I am interested in elucidating the structural mechanism of the bacterial proteases FtsH and ClpXP upon substrate degradation. Based on the crystallographic structures of these proteins, the proteases labeled at specific positions with acceptor and donor fluorophores trigger a Foerster Resonance Energy Transfer upon conformational change. Using TIRF microscopy, we observe these events at a single-molecular level (sm-FRET). With this method, we are studying the dynamics of the proteases upon substrate degradation, and the dynamics dependence in the folding state of the substrates. Coupled to a magnetic tweezer, the sm-FRET setup will further give us force informations underlying these degradation processes.
I am working on the integron, a bacterial genetic platform predominantly involved in the spread of bacterial antibiotic multiresistance. It assembles resistance genes using a unique mechanism of recombination of a single strand of DNA, that forms a hairpin and thus resembles a canonical recombination site. Using two optical tweezers to trap a single molecule of single stranded DNA at both ends and probing its mechanical properties, I am structurally characterizing the integron recombination sites. I combine this approach with single molecule fluorescence to elucidate interaction with proteins like the recombinase and single strand binding proteins.
DNA replication initiation is mediated by initiator proteins at the origin of replication. We want to find out the melting mechanism of the origin of replication by the DnaA and G38P initiators in the bacteria Aquifex aeolicus and the phage SPP1, respectively. For this attend, we use the smFRET technique to observe the dynamic process of these DNA binding events at the molecular level.
I am working on a setup aiming to combine the external mechanical stimuli of magnetic tweezers with the resolution of small distances from single-molecule FRET measurements. The combined information from fluorescence and controlled mechanical interaction of biomolecules can then be used to answer questions of DNA repair and the relation of forces and action of protein biomachines.
I am working on so called “Zero-Mode Waveguides” – a unique approach in Single-molecule detection. It is basically holes in a metal film on a glass surface. These holes are illuminated from the bottom with a common fluorescence microscope. Single molecules tagged with fluorophores will be anchored to the bottom of the holes and their fluorescence can be observed. Due to the small radius of the holes (50-150nm) the light can’t propagate and decays exponentially. This way a very small excitation volume is created – around 3 orders of magnitude smaller than in a common TIRF setup. This allows us to increase the concentration of fluorophores to a biological relevant level for example. Zero-Mode Waveguides also have the advantage of parallel detection of hundreds of molecules and good confinement. My task in particular is to design these waveguides, manufacture them and use them for biological problems.
My project focuses on building up a setup for 3D manipulations of hydrogels with laser. This hydrogels are a great biomaterial for 3D culturing of cells in user-defined environment on the nano or micro scale. However, in order to study cell differentiation or cell response to chemical and mechanical stimuli it would be desirable to directly change those properties in the local environment of individual cells. To increase the penetration depth and resolution, lower the absorption and avoid damages to living cells the gel manipulation asks for a 2-photon excitation.
To be able to observe movements and conformational changes of proteins with high time resolution, to generate in vivo lifetime images for cell experiments and to analyze the behavior of fluorescent dyes under different conditions, we are going to build a customized scanning confocal microscope. The picosecond pulsed lasers in this setup allows us to correlate raw FRET intensities with modifications in the fluorescence lifetime, giving us access to a greater information space.
Responsibility: Andreas Hartmann
Many chemo-mechanical processes in cells involve either conformational changes in enzymes, e.g. molecular motors, or result in conformational changes of the substrate, e.g. double-strand opening through helicases. Förster resonance energy transfer (FRET) is very sensitive for length changes on the nanometer scale and thus matches nicely the size of most biomachines. Single-molecule FRET (smFRET) allows the observation of individual biomachines involved in DNA repair to prevent cancer, DNA replication or protein degradation to understand crucial steps in cell duplication. With our smFRET setup we can observe on average hundreds of individual molecules at the same time during work yielding insights on how these biomachines work or not.
Responsibility: Michael Schlierf
Magnetic tweezers mechanically manipulate single molecules, preferably biomolecules. By chemically attaching magnetic particles to molecules and fixating the molecules on sample surfaces we can use magnets to pull and twist individual molecules. The forces involved are all in the picoNewton range which biomolecules exert in many biological processes, such as unwinding DNA or unfolding proteins. The magnetic beads simultaneously act as probes to localize the molecules and measure their extension or response to the external forces from the magnets. By additionally attaching fluorescent probes for single-molecule FRET experiments to the molecules we can also track distances in the reference frame of the molecule on length scales below 10 nanometers. This combination of techniques can therefore characterize many parameters of a given single-molecule experiment simultaneously.
Responsibility: Marko Swoboda
by MS, Hongbin Li and Julio Fernandez
Single-molecule force spectroscopy has become a prominent technique in studying protein folding. During the last decade AFM-based single-molecule force spectroscopy mainly focused on the mechanical unfolding energy landscape. One common model for the analysis of single-molecule force spectroscopy experiments is the so-called Bell model which describes the mechanical unfolding rate exponentially dependent und the unfolding force. In the collaboration with Hongbin Li and Julio M. Fernandez @ Columbia, for the first time we were able to show that within the experimental errors the unfolding kinetics of single ubiquitin domains can be well described by the Bell model.
by MS and Matthias Rief
Temperature variation in single-molecule force spectroscopy experiments should per se vary the mechanical unfolding behaviour only little. However, a study of the mechanical unfolding behaviour of the filamin crosslinker ddFLN4 showed that the underlying energy landscape of this protein is strongly temperature dependent. At low temperatures (5°C) the energy landscape showed a narrow potential while the width of this potential increased by nearly a factor of 3 at 37°C. The molecular spring constant changed therefore by a factor of 7 indicating a prominent softening of ddFLN4. Such a significant change in the underlying mechanical potential may indicate a change in the critical stabilizing interactions.
by MS and Matthias Rief
Limited resolution and ill-defined loading rates have precluded a detailed study of the mechanical unfolding barrier in the past. Although the commonly used Bell model reproduces average unfolding forces, the actual shape change of the unfolding force distributions by varying the pulling velocity could not be reproduced satisfactorily. We studied in detail the dependence of the unfolding force distributions of the filamin domain ddFLN4 under various loading rates. A detailed analysis with a Kramers diffusion approach leads to a funnel shaped energy landscape of ddFLN4, while the commonly used Bell model yields a misleading view of the unfolding energy landscape.
by MS, Felix Berkemeier and Matthias Rief
We were interested in studying protein folding with AFM-based single-molecule force spectroscopy experiments. Refolding forces are expected to be in the very low force regime between 0 and 20 pN. In order to study single folding events under external force, a technique called lock-in force spectroscopy was developed. With an increased resolution by one to two orders of magnitude, we were able to observe direct protein folding events under force. A detailed analysis of the protein folding forces needed a new model to describe folding kinetics under external force. With the help of this model we were able to extract information about the structure of the unfolded polypeptide chain prior to folding.
by MS and Matthias Rief
The anisotropy of the folding-energy landscape of proteins under force can be tested with cysteine engineering. The shorter the actively contracting polypeptide (see scheme, from blue to green), the higher the force at which the protein folds. The anisotropy of the folding mechanics can be described surprisingly simply with the help of a minimal model, mainly considering the entropic elasticity of the polypeptide.