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Designer amphiphilic proteins as building blocks for the intracellular formation of organelle-like compartments

Nature Materials volume 14, pages 125–132 (2015)Cite this article

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Abstract

Nanoscale biological materials formed by the assembly of defined block-domain proteins control the formation of cellular compartments such as organelles. Here, we introduce an approach to intentionally ‘program’ the de novo synthesis and self-assembly of genetically encoded amphiphilic proteins to form cellular compartments, or organelles, in Escherichia coli. These proteins serve as building blocks for the formation of artificial compartments in vivo in a similar way to lipid-based organelles. We investigated the formation of these organelles using epifluorescence microscopy, total internal reflection fluorescence microscopy and transmission electron microscopy. The in vivo modification of these protein-based de novo organelles, by means of site-specific incorporation of unnatural amino acids, allows the introduction of artificial chemical functionalities. Co-localization of membrane proteins results in the formation of functionalized artificial organelles combining artificial and natural cellular function. Adding these protein structures to the cellular machinery may have consequences in nanobiotechnology, synthetic biology and materials science, including the constitution of artificial cells and bio-based metamaterials.

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Figure 1: The design and expression of artificial amphiphilic proteins with the potential for self-assembly allows the formation of cellular compartments in E. coli.
Figure 2: The influence of the translational order and ratio of amphiphilic block domains on the subcellular distribution and higher-order structure formation in vivo.
Figure 3: The self-assembly of mEGFP–E20F20 proteins to form vesicular-like structures in vitro and in vivo visualized by cryo-TEM, HPF- and conventional TEM.
Figure 4: TIRF and TIRF–SIM fluorescence microscopy images resolve the structure of the artificial cellular compartments in E. coli cells.
Figure 5: The site-selective modification of cellular compartments.

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Acknowledgements

We thank R. Thomann for in vitro TEM images and the element-specific imaging and high-contrast imaging measurements. For the LC–MS/MS analysis we thank M. Samalikova and J. Dengjel. We are grateful to P. G. Schultz, TSRI, La Jolla, California, USA for providing the plasmid pEVOLpAzF. P.K. acknowledges the financial support of the Hungarian Scientific Research Fund (OTKA, grant numbers K-100134, NN-110214) and the ‘Lendület’ Program of the Hungarian Academy of Sciences (LP2013-55/2013) is greatly acknowledged. We are grateful to the Freiburg Institute for Advanced Studies (FRIAS), the Institute for Macromolecular Chemistry, the Institute for Pharmaceutical Sciences, the Institute for Micro System Engineering (IMTEK), the competence network of functional nanostructures (KFN), the Baden-Württemberg Stiftung, the Ministry of Science, Research and the Arts (MWK) Baden-Württemberg, the German Science Foundation (DFG): SPP1623 and EXC 294 BIOSS Centre for Biological Signalling Studies, the BMBF (BMBF Forschungspreis 2014) and the Rectorate of the University of Freiburg for support.

Author information

Author notes

  1. Matthias C. Huber and Andreas Schreiber: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute for Macromolecular Chemistry, University of Freiburg, Stefan-Meier-Str. 31 D-79104 Freiburg, Germany,

    Matthias C. Huber, Andreas Schreiber & Stefan M. Schiller

  2. Institute for Pharmaceutical Sciences, University of Freiburg, Albertstr. 25 D-79104 Freiburg, Germany,

    Matthias C. Huber, Andreas Schreiber & Stefan M. Schiller

  3. Freiburg Institute for Advanced Studies (FRIAS), School of Soft Matter Research, University of Freiburg, Albertstr. 19 D-79104 Freiburg, Germany,

    Matthias C. Huber, Andreas Schreiber & Stefan M. Schiller

  4. Faculty of Chemistry and Pharmacy, University of Freiburg, Fahnenbergplatz D-79104 Freiburg, Germany,

    Matthias C. Huber, Sabine Barnert, Rolf Schubert & Stefan M. Schiller

  5. Faculty of Biology, University of Freiburg, Schänzlestrasse 1 D-79085 Freiburg, Germany,

    Andreas Schreiber & Stefan M. Schiller

  6. Department of Microsystems Engineering, Bio- and Nano-Photonics, University of Freiburg, Georges-Köhler-Allee 102 D-79110 Freiburg, Germany,

    Philipp von Olshausen

  7. BIOSS Centre for Biological Signalling Studies, University of Freiburg, Schänzlestrasse 18 D-79104 Freiburg, Germany,

    Philipp von Olshausen, Oliver Kretz, Rolf Schubert, Stefan Eimer & Stefan M. Schiller

  8. Chemical Biology Research Group, Hungarian Academy of Sciences, CNS, IOC, Magyar tudósok krt. 2 H-1117 Budapest, Hungary,

    Balázs R. Varga & Péter Kele

  9. Institute for Neuroanatomy University of Freiburg, Albertstr. 17 D-79104 Freiburg, Germany,

    Barbara Joch

  10. Department of Pharmaceutical Technology and Biopharmacy, Institute of Pharmaceutical Sciences, University of Freiburg, Hermann-Herder-Str. 9 D-79104 Freiburg, Germany,

    Sabine Barnert & Rolf Schubert

  11. IMTEK Department of Microsystems Engineering, University of Freiburg, Georges-Köhler-Allee 103 D-79110 Freiburg, Germany,

    Stefan M. Schiller

  12. Center for Biosystems Analysis (ZBSA), University of Freiburg, Habsburger Str. 49 D-79104 Freiburg, Germany,

    Stefan M. Schiller

Authors
  1. Matthias C. Huber
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  11. Stefan M. Schiller
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Contributions

S.M.S. conceived the project. A.S. performed the in vitro and in vivo characterization of the newly cloned proteins and their formed structures using fluorescence microscopy and TEM. Further, A.S. conducted the bio-orthogonal modification of the artificial organelles in vivo. M.C.H. designed and cloned the different protein constructs, and carried out the coexpression experiments and in vivo characterization using fluorescence microscopy. A.S. and M.C.H. contributed equally to the paper. P.v.O. performed the TIRF and TIRF–SIM measurements. P.K. and B.R.V. designed and synthesized the copper-free clickable fluorescent dye. O.K. conducted the HPF experiments. B.J. did the in vivo TEM micrographs. S.B. conducted the cryo in vitro TEM analysis. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Stefan M. Schiller.

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The authors declare no competing financial interests.

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Huber, M., Schreiber, A., von Olshausen, P. et al. Designer amphiphilic proteins as building blocks for the intracellular formation of organelle-like compartments. Nature Mater 14, 125–132 (2015). https://doi.org/10.1038/nmat4118

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