Professor Richard Berry D. Phil.

Abstract:

The main motivation of this work was to address the challenge of single-molecule functional study of membrane proteins under stable and independently controlled electrical and chemical membrane potentials. Although transmembrane potential is often essential for the function of membrane proteins, current in vitro systems provide only limited options for studying them under biologically relevant conditions. Our experimental assay is based on the droplet-on-hydrogel bilayer technique (Leptihn et al. Nat Protoc 8:1048–1057, 2013), where a lipid bilayer forms between a sub-millimetre water droplet and a thin hydrogel layer on a glass cover slip, enabling high-resolution microscopy in total internal reflection mode. To extend the application of this assay beyond channels to other membrane proteins, we introduce a custom-built, electronically controlled perfusion system that is designed to directly connect to the droplet above the lipid bilayer. This system can supply a stable voltage to the bilayer and is suitable for delivery of fragile membrane proteins embedded in proteoliposomes via charged fusion (Ishmukhametov et al. Nat Commun 7:13025, 2016), introducing changes of chemical potentials, and timed introduction of labels or substrate into the droplet. This work represents one of the steps towards single-molecule functional study of F1Fo ATP synthase under variable transmembrane potentials. High-resolution single-molecule observation of its rotation steps on the microsecond timescale could provide valuable insights into the mechanisms of energy transport across the molecule.

Abstract:

Zorya is a recently identified and widely distributed bacterial immune system that protects bacteria from viral (phage) infections. Three Zorya subtypes have been identified, each containing predicted membrane-embedded ZorA–ZorB (ZorAB) complexes paired with soluble subunits that differ among Zorya subtypes, notably ZorC and ZorD in type I Zorya systems1, 2. Here we investigate the molecular basis of Zorya defence using cryo-electron microscopy, mutagenesis, fluorescence microscopy, proteomics and functional studies. We present cryo-electron microscopy structures of ZorAB and show that it shares stoichiometry and features of other 5:2 inner membrane ion-driven rotary motors. The ZorA5B2 complex contains a dimeric ZorB peptidoglycan-binding domain and a pentameric α-helical coiled-coil tail made of ZorA that projects approximately 70 nm into the cytoplasm. We also characterize the structure and function of the soluble Zorya components ZorC and ZorD, finding that they have DNA-binding and nuclease activity, respectively. Comprehensive functional and mutational analyses demonstrate that all Zorya components work in concert to protect bacterial cells against invading phages. We provide evidence that ZorAB operates as a proton-driven motor that becomes activated after sensing of phage invasion. Subsequently, ZorAB transfers the phage invasion signal through the ZorA cytoplasmic tail to recruit and activate the soluble ZorC and ZorD effectors, which facilitate the degradation of the phage DNA. In summary, our study elucidates the foundational mechanisms of Zorya function as an anti-phage defence system.