Structural Light Microscopy - Single Molecule Spectroscopy
Previous and Current Research
Research in our laboratory combines biochemistry/molecular biology/cell biology and modern chemical biology methods with advanced fluorescence and single molecule techniques 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, single molecule 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. While such experiments are even possible in the natural environment of the entire cell, single molecule fluorescence studies require labeling with special fluorescent dyes which still hampers the broad application of this technique. In our laboratory we are utilizing a large spectrum of chemical biology and state of the art protein engineering tools to overcome this limitation, with genetically encoding unnatural amino acids as one of our primary strategy. With a focus on studying biological questions, 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.
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, such as DNA packing, epigenetics and nuclear transport processes, are largely built from IDPs. We aim to explore the physical and molecular rationale behind the fundamental role of IDPs by combining molecular biology and protein engineering tools with single molecule biophysics. Our long-term goal is to develop general strategies to combine protein structure and dynamics into a four dimensional view of biological function within its natural complex environment.
Figure: 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.