Optical Methods for Investigating Proteins

While mechanical methods offer unique information about the functioning of proteins under stress, biological processes in vitro and in living cells can be investigated with unprecedented spatial and temporal resolution using optical microscopy. Ultra-sensitive fluorescence spectroscopy can be used for single molecule microscopy with all of the advantages of single molecule measurements discussed above and is exceptional for dynamic live-cell imaging. The optical microscopy groups provide expertise in a large range of ultra-sensitive fluorescence techniques including: wide-field real-time live-cell imaging, dual-color two- and three-dimensional particle tracking, confocal three-color microscopy, Optical Structured Illumination Sectioning, Total-Internal-Reflection Fluorescence Microscopy, Fluorescence Correlation and Cross Correlation Spectroscopy, Pulsed Interleaved Excitation (PIE), Polarization dependent Orientational Analysis, Fluorescence Recovery after Photobleaching (FRAP), sub-Abbé microscopy, and ultrafast IR spectroscopy. The methods focus on ultra-high sensitivity, ultra-high spatial resolution, and ultra-high temporal resolution. These groups will continue to develop ultra-sensitive fluorescence and ultrafast IR techniques, adapt them for use in in vitro and live-cell experiments and, in collaboration with various partners of the cluster, apply the optimal methods to the most challenging questions in protein interactions.

 

Ultra-sensitivity Microscopy, in vitro: Single Molecule Protein Folding Studies

Investigation of the folding process (areas B and F) requires a method that is sensitive to distance changes on the nanometer scale, a range which is ideal for fluorescence resonance energy transfer (FRET), and can be performed on single molecules. The laboratory of Prof. Dr. Christoph Bräuchle has recently developed a new method (using PIE) to increase the sensitivity and the range of FRET distances that can be measured and have expanded their capabilities to three-color FRET, which is capable of determining three distances simultaneously.

The Bräuchle group has collaborations with the groups of Hartl (MPI) and Buchner (TUM) to investigate the function of chaperons in protein folding. Experiments using single pair FRET to investigate the folding pathway of the Maltose Binding Protein (MBP) in the presence and absence of the chaperon protein GroEL have revealed a new conformation of unfolding MBP bound to GroEL. This novel result suggests the chaperon actively opens other folding pathways and is a first step in unraveling the function of chaperon proteins.

For studying the kinetics of protein folding, their expertise in particle tracking and single-molecule FRET will be combined to track individual proteins as they are folding. A microfluidic chamber induces folding by mixing buffers on the microsecond timescale and the kinetics of folding measured by tracking the same single molecules for milliseconds up to seconds. This would give unprecedented detail of the dynamics of protein folding measured during the folding of a single protein.

 

Ultra-sensitivity Microscopy, in vivo: Live Cell Imaging

In vitro single molecule experiments have revealed much about the dynamics and function of proteins and will be a mainstream method for investigating protein interactions during the next decades. For biological relevance, it is essential to perform such single-molecule investigations within living cells. Over the last years, the Bräuchle lab has acquired great expertise with live-cell imaging experiments. They performed high-sensitivity single particle experiments on the entry pathway of various native and artificial viruses. This expertise is being used to investigate protein transport, function, and interactions within living cells as well as tracking the uptake of small molecules and peptides studied in area E (Carell, Sieber, Mayer). Furthermore, in collaboration with Jansen (area D), they are investigating the transport mechanism of mRNA in budding yeast cells. Using dual-color particle tracking the transport of mRNA is followed in one channel while various transport proteins are monitored in the other channel. Thus, the proteins involved in transport will be identified, how these proteins interact with the mRNA and other cellular components will be investigated, and the dynamics of the transport process quantified. The ability to track biomolecules in living cells makes it possible to investigate “a day in the life” of various proteins.

Additional live cell studies will be performed in close collaboration with the Leonhardt group (area D) who employ photobleaching and photoactivation techniques in an “in vivo biochemistry” approach.

 

High Spatial-Resolution Imaging

Exciting new developments have recently broken Abbé’s diffraction barrier and allow for the first time microscopic studies at a resolution below 200 nm. It is now possible to study subcellular structures that were previously inaccessible to light microscopy, opening a new dimension to live-cell imaging. In collaboration with the group of Prof. John Sedat at UCSF, this new technology will be available for this cluster. The unprecedented higher optical resolution will help to close the resolution gap to electron microscopy and will soon become a key technology of Life Sciences.

 

Ultrafast techniques

Ultrafast IR techniques are the most promising tools to probe the dynamics of conformational changes and primary steps of folding processes on the molecular level (areas B and F). In the present project, the Zinth group will apply ultrafast IR techniques to the study of native proteins and newly synthesized peptide systems (Carell), where fast structural changes occur and where they can be induced on the sub-picosecond time scale. The molecular systems comprise peptides combined with ultrafast trigger molecules and chromoproteins or protein-nucleic acid complexes (research area D). Ultrashort light pulses will be used to initiate fast reaction dynamics or to alter the shape of a trigger molecule. This local structural change is transferred to the protein/peptide molecule and induces structural variations of the peptide moiety. The dynamics of this structural rearrangement will be recorded with vibrational spectroscopy over the complete time range, starting with the fastest processes on sub-picoseconds time domain. The spectroscopic information will be complemented with molecular dynamic simulations to obtain a complete picture of the reaction.

One of the most important future developments in structural biology is time-resolved X-ray crystallography of proteins. It will ultimately offer the possibility to obtain real-time movies of protein motion.

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