Periodic Reporting for period 3 - MultiMotif (Motif repeats in intrinsically disordered regions of the clathrin mediated endocytosis pathway)
Periodo di rendicontazione: 2022-05-01 al 2023-10-31
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.
This project 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.
This project 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.
We are setting up a home-built single molecule fluorescence spectrometer, and developing approaches for integrating parameters obtained from nuclear magnetic resonance (NMR) and single molecule fluorescence spectroscopy to study intrinsically disordered proteins.
NMR and single molecule fluorescence count amongst the techniques that are best suited to study intrinsically disordered proteins (IDPs), which are dynamic in nature. While NMR parameters inform about local structural propensities of the IDP, single molecule fluorescence approaches, in particular Förster resonance energy transfer (FRET/smFRET), can specifically probe long-range distances that can often provide the essential necessary information required to explain molecular behaviour. We are expecting to apply combinations of NMR and smFRET to shed light on the complex interaction network in clathrin-mediated endocytosis, built up with the help of IDPs.