Design und mechanische Einzelmolekül-Charakterisierung funktionaler supramolekularer Proteinstrukturen
Zusammenfassung der Projektergebnisse
Proteins are highly sophisticated nano-machines that can act as enzymes, channels, molecular motors as well as structural scaffolds. The function of proteins is intimately linked to their threedimensional structure and conformational flexibility. In this project we combined protein engineering approaches with single molecule force spectroscopy to measure force-induced conformational changes directly within single proteins. To this end, we developed strategies to contact individual enzymes at almost arbitrary sites using atomic force microscopy. Three different protein systems were studied: - Calmodulin is the primary calcium sensing protein in our body. Calcium binding induces conformational changes so that calmodulin is able to bind to more than 300 known target proteins. Using single molecule force spectroscopy, we could show that the folding of calmodulin domains is strongly calcium dependent. Moreover, we could directly show the equilibrium binding and unbinding of target peptides to and from calmodulin. By tethering the target peptide to the terminus of calmodulin, we could identify differences in the binding modes among different target peptides. While skMLCK binds strongly and cooperatively to both calmodulin domains simultaneously, CamKK can also bind strongly to a single calmodulin domain. These results are important for understanding the selectivity of calmodulin amongst its various binding partners. - Maltose binding protein (MBP) undergoes a large conformational change upon binding of maltose. We used single molecule force spectroscopy to study the dependence of the conformational stability on maltose binding. By designing and investigating various pulling directions, we found that the conformational stability is only increased when the vector of force application runs directly through the ligand binding site. In contrast, MBP unfolding forces along an N-C terminal pulling direction did not depend on the presence of ligand. Using mutagenesis experiments, we also demonstrated that the mechanical stabilization effect is due to only a few key interactions of the protein with its ligand. Moreover, we could show that MBP is assembled from 4 stable building blocks that can fold independently. - In a final series of experiments we investigated the mechanical stability of green fluorescent protein (GFP). We designed various mutants allowing to pull GFP along almost arbitrary directions. In combination with MD simulations performed by Dave Thirumalai (University of Maryland) we could show that GFP exhibits a bifurcation in its unfolding pathway where the molecule either unravels from its N-terminus or from the C-terminus. Moreover, we developed a semianalytical model based on elastic networks that allowed to understand the mechanical anisotropy of GFP. Those results indicate that mechanical protein stability is rather dominated by the topology of the fold than by sequence details.
Projektbezogene Publikationen (Auswahl)
- Revealing the bifurcation in the unfolding pathways of GFP by using single-molecule experiments and simulations. 2007, PNAS, 51, 20268-73
M. Mickler, R. I. Dima, H. Dietz, C. Hyeon, D. Thirumalai, M. Rief
- Elastic bond network model for protein unfolding mechanics. 2008, Phys Rev Lett, 9, 098101
H. Dietz, M. Rief
- Ligand binding mechanics of maltose binding protein. 2009, J Mol Biol, 5, 1097-105
M. Bertz, M. Rief
- Ligand-dependent equilibrium fluctuations of single calmodulin molecules. 2009, Science, 5914, 633-7
J. P. Junker, F. Ziegler, M. Rief
- Single-molecule force spectroscopy distinguishes target binding modes of calmodulin. 2009, PNAS, 34, 14361-6
J. P. Junker, M. Rief
- The titin-telethonin complex is a directed, superstable molecular bond in the muscle Z-disk. 2009, PNAS, 32, 13307-133310
M. Bertz, M. Wilmanns, M. Rief
