Single-molecule fluorescence studies of KirBac1.1

Thesis

Inwardly rectifying potassium (Kir) channels are essential for controlling the excitability of eukaryotic cells, forming a key part of the inter-cellular signalling system in multi-cellular organisms. However, as prokaryotic (KirBac) channels are less technically challenging to study in vitro and have been shown to be directly homologous to eukaryotic channels, they are often studied in lieu of their mammalian counterparts. A vital feature of Kir and KirBac channels is their mechanism for opening and closing, or their gating: this study predominantly features observations of open and/or closed channel populations. A well-characterised member of the KirBac family, KirBac1.1, has been successfully expressed, purified into detergent micelles, and doubly labelled with fluorescent maleimide dyes in order to enable observation of confocal-in-solution Förster Resonance Energy Transfer (FRET) at the single molecule level. Results demonstrate single-molecule FRET signals from KirBac1.1 and therefore represent the first single-molecule FRET observations from a KirBac channel. Perturbation of the open-closed dynamic equilibrium was performed via activatory point mutations, changes in pH, and ligand binding. A protocol for reconstitution into nanodiscs was optimised in order to more closely approximate native conditions, and the single-molecule FRET observations repeated. This thesis presents a comparison between measurements made using the detergent solubilisation system and those made using nanodiscs.

Authors: Sadler, E; Emma E Sadler

• Publication date: 2015
• DOI: 10.5287/ora-gjygp9de0
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: Oxford University, UK
• Language: English
• Keywords: nanodiscs; detergent; dynamic equilibrium; single-molecule; KirBac; FRET; excitable membranes; potassium channels; confocal; Kir
• Subjects: Biophysics; Condensed Matter Physics; Microscopy; Membrane proteins