The protein disorder paradox: What do natively unfolded proteins look like inside living cells?
Final Report Abstract
The general goal of this project was to answer the question: ‘What do natively unfolded proteins look like inside live cells?’ Having primarily focused on the prototypic disordered human amyloid protein alpha-Synuclein (aSyn)-, as originally proposed -, we are now in a position to answer this question in the following way: • Overall, the conformational ensemble properties of disordered aSyn states populated under isolated in vitro conditions are largely preserved in different cell types. • Structural differences primarily concern the most compact ensemble configurations, which are additionally enforced in the contexts of different intracellular environments. • Importantly, this also includes anti-aggregation conformations, in which hydrophobic residues of the aSyn NAC region are protected from solvent exposure via self-shielding, intra-molecular contacts between the N- and C- termini of the protein. • Extended intracellular lifetimes of delivered aSyn samples suggest that disordered proteins are not more prone to proteolytic degradation by endogenous proteases and that natively unfolded protein states stably persist in different intracellular environments. • While the overall conformational properties of exogenously delivered aSyn are similar to the known in vitro reference states, important differences concern the ubiquitous occurrence of post-translational N-terminal acetylation found in all mammalian cell lines, and the concomitant increase in residual helicity within the first 10 residues of aSyn (from 10% to 30%). • This increase in helicity has been shown to promote binding to reconstituted membrane vesicles in vitro, although we did not obtain indications for membrane interactions in cells. • aSyn residues that display transient weak interactions with cytoplasmic components include Tyr39, which is also a key residue in the amyloid formation process. • In ‘healthy’ cells, endogenous enzymatic activities efficiently repair damaged forms of aSyn, which includes proteins states that occur in response to conditions known to promote intracellular aggregation, such as cellular oxidative stress.
Publications
- Cellular Structural Biology (Review). Current Opinion in Structural Biology,15(3) 321-9
Ito Y, Selenko P
- Simultaneous detection of protein phosphorylation and acetylation by high-resolution NMR spectroscopy. Journal of the American Chemical Society, 132(42) 14704-5, 2010
Liokatis S, Dose A, Schwarzer D, Selenko P
- Paramagnetic relaxation enhancement to improve sensitivity of fast NMR methods: application to intrinsically disordered proteins. J. Biomol. NMR, 51(4) 487-95
Theillet FX, Binolfi A, Liokatis S, Verzini Selenko P
- Bacterial in-cell NMR of human alpha-synuclein: A disordered monomer by nature? Biochemical Society Transactions, 40(5) 950-4, 2012
Binolfi A, Theillet FX, Selenko P
(See online at https://doi.org/10.1042/BST20120096) - In-cell NMR in mammalian cells: Part 1. Methods in Molecular Biology, 895 43-54, 2012
Bekei B, Rose HM, Herzig M, Dose A, Schwarzer D, Selenko P
- In-cell NMR in mammalian cells: Part 2. Methods in Molecular Biology, 895 55-66, 2012
Bekei B, Rose HM, Herzig M, Selenko P
- In-cell NMR in mammalian cells: Part 3. Methods in Molecular Biology, 895 67-83, 2012
Bekei B, Rose HM, Herzig M, Stephanowitz H, Krause E, Selenko P
- Site-specific NMR mapping and time-resolved monitoring of serine and threonine phosphorylation in reconstituted kinase reactions and mammalian cell extracts. Nature Protocols, 8(7) 1416-32, 2013
Theillet FX, Rose HM, Liokatis S, Binolfi A, Thongwichian R, Stuiver M, Selenko P
(See online at https://doi.org/10.1038/nprot.2013.083)
