Single-molecule force spectroscopy of biological complexes

Biomolecular interactions form the basis of all living organisms and their detailed investigation on the molecular level is crucial to the understanding of complex biological systems. With the advent of single-molecule (SM) techniques in response to the growing interest in the molecular nature of interactions, a whole new layer of knowledge has emerged. Hence, bulk-derived characteristics of molecular complexes need to be complemented by more detailed information addressing individual molecules and not only their statistical representatives. This way also interactions that are transient, weak or less abundant in the population are considered. This work focuses on the single-molecule elucidation of different types of biomolecular interactions - from protein-ligand to protein-DNA, to protein-protein ones. The focus of the presented research is on force measurements, considered both in an absolute manner as well as in comparison to other reference interactions. Comparative analysis is more informative in many cases in which a ranking of interactions against each other is of interest rather than their absolute strengths. Several SM techniques are explored and their complementarity in targeting specific aspects of single-molecule accessibility is discussed. I present a way to eliminate multiple events’ bias in AFM measurements of biotinstreptavidin bond rupture. Despite numerous studies, available data regarding the binding force of the complex are not fully consistent and contain a lot of open questions. Here, the introduced DNA tether provided an intrinsic fingerprint, thus ensuring SM-accessibility. The same assay allowed to address the MeCP2-DNA binding, thought to lead to DNA cross-linking and looping. We observed DNA clustering upon addition of the protein and turned to magnetic tweezers to further analyse the mechanism of MeCP2 action. This instance demonstrates the challenges in proper experimental design in both techniques when it is desired to achieve a truly SM resolution not only in sensing but also in the behavior of the investigated system. Finally, I characterized the GFP-Nanobody binding as an exemplary protein-antibody interaction. The energy landscape of the complex was explored by the AFM. Interestingly, the force measurements revealed several regimes related to various pulling geometries, as well as force dependence on the type of GFP despite identical epitopes. Then, multiplexed single-molecule measurements by means of Molecular Force Assay demonstrated the usefulness of this pair as a reference in comparative studies. In this thesis I show that even with dedicated techniques achieving SM resolution may not be a straightforward task. Studying molecular systems often requires a very individualized approach so that native-like conditions can be mimicked while the focus is strictly confined to one molecule only. To sum up, we have designed assays to analyze biomolecular interactions on the SM level and demonstrate how to ensure SM resolution by making use of intrinsic features of biomolecules. The presented work contributes to the expansion of the existing SM techniques in the field of protein research and provides binding force data for the GFP-Nanobody complex - a promising molecular reference.