Intrinsically disordered proteins in endocytosis
Endocytosis is responsible for the entry of molecules into the eukaryotic cell, as for example nutrients, signaling molecules and their receptors, but also pathogens. This mechanism is thus very important and relies on the small protein clathrin (clathrin-dependent endocytosis), which forms the structural scaffold shaping the membrane and finally resulting in the uptake of a coated vesicle. However, a lot of other proteins are necessary for this highly regulated uptake process, amongst others adaptor proteins that contain long intrinsically disordered regions (IDR), i.e. regions without a stable three-dimensional structure. These regions are interspersed with small sequence stretches, called linear motifs, which interact with other proteins from the endocytosis machinery : other adaptors for example or clathrin. Although these interactions are crucial for endocytosis, they are not very well understood due to the flexibility and dynamics of the protein sequences they are embedded in.
Our team aims at developing integrated approaches using single molecule fluorescence and NMR spectroscopy to study these intrinsically disordered adaptor proteins and understand the molecular mechanism by which their linear motifs regulate the process of endocytosis. Understanding the way of function of these motifs is important not only for endocytosis, but also many other biological processes that also rely on using linear motifs.
Integrating single molecule fluorescence and NMR spectroscopy
Nuclear magnetic resonance (NMR) and single molecule fluorescence count amongst the techniques that are best suited to study intrinsically disordered proteins (IDP), which are dynamic in nature.
While NMR parameters, such as chemical shifts or residual dipolar couplings, inform about local structural propensities of the IDR and the angular averaging of individual bond vectors with atomic resolution, single molecule fluorescence approaches, in particular Förster resonance energy transfer (FRET/smFRET) can specifically probe distances that are beyond reach for NMR, and that can often provide the essential necessary information required to explain molecular behaviour. At the same time, both NMR and fluorescence spectroscopies are determined by intrinsic parameters that govern different time scales and appropriate experimental approaches have been devised to enable the analysis of protein dynamics from picoseconds to milliseconds and beyond. In combination, this allows the investigation of fast dynamics such as those described by a rapidly interconverting intrinsically disordered polypeptide chain, but also slower dynamic features arising through transient population of secondary structures as well as slower, physiologically important, domain motions, for instance.
Our team is setting up a home-built single molecule fluorescence spectrometer, and we are developing qualitative and quantitative approaches for integrating parameters of both techniques into a common dynamic structural model.
Research SectionStructural Biology
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