Lemke Group
Structural light microscopy - single molecule spectroscopy
Interfacing a large set of tools with our home-built highly sensitive equipment allows us to study structure and dynamics of even heterogeneous biological systems in 4D.
Previous and current research
Research in our laboratory combines advanced fluorescence single molecule techniques and superresolution microscopy with modern chemical biology tools to elucidate the nature of protein disorder in biological systems and disease mechanisms.
Currently, more than 50,000 protein structures with atomic resolution are available from the protein databank and due to large efforts (mainly crystallography and NMR) their number is rapidly growing. However, even if all 3D protein structures were available, our view of the molecular building blocks of cellular function would still be rather incomplete, as we now know that many proteins are intrinsically disordered, which means that they are unfolded in their native state. Interestingly, the estimated percentage of intrinsically disordered proteins (IDPs) grows with the complexity of the organism (prokaryotes ≈ 5% and eukaryotes ≈ 50%). In a modern view of systems biology, these disordered proteins are believed to be multi-functional signaling hubs central to the interactome (the whole set of molecular interactions in the cell). Their ability to adopt multiple conformations is considered a major driving force behind their evolution and enrichment in eukaryotes.
While the importance of IDPs in biology is now well established, many common strategies for probing protein structure are incompatible with molecular disorder and the highly dynamic nature of those systems. In addition, in any complex biological system a mosaic of molecular states and reaction pathways exist simultaneously, further complicating the situation to measure these systems. For example, some proteins might behave differently than the average, giving rise to new and unexpected phenotypes. One such example are the infamous Prion proteins, where misfolding of only subpopulations of proteins can trigger a drastic signaling cascade leading to completely new phenotypes. Conventional ensemble experiments are only able to measure the average behavior of such a system, ignoring coexisting populations and rare events. This can easily lead to generation of false or insufficient models, which may further impede our understanding of the biological processes and disease mechanisms. In contrast to conventional approaches, single molecule and superresolution techniques, which directly probe the distribution of molecular events, can reveal important mechanisms that otherwise remain obscured. In particular, single molecule fluorescence studies allow probing of molecular structures and dynamics at near atomic scale with exceptional time resolution.
Future projects and goals
Recent studies have shown that even the building blocks of some of the most complex and precise machines with an absolute critical role to survival of the cell are built from IDPs rather than “structured “proteins. The “biggest” example are the nuclear pore complexes (NPC), which, with an approximate molecular weight of 120 megaDalton, constitute the largest molecular machine in the cell and malfunctions of this machinery is linked to diseases ranging from leukemia to HIV. The NPC is built form hundreds of proteins and can regulate the transport of ≈ 1000 proteins per second per single pore. Anchored in the membrane by several scaffold proteins, the core of the NPC is formed by IDPs which form a transport conduit of unknown structure. Key to the transport mechanism is a complex protein network between IDPs and folded proteins, but how such floppy protein networks are formed and maintain high specificity, high exchange rates, low error rates and diversity at the same time, challenges our views on how nature designs biological machineries. Examples such as the NPC, but also DNA packing (Chromatin) and epigenetic mechanisms call for new approaches in biology: We aim to explore the physical and molecular rationale behind the fundamental role of IDPs by combining protein engineering tools with diverse high/superresolution and single molecule fluorescence techniques. For example, single molecule fluorescence studies require labeling with special fluorescent dyes which still hampers the broad application of this technique. In our laboratory we actively extend the spectrum of chemical biology and protein engineering tools to overcome this limitation, with genetically encoding unnatural amino acids as one of our primary strategy. We also continue to develop new methods and recruit techniques from other disciplines (such as microfluidics), whenever they promise to assist our overall goal of improving our biological understanding.