Production of Multicomponent Protein Templates for the Positioning and Stabilization of Enzymes

Part of the Methods in Molecular Biology book series (MIMB, volume 2073)


Harnessing the ability of proteins to self-assemble into complex structures has enabled the creation of templates for applications in nanotechnology. Protein templates can be used to position functional molecules in regular patterns with nanometer precision over large surface areas. A difficult but successful approach to building customizable protein templates involves designing novel protein-protein interfaces to join protein building blocks into ordered arrangements. This approach was illustrated recently by engineering the protein interfaces of a molecular chaperone to produce filamentous templates composed of repeating subunits. In this chapter, we describe how these multicomponent protein templates can be produced recombinantly, assembled into filaments, and used as material templates. The templates enable the positioning and alignment of functional molecules at varying distances along the length of the filament, which can be demonstrated using a Förster resonance energy transfer (FRET) assay. In addition, we describe a method to quantify the chaperone ability of these filaments to stabilize and protect other proteins from thermal-induced aggregation—a useful property for bionanotechnology applications that involve molecular scaffolds for positioning and stabilizing enzymes.

Key words

Filament Self-assembly Protein interface Coiled-coil Scaffold Template Chaperone Biomaterials 



This work was supported by the Air Force Office of Scientific Research (FA9550-14-1-0026).


  1. 1.
    Glover DJ, Clark DS (2016) Protein calligraphy: a new concept begins to take shape. ACS Cent Sci 2:438–444CrossRefGoogle Scholar
  2. 2.
    Kostiainen MA, Hiekkataipale P, Laiho A, Lemieux V, Seitsonen J, Ruokolainen J, Ceci P (2013) Electrostatic assembly of binary nanoparticle superlattices using protein cages. Nat Nanotechnol 8:52–56CrossRefGoogle Scholar
  3. 3.
    Schoen AP, Schoen DT, Huggins KNL, Arunagirinathan MA, Heilshorn SC (2011) Template engineering through epitope recognition: a modular, biomimetic strategy for inorganic nanomaterial synthesis. J Am Chem Soc 133:18202–18207CrossRefGoogle Scholar
  4. 4.
    Oh D, Qi J, Lu Y-C, Zhang Y, Shao-Horn Y, Belcher AM (2013) Biologically enhanced cathode design for improved capacity and cycle life for lithium-oxygen batteries. Nat Commun 4:2756CrossRefGoogle Scholar
  5. 5.
    Wörsdörfer B, Woycechowsky KJ, Hilvert D (2011) Directed evolution of a protein container. Science 331:589–592CrossRefGoogle Scholar
  6. 6.
    Lau YH, Giessen TW, Altenburg WJ, Silver PA (2018) Prokaryotic nanocompartments form synthetic organelles in a eukaryote. Nat Commun 9:1311CrossRefGoogle Scholar
  7. 7.
    King NP, Bale JB, Sheffler W, McNamara DE, Gonen S, Gonen T, Yeates TO, Baker D (2014) Accurate design of co-assembling multi-component protein nanomaterials. Nature 510:103–108CrossRefGoogle Scholar
  8. 8.
    Lai Y-T, Reading E, Hura GL, Tsai K-L, Laganowsky A, Asturias FJ, Tainer JA, Robinson CV, Yeates TO (2014) Structure of a designed protein cage that self-assembles into a highly porous cube. Nat Chem 6:1065–1071CrossRefGoogle Scholar
  9. 9.
    Bale JB, Gonen S, Liu Y et al (2016) Accurate design of megadalton-scale two-component icosahedral protein complexes. Science 353:389–394CrossRefGoogle Scholar
  10. 10.
    Shen H, Fallas JA, Lynch E et al (2018) De novo design of self-assembling helical protein filaments. Science 362:705–709CrossRefGoogle Scholar
  11. 11.
    Glover DJ, Giger L, Kim SS, Naik RR, Clark DS (2016) Geometrical assembly of ultrastable protein templates for nanomaterials. Nat Commun 7:11771CrossRefGoogle Scholar
  12. 12.
    Whitehead TA, Boonyaratanakornkit BB, Höllrigl V, Clark DS (2007) A filamentous molecular chaperone of the prefoldin family from the deep-sea hyperthermophile Methanocaldococcus jannaschii. Protein Sci 16:626–634CrossRefGoogle Scholar
  13. 13.
    Lim S, Glover DJ, Clark DS (2018) Prefoldins in Archaea. Adv Exp Med Biol 1106:11–23CrossRefGoogle Scholar
  14. 14.
    Glover DJ, Giger L, Kim JR, Clark DS (2012) Engineering protein filaments with enhanced thermostability for nanomaterials. Biotechnol J 8:228–236CrossRefGoogle Scholar
  15. 15.
    Glover DJ, Clark DS (2015) Oligomeric assembly is required for chaperone activity of the filamentous γ-prefoldin. FEBS J 282:2985–2997CrossRefGoogle Scholar
  16. 16.
    Lim S, Jung GA, Muckom RJ, Glover DJ, Clark DS (2019) Engineering bioorthogonal protein-polymer hybrid hydrogel as a functional protein immobilization platform. Chem Commun 55:806–809Google Scholar
  17. 17.
    Glover DJ, Lim S, Xu D, Sloan NB, Zhang Y, Clark DS (2018) Assembly of multicomponent protein filaments using engineered subunit interfaces. ACS Synth Biol 7:2447–2456CrossRefGoogle Scholar
  18. 18.
    Kida H, Sugano Y, Iizuka R, Fujihashi M, Yohda M, Miki K (2008) Structural and molecular characterization of the prefoldin beta subunit from Thermococcus strain KS-1. J Mol Biol 383:465–474CrossRefGoogle Scholar

Copyright information


Authors and Affiliations

  1. 1.Department of Chemical and Biomolecular EngineeringUniversity of CaliforniaBerkeleyUSA
  2. 2.School of Biotechnology and Biomolecular SciencesThe University of New South WalesSydneyAustralia

Personalised recommendations