Oral Presentation 50th Lorne Proteins Conference 2025

Droplet-on-hydrogel bilayer based assay for functional study of membrane proteins (115007)

Zuzana Coculova 1 , Richard Berry 2
  1. UNSW, Kensington, NSW, Australia
  2. Physics, University of Oxford, Oxford, United Kingdom

Protein motors are incredible biological machines that convert chemical energy into mechanical work, thereby driving a wide range of essential biological processes. Our work is focused on single-molecule methods for functional studies of various molecular motors. In the first part, we introduce a droplet-on-hydrogel bilayer based assay suitable for observing rotary motor F1Fo in conditions mimicking the protein’s natural environment. This part of the work was performed with Professor Richard Berry at the University of Oxford. In the second part, we review various experimental assays suitable for studying linear motors moving along a DNA track. 

Functional study of membrane proteins, especially when transmembrane potential is required, remains challenging. Various creative approaches - using either liposomes, lipid nanodiscs, or a wide range of different planar biomembrane systems - have contributed to building our understanding of these molecular machines, but the picture is not complete yet. Our single-molecule assay adapts the droplet-on-hydrogel bilayer technique [1] with independent control of chemical and electrical transmembrane potential. The bilayer is formed between a 200 nl water droplet and a supporting layer of hydrogel, granting a stable, functional, and accessible lipid bilayer. Protein incorporation can be facilitated by fusion of positively charged proteoliposomes with the negatively charged lipid bilayer [2].  We present a low-cost custom-built electronically controlled perfusion system suitable for delivery of proteins, labels, substrates, or ions to the droplet above the bilayer. Experiments presented in this work show a stable perfusion with 27 ± 3 nl steps. 

Recent advances in synthetic biology allow insight into the mechanics of molecular motors through single-molecule studies of artificial molecular motors. Focusing on the Tumbleweed [3], a synthetic motor with three discrete ligand‐dependent DNA‐binding domains that allow movement along a synthesized DNA track, we discuss different approaches to DNA immobilization to observe stepping of this linear molecular motor.

 

  1. [1] Leptihn, S. et al. Nature Protocol. 8 (6): 1048–1057 (2013)
  2. [2] Ishmukhametov, R. et al. Nature Communications. 7: 13025 (2016)
  3. [3] Bromley, E.H.C. et al. HFSP Journal. 3 (3): 204-212 (2010)