PDF hosted at the Radboud Repository of the Radboud University Nijmegen The following full text is a publisher's version. For additional information about this publication click this link. http://hdl.handle.net/2066/161485 Please be advised that this information was generated on 2017-12-06 and may be subject to change. Engineering compartmentalised life-like molecular systems Marlies Nijemeisland Engineering compartmentalised life-like molecular systems Proefschrift ter verkrijging van de graad van doctor aan de Radboud Universiteit Nijmegen op gezag van de rector magnificus prof. dr. J.H.J.M. van Krieken, volgens besluit van het college van decanen in het openbaar te verdedigen op donderdag 12 januari 2017 om 12.30 uur precies door Marlies Nijemeisland geboren op 22 september 1984 te Zelhem Promotoren: Prof. dr. ir. Jan C.M. van Hest Prof. dr. Wilhelm T.S. Huck Manuscriptcommissie: Prof. dr. Roeland J.M. Nolte (voorzitter) Prof. dr. Jan H. van Esch (Technische Universiteit Delft) Dr. ir. Tom F.A. de Greef (Technische Universiteit Eindhoven) ISBN: 978-94-92380-06-7 © 2016 Marlies Nijemeisland This work was financially supported by the Radboud University (Bionic Cell project), the Ministry of Education, Culture and Science (Gravitation program 024.001.035) and funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-20012)/ERC- StG 307679 “StomaMotors”. Cover design: Ria Nijemeisland-Heusinkveld Press: Gildeprint Table of Contents Chapter 1 General introduction and thesis outline ......................................... 1 1.1 Introduction ........................................................................................... 2 1.2 Compartments ........................................................................................ 2 1.2.1 Membrane vesicles ....................................................................... 3 1.2.2 Multi-compartment systems ......................................................... 8 1.3 Far-from-equilibrium .............................................................................. 12 1.3.1 Far-from-equilibrium systems ....................................................... 12 1.3.2 Energy sources to maintain systems far-from-equilibrium ............. 15 1.4 Functional molecular systems ................................................................ 17 1.4.1 Metabolic pathways .................................................................... 18 1.4.2 Molecular and nanomotors .......................................................... 21 1.5 Aim of the research ............................................................................... 24 1.6 References ............................................................................................. 26 Chapter 2 Light-driven ATP synthesis by an ATP synthase/ delta-rhodopsin liposome system ................................................ 35 2.1 Introduction .......................................................................................... 36 2.2 Results and discussion ........................................................................... 38 ɛΔc 2.2.1 Purification of CF1F0 ATP synthase and TF0F1 ATP synthase .. 38 2.2.2 Expression and purification of deltarhodopsin .............................. 42 2.2.3 Reconstitution in pre-formed liposomes ....................................... 43 2.2.4 Light-induced ATP synthesis ....................................................... 45 2.2.5 ATP synthesis by acid-base transition ......................................... 48 2.2.6 ATP hydrolysis by proteosomes ................................................... 51 2.3 Conclusions ........................................................................................... 52 2.4 Acknowledgments .................................................................................. 53 2.5 Experimental ......................................................................................... 54 2.6 References ............................................................................................. 61 Chapter 3 An out-of-equilibrium enzymatic network .................................... 67 3.1 Introduction .......................................................................................... 68 3.2 Results and discussion ........................................................................... 73 3.2.1 Evaluation of the metabolic network ........................................... 73 3.2.3 Light-controlled enzymatic network ............................................. 81 3.3 Conclusions ........................................................................................... 84 3.4 Acknowledgements ................................................................................ 84 3.5 Experimental ......................................................................................... 85 vi 3.6 References ............................................................................................. 88 Chapter 4 A mild method for loading of enzymes in asymmetric polymeric nanoreactors ............................................ 93 4.1 Introduction .......................................................................................... 94 4.2 Results and discussion ........................................................................... 96 4.2.1 Nanoreactor formation ................................................................ 96 4.2.2 Encapsulation of an enzymatic cascade ....................................... 98 4.2.3 Movement analysis .................................................................... 106 4.3 Conclusions ......................................................................................... 110 4.4 Acknowledgements .............................................................................. 110 4.5 Experimental ....................................................................................... 111 4.6 References ........................................................................................... 117 Chapter 5 Compartmentalisation of an enzymatic network ........................ 121 5.1 Introduction ........................................................................................ 122 5.2 Results and discussion ......................................................................... 123 5.2.1 Compartmentalisation of the metabolic network ........................ 123 5.2.2 Analysis of movement of the nanoreactors ................................. 127 5.3 Conclusions ......................................................................................... 132 5.4 Acknowledgements .............................................................................. 132 5.5 Experimental ....................................................................................... 133 5.6 References ........................................................................................... 140 Chapter 6 Conclusions and future perspectives .......................................... 143 6.1 Conclusions ......................................................................................... 144 6.2 Future perspectives .............................................................................. 146 6.2.1 Life-like materials ...................................................................... 146 6.2.2 Far-from-equilibrium systems .................................................... 148 6.3 References ........................................................................................... 150 Summary ................................................................................................. 152 Samenvatting ........................................................................................... 156 Acknowledgements ................................................................................... 160 About the author ..................................................................................... 163 vii Chapter 1 General introduction and thesis outline Chapter 1 1.1 Introduction The cell is the basic unit for all forms of life, it contains genetic and biochemical machineries of extreme complexity. Every organism consists of cells or is a single cell by itself. Cells can vary in appearance and function and have distinct characteristics for maintaining homeostasis: self-feeding or nutrition, proliferation, or for example chemical signalling. The structure of cells as well their physico-chemical properties, are a source of inspiration to construct functional life-like molecular materials (1, 2). Two goals are defined in this field: (i) construction of life-like molecular systems using chemical and/or biological building blocks in order to mimic biomolecular architectures found in the cellular environment with the aim to better understand and affect biological processes. (ii), assembly of smart (nano)materials that are able to perform various types of functions such as self-repair and adaptation. These systems can be more versatile than life-like molecular systems as they are based on chemical or biological building blocks and accept a wider range of conditions. Reducing the complexity has the advantage that biomimetic function is easier to engineer and with more control. A disadvantage is that without repair mechanisms and maintenance, simple artificial nano-scale devices will function for a limited amount of time due to degradation of the components. This chapter gives an overview of the different concepts that have been