Continuous throughput and long-term observation of single-molecule FRET without immobilization.
Tyagi, S., VanDelinder, V., Banterle, N., Fuertes, G., Milles, S., Agez, M. & Lemke, E.A.
Nat Methods. 2014 Mar;11(3):297-300. doi: 10.1038/nmeth.2809. Epub 2014 Jan 19.
We present an automated microfluidic platform that performs multisecond observation of single molecules with millisecond time resolution while bypassing the need for immobilization procedures. With this system, we confine biomolecules to a thin excitation field by reversibly collapsing microchannels to nanochannels. We demonstrate the power of our method by studying a variety of complex nucleic acid and protein systems, including DNA Holliday junctions, nucleosomes and human transglutaminase 2.
Minimal Tags for Rapid Dual-Color Live-Cell Labeling and Super-Resolution Microscopy.
Nikic, I., Plass, T., Schraidt, O., Szymanski, J., Briggs, J.A., Schultz, C. & Lemke, E.A.
Angew Chem Int Ed Engl. 2014;53(8):2245?2249. doi: 10.1002/anie.201309847.
The growing demands of advanced fluorescence and super-resolution microscopy benefit from the development of small and highly photostable fluorescent probes. Techniques developed to expand the genetic code permit the residue-specific encoding of unnatural amino acids (UAAs) armed with novel clickable chemical handles into proteins in living cells. Here we present the design of new UAAs bearing strained alkene side chains that have improved biocompatibility and stability for the attachment of tetrazine-functionalized organic dyes by the inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC). Furthermore, we fine-tuned the SPIEDAC click reaction to obtain an orthogonal variant for rapid protein labeling which we termed selectivity enhanced (se) SPIEDAC. seSPIEDAC and SPIEDAC were combined for the rapid labeling of live mammalian cells with two different fluorescent probes. We demonstrate the strength of our method by visualizing insulin receptors (IRs) and virus-like particles (VLPs) with dual-color super-resolution microscopy.
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.
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.