Lemke GroupPublications
Facilitated aggregation of FG nucleoporins under molecular crowding conditions.
Milles, S., Huy Bui, K., Koehler, C., Eltsov, M., Beck, M. & Lemke, E.A.
EMBO Rep. 2013 Feb;14(2):178-83. doi: 10.1038/embor.2012.204. Epub 2012 Dec 14.
Intrinsically disordered and phenylalanine-glycine-rich nucleoporins (FG Nups) form a crowded and selective transport conduit inside the NPC that can only be transited with the help of nuclear transport receptors (NTRs). It has been shown in vitro that FG Nups can assemble into two distinct appearances, amyloids and hydrogels. If and how these phenomena are linked and if they have a physiological role still remains unclear. Using a variety of high-resolution fluorescence and electron microscopic (EM) tools, we reveal that crowding conditions mimicking the NPC environment can accelerate the aggregation and amyloid formation speed of yeast and human FG Nups by orders of magnitude. Aggregation can be inhibited by NTRs, providing a rationale on how the cell might control amyloid formation of FG Nups. The superb spatial resolving power of EM also reveals that hydrogels are enlaced amyloid fibres, and these findings have implications for existing transport models and for NPC assembly.
PubMed
Intramolecular three-colour single pair FRET of intrinsically disordered proteins with increased dynamic range.
Milles, S., Koehler, C., Gambin, Y., Deniz, A.A. & Lemke, E.A.
Mol Biosyst. 2012 Oct;8(10):2531-4. doi: 10.1039/c2mb25135c.
Single molecule observation of fluorescence resonance energy transfer can be used to provide insight into the structure and dynamics of proteins. Using a straightforward triple-colour labelling strategy, we present a measurement and analysis scheme that can simultaneously study multiple regions within single intrinsically disordered proteins.
PubMed
Amino acids for diels-alder reactions in living cells.
Plass, T., Milles, S., Koehler, C., Szymanski, J., Mueller, R., Wiessler, M., Schultz, C. & Lemke, E.A.
Angew Chem Int Ed Engl. 2012 Apr 23;51(17):4166-70. doi: 10.1002/anie.201108231.Epub 2012 Mar 30.
Under tension: A set of genetically encoded unnatural amino acids can be used for biocompatible site-specific labeling of proteins with fluorogenic dyes. The new compounds have norbornene and trans-cyclooctene units that react with tetrazine derivatives in an inverse-electron-demand Diels-Alder cycloaddition (left in picture). The technique offers fast labeling that is orthogonal to labeling through azide-cyclooctyne click reaction (right).
PubMed
Click strategies for single-molecule protein fluorescence.
Milles, S., Tyagi, S., Banterle, N., Koehler, C., Vandelinder, V., Plass, T., Neal, A.P. & Lemke, E.A.
J Am Chem Soc. 2012 Mar 21;134(11):5187-95. Epub 2012 Mar 5.
Single-molecule methods have matured into central tools for studies in biology. Foerster resonance energy transfer (FRET) techniques, in particular, have been widely applied to study biomolecular structure and dynamics. The major bottleneck for a facile and general application of these studies arises from the need to label biological samples site-specifically with suitable fluorescent dyes. In this work, we present an optimized strategy combining click chemistry and the genetic encoding of unnatural amino acids (UAAs) to overcome this limitation for proteins. We performed a systematic study with a variety of clickable UAAs and explored their potential for high-resolution single-molecule FRET (smFRET). We determined all parameters that are essential for successful single-molec studies, such as accessibility of the probes, expression yield of proteins, and quantitative labeling. Our multiparameter fluorescence analysis allowed us to gain new insights into the effects and photophysical properties of fluorescent dyes linked to various UAAs for smFRET measurements. This led us to determine that, from the extended tool set that we now present, genetically encoding propargyllysine has major advantages for state-of-the-art measurements compared to other UAAs. Using this optimized system, we present a biocompatible one-step dual-labeling strategy of the regulatory protein RanBP3 with full labeling position freedom. Our technique allowed us then to determine that the region encompassing two FxFG repeat sequences adopts a disordered but collapsed state. RanBP3 serves here as a prototypical protein that, due to its multiple cysteines, size, and partially disordered structure, is not readily accessible to any of the typical structure determination techniques such as smFRET, NMR, and X-ray crystallography.
PubMed
Single molecule study of the intrinsically disordered FG-repeat nucleoporin 153.
Milles, S. & Lemke, E.A.
Biophys J. 2011 Oct 5;101(7):1710-9. doi: 10.1016/j.bpj.2011.08.025.
Nucleoporins (Nups), which are intrinsically disordered, form a selectivity filter inside the nuclear pore complex, taking a central role in the vital nucleocytoplasmic transport mechanism. These Nups display a complex and nonrandom amino-acid architecture of phenylalanine glycine (FG)-repeat clusters and intra-FG linkers. How such heterogeneous sequence composition relates to function and could give rise to a transport mechanism is still unclear. Here we describe a combined chemical biology and single-molecule fluorescence approach to study the large human Nup153 FG-domain. In order to obtain insights into the properties of this domain beyond the average behavior, we probed the end-to-end distance (R(E)) of several approximately 50-residues long FG-repeat clusters in the context of the whole protein domain. Despite the sequence heterogeneity of these FG-clusters, we detected a reoccurring and consistent compaction from a relaxed coil behavior under denaturing conditions (R(E)/R(E,RC) = 0.99 +/- 0.15 with R(E,RC) corresponding to ideal relaxed coil behavior) to a collapsed state under native conditions (R(E)/R(E,RC) = 0.79 +/- 0.09). We then analyzed the properties of this protein on the supramolecular level, and determined that this human FG-domain was in fact able to form a hydrogel with physiological permeability barrier properties.
PubMed