"Unfolding" the Nucleo-Cytoplasmic Transport Machinery one molecule at a time

• Antragsteller: Professor Dr. Edward A. Lemke
• Fachliche Zuordnung: Biophysik
• Förderung: Förderung von 2010 bis 2016
• Projektkennung: Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 157900825

• Erstellungsjahr: 2017

Zusammenfassung der Projektergebnisse

Intrinsically disordered and phenylalanine‐glycine rich nucleoporins (FG‐Nups) form a selective permeability barrier inside the nuclear pore complex (NPC): Large molecules can only cross the central channel of the NPC when piggybacked by nuclear transport receptors (NTRs) that specifically interact with FG‐Nups. These FG‐Nups, however, display complex and non‐random amino acid architecture and possess repeatedly occurring FG‐motifs flanked by distinct amino acid stretches. How such heterogeneous sequence composition relates to function and how homotypic interactions between such disordered stretches, and transient heterotypic interactions with folded transport receptors could give rise to a transport mechanism is still unclear. Paradoxically, nuclear transport is very fast and specific at the same time. We have now developed an integrated chemical biology‐ fluorescence approach to study the molecular plasticity of FG‐Nups on the single‐molecule level using multi‐parameter fluorescence spectroscopy. In a bottom up fashion our studies ranged from characterization of basic physicochemical properties, to mapping and structurally characterizing diverse supramolecular states, such as amyloids and hydrogels. Ultimately, we discovered that rapidly fluctuating FG‐Nup populates an ensemble of conformations that are prone to bind NTRs with remarkably fast diffusion‐limited on‐rates. This is achieved using multiple, minimalistic, low affinity binding motifs that are in rapid exchange when engaging with the NTR, allowing the FG‐Nup to maintain an unexpectedly high plasticity in its bound state. The binding mechanism appears distinct from classical folding upon protein binding mechanisms, and describes a novel pathway on how ultrafast interaction between biomolecules can be mediated and explains how ncuelar transport can be fast and selective. Furthermore, we have developed a novel microfluidic technology that can bypass many problems commonly associated with performing single molecule studies in biology. A particular focus was a new platform build from an elastomer that allows long‐term visualization of single biomolecules with high signal to noise without the need for immobilization. This platform increases throughput and quality of our in vitro single molecule measurements. Paired with the development our new multicolor labeling technologies, these efforts will dramatically accelerate our abilities to study protein plasticity, and advance single molecule science in general. Our studies are greatly supported by our progress in protein engineering. We have now developed a semi‐synthetic strategy based on novel artificial amino acids that are easily and site‐specifically introduced into any protein by the natural machinery of the living cell. We showed that ring‐strained cyclooctyne or cyclooctene functional group can be stably incorporated into any protein and readily react with commercially available single molecule fluorophores without the need of special reagents, catalyst or non‐physiological buffer conditions. The speed and specificity of this method will further improve our ability to study proteins in vitro and in vivo with residue specific resolution.

Projektbezogene Publikationen (Auswahl)

• Genetically encoded copper‐free click chemistry. Angew Chem Int Ed Engl. 18;50(17):3878‐81. (2011)
  Plass T, Milles S, Koehler C, Schultz C, Lemke EA
  
• Single molecule study of the intrinsically disordered FG‐repeat Nucleoporin 153. Biophys J 101(7):1710‐9 (2011)
  Milles S, Lemke EA
  
• Amino Acids for Diels‐Alder Reactions in Living Cells. Angew Chem Int Ed Engl. 23;51(17):4166‐70. (2012)
  Plass T, Milles S, Koehler C, Szymanski J. Mueller R, Wieβler M, Schultz C, Lemke EA
  
• Click Strategies for Single‐Molecule Protein Fluorescence. J Am Chem Soc 134, 5187‐95 (2012)
  Milles S, Tyagi S, Banterke N, Koehler C, VanDelinder V, Neal AP, Lemke EA
  
• Intramolecular three‐colour single pair FRET of intrinsically disordered proteins with increased dynamic range. Mol Biosyst Jun 27 (2012)
  Milles S, Koehler C, Gambin Y, Deniz AA, Lemke EA
  
• Continuous throughput and long‐term observation of single‐molecule FRET without immobilization. Nature Methods, Mar;11(3):297‐300 (2014)
  Tyagi S, VanDelinder V, Banterle N, Fuertes G, Milles S, Agez M, Lemke EA
  
• Dual‐color super‐resolution microscopy via genetic code expansion technology and tuned click reactions. Angew Chem Int Ed Engl, 2014, Feb, 53(8), 2245‐2249 (2014)
  Nikic I, Plass T, Schraidt O, Szymanski J, Briggs JA, Schultz C, Lemke EA
  
• Mapping multivalency and differential affinities within large intrinsically disordered protein complexes with segmental motion analysis. Angew Chem Int Ed Engl. Jul 7;53(28):7364‐7 (2014)
  Milles S, Lemke EA
  
• Plasticity of an ultrafast interaction between nucleoporin and transport receptors. Cell
  Milles S, Mercadante D, Aramburu IV, Jensen MR, Banterle N, Koehler C, Tyagi S, Clarke J, Shammas S, Blackledge M, Gräter F, Lemke EA
  
• US patent (9085514, issued 21/07/2015: Unnatural amino acids comprising a cyclooctynyl analog group and uses thereof
  Lemke EA, Plass T, Milles S, Koehler C, Schultz C
  
• Genetic code expansion for multiprotein complex engineering, Nature Methods, Dec;13(12):997‐100 (2016)
  Koehler C, Sauter PF, Wawryszyn M, Estrada Girona G, Gupta K, Landry JJM, Hsi‐Yang Fritz M, Radic K, Hoffmann J‐E, Chen ZA, Zou J, Tan PS, Galik B, Junttila S, Stolt‐Bergner P, Pruneri G, Gyenesei A, Schultz C, Biskup MB, Besir H, Benes V, Rappsilber J, Jechlinger M, Korbel JO, Berger I, Braese S, Lemke EA