Finding order in chaos: scientists determine the structure of glass-shaping protein in sponges
B CUBE hosts the 4th Scientific Advisory Board meeting
Schlierf Group - Technology Development
FRET and farFRET
Single-molecule Förster resonance energy transfer (smFRET) has become a powerful nanoscopic tool in studies of biomolecular structures and nanoscale objects. smFRET allows access to structural as well as kinetic data of conformational changes of biomolecules. High-resolution measurements require stable and long-living organic fluorophores. We develop methodologies to enhance photo stability of organic fluorophores. Cconventional smFRET measurements are generally blind to distances above 10 nm thus impeding the study of long-distance phenomena. Here, we develop farFRET, a technique that extends the range in smFRET measurements beyond the 10 nm line by enhanced energy transfer using multiple acceptors.
Team members on the project: Georg, Andreas, Marko, Hsin-Mei
- Swoboda, ..., Plumere, Schlierf (2012) Enzymatic Oxygen Scavenging for Photostability without pH Drop in Single-Molecule Experiments, ACS Nano
- Krainer, Hartmann, Schlierf (2015) farFRET: Extending the Range in Single-Molecule FRET Experiments beyond 10 nm, Nano Letters
Typically single-molecule techniques are applied in isolation, that is, either force or fluorescence. However, in these kinds of experiments the accessible information space is often limited to a simplifying one-dimensional coordinate. Thus, the content available from single-molecule measurements can be significantly enhanced by a combination of orthogonal methods. We develop hybrid single-molecule techniques combining magnetic tweezers and Förster resonance energy transfer (FRET) measurements. Through applying external forces to a paramagnetic sphere, we can induce conformational changes in DNA nanostructures, which can further be processed by enzymes. A synchronized orthogonal readout in different observation channels (force and fluorescence) will facilitate deciphering the complex mechanisms of biomolecular machines.
Team members on the project: Marko (in collaboration with Seidel group)
- Swoboda, Grieb, Hahn, Schlierf (2014) Measuring Two at the Same Time: Combining Magnetic Tweezers with Single-Molecule FRET, EXS
- Kemmrich, Swoboda, Kauert, Grieb, Hahn, Schwarz, Seidel, Schlierf (2015) Simultaneous Single-Molecule Force and Fluorescence Sampling of DNA Nanostructure Conformations Using Magnetic Tweezers, Nano Letters
Hydrogels are a great biomaterials for 3D culturing of cells in user-defined environment. 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. Here, we develop methods for 2D and 3D growth of hydrogels using two-photon polymerisation. Two-photon excitation of the photo-crosslinks allow a precise control of structural resolution - also in 3D - and avoid cell damage due to low absorption.
Team members on the project: Christiane (in collaboration with Mikhail Tsurkan (Werner group, IPF))
Patent: DE 10 2014 104 735.4
TIR smFRET Setup
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, DNA replication or protein degradation. With our TIR smFRET setup we can observe on average hundreds of individual molecules at the same time during work yielding insights on how these molecules change conformations on sub-second to second time scale.
TCSPC Setup for smFRET (Time-Correlated Single Photon Counting)
Faster conformational changes on the sub-millisecond to millisecond time scale can be monitored with photodiode based confocal single-molecule FRET microscopes. Picosecond pulsed lasers further allow pulsed interleaved excitation or alternating laser excitation to sort singly-labeled molecules from double-labeled molecules and give access to fluorescence lifetime space. in this setup we are further able to measure fluorescence correlation (FCS) and fluorescence anisotropy.
Magnetic tweezers mechanically manipulate single molecules, preferably biomolecules. By chemically attaching magnetic particles to molecules and fixating the molecules on sample surfaces we use magnets to pull and twist individual molecules. The forces involved are all in the sub-picoNewton to picoNewton range which biomolecules experience in many biological processes. 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 a few nanometers and in orthogonal direction.
Manipulation of single molecules with sub-pN and nanometer resolution is possible with our optical tweezers instrument. We use two focussed laser beams to trap microspheres and move them relative to each other. Once a single protein or DNA molecule is tethered between these spheres we can exert small forces of a few pN and force the molecule to unfold. Equilibrium measurements of tens to hundreds of seconds allow to monitor conformational kinetics and distances with high precision to reconstruct underlying energy landscapes.
Typical fluorescence microscopes can reach a resolution of approximately 200 nm, and consequently two proteins located within 200 nm distance appear at the same position. For the co-localization of two proteins, various other fluorescence-based techniques have been developed, including Förster resonance energy transfer (FRET) or split-GFP. These techniques yield information about the relative distance between two proteins up to 10 nm distance, but lack information beyond 10 nm to 200 nm. STORM and PALM allow single-molecule localisation with down to 20nm resolution. Here, we use a commercial Nikon N-STORM microscope for STORM and PALM experiments.