemble measurements. Finally, even monodentate binding to a single ligand may be affected by subnanometer variations in its position, highlighting the importance of local microenvironments that vary even over molecular distances. While this single‐molecule approach can provide viable information on the effects of ligand arrangements on bidentate protein binding, in‐depth investigations into the nature of local microenvironments will be required to exploit its full potential.
Document Details
- Document Type
- Pub Defense Publication
- Publication Date
- Sep 09, 2020
- Source ID
- 10.1002/sstr.202000038
Entities
People
- Adrian Keller
- Antonio Suma
- Charlotte Kielar
- Guido Grundmeier
- Jingyuan Huang
- Meiying Cui
- Vincenzo Carnevale
- Yixin Zhang
Organizations
- German Research Foundation
- National Science Foundation
- Technische Universität Dresden
- Temple University
- United States Army Research Laboratory
- University of Paderborn