Institut de Biologie StructuraleGrenoble / France

Contact person(s) related to this article / MILLES Sigrid

S. Milles ERC Team

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.

Candidates for an internship, PhD or a postdoc in the team should contact Sigrid Milles.

Related publications

Naudi-Fabra S, Blackledge M, Milles S.
Synergies of Single Molecule Fluorescence and NMR for the Study of Intrinsically Disordered Proteins
Biomolecules 2022, 12, 27

Naudi-Fabra S, Tengo M, Jensen MR, Blackledge M, Milles S.
Quantitative Description of Intrinsically Disordered Proteins Using Single-Molecule FRET, NMR, and SAXS.
J Am Chem Soc. 2021 Dec 8 ;143(48):20109-20121

Guseva S, Milles S, Jensen MR, Salvi N, Kleman JP, Maurin D, Ruigrok RWH, Blackledge M.
Measles virus nucleo- and phosphoproteins form liquid-like phase-separated compartments that promote nucleocapsid assembly.
Sci Adv. 2020 Apr 1 ;6(14):eaaz7095

Milles S, Jensen MR, Lazert C, Guseva S, Ivashchenko S, Communie G, Maurin D, Gerlier D, Ruigrok RWH, Blackledge M.
An ultraweak interaction in the intrinsically disordered replication machinery is essential for measles virus function
Sci Adv. 2018 Aug 22 ;4(8):eaat7778

Milles S, Mercadante D, Aramburu IV, Jensen MR, Banterle N, Koehler C, Tyagi S, Clarke J, Shammas SL, Blackledge M, Gräter F, Lemke EA.
Plasticity of an ultrafast interaction between nucleoporins and nuclear transport receptors
Cell. 2015 Oct 22 ;163(3):734-45

Delaforge E, Milles S, Bouvignies G, Bouvier D, Boivin S, Salvi N, Maurin D, Martel A, Round A, Lemke EA, Jensen MR, Hart DJ, Blackledge M.
Large-Scale Conformational Dynamics Control H5N1 Influenza Polymerase PB2 Binding to Importin α
J Am Chem Soc. 2015 Dec 9 ;137(48):15122-34.

Milles S, Lemke EA.
Mapping multivalency and differential affinities within large intrinsically disordered protein complexes with segmental motion analysis
Angew Chem Int Ed Engl. 2014 Jul 7 ;53(28):7364-7.

Milles S, Tyagi S, Banterle N, Koehler C, VanDelinder V, Plass T, Neal AP, Lemke EA.
Click strategies for single-molecule protein fluorescence
J Am Chem Soc. 2012 Mar 21 ;134(11):5187-95