Massey Documents by Type

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CryoEM structure of the outer membrane secretin channel pIV from the f1 filamentous bacteriophage
(Springer Nature Limited, 2021-11-02) Conners R; McLaren M; Łapińska U; Sanders K; Stone MRL; Blaskovich MAT; Pagliara S; Daum B; Rakonjac J; Gold VAM
The Ff family of filamentous bacteriophages infect gram-negative bacteria, but do not cause lysis of their host cell. Instead, new virions are extruded via the phage-encoded pIV protein, which has homology with bacterial secretins. Here, we determine the structure of pIV from the f1 filamentous bacteriophage at 2.7 Å resolution by cryo-electron microscopy, the first near-atomic structure of a phage secretin. Fifteen f1 pIV subunits assemble to form a gated channel in the bacterial outer membrane, with associated soluble domains projecting into the periplasm. We model channel opening and propose a mechanism for phage egress. By single-cell microfluidics experiments, we demonstrate the potential for secretins such as pIV to be used as adjuvants to increase the uptake and efficacy of antibiotics in bacteria. Finally, we compare the f1 pIV structure to its homologues to reveal similarities and differences between phage and bacterial secretins.
The role of dynamin-related proteins in vacuole biogenesis in fission yeast (Schizosaccharomyces pombe) : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand
(Massey University, 2008) Röthlisberger, Sarah Ruth
Dynamins are GTPases concerned with membrane tubulation and scission (Praefcke and McMahon, 2004). In the fission yeast, Schizosaccharomyces pombe, the dynamin-related proteins (DRPs) Vps1 and Dnm1 act redundantly in peroxisome biogenesis (Jourdain et al., 2008) but nothing is known about their other cellular roles. Fission yeast cells contain ~20 small, spherical vacuoles that undergo fission or fusion in response to environmental signals (Bone et al., 1998). S. pombe cells lacking Vps1 had smaller vacuoles with reduced capacity for fusion in response to hypotonic stress but enhanced fission in response to hypertonic conditions. Unlike wild type, vps1Δ vacuoles showed no change in diameter in response to temperature stress. Vps1-Cgfp localised to the vacuolar membrane both in living cells and in isolated vacuoles. vps1Δ cells showed close to wild type levels of vacuole protein processing and normal actin organisation and endocytosis. Overexpression of Vps1 caused a global transformation of vacuoles from spherical to tubular. Spherical vacuoles were restored by repression of vps1 expression or by induction of vacuole fusion. Tubulation was blocked in the presence of GTPγS and in a vps1 mutant that lacked the entire GTPase domain. Vacuole tubulation was more extensive in the absence of a second DRP, Dnm1. The absence of Dnm1 abolished the hyper fission phenotype of vps1Δ, whereas overexpression of Dnm1 induced vacuole fission. These results are consistent with a model of vacuole fission in which Vps1 creates a tubule of an appropriate diameter for subsequent scission by another DRP. Preliminary evidence suggests that Dnm1 serves the latter role.
Molecular dynamics simulations of protein-membrane interactions focusing on PI3Kα and its oncogenic mutants : a thesis presented in fulfilment of the requirements for the degree of Doctor of Philosophy in Computational Biochemistry at Massey University, Albany, New Zealand
(Massey University, 2017) Irvine, William A
The interactions between proteins and membranes are key to many aspects of biological function. Molecular dynamics simulations can provide insight into both atomic-level structural details and energetics of protein-membrane interactions. This thesis describes the development of a physiologically accurate brain lipid bilayer, and its use in molecular dynamics simulations to characterise how proteins that are important drug targets interact with the cell membrane. A method for rapidly identifying the orientation of a protein that interacts most favourably with a membrane was also developed and tested. The first chapter provides an introduction to molecular dynamics and its role in the context of this research. The second chapter details the development of a cellular membrane with a physiologically representative brain lipid composition. This was done through the testing of simple systems prior to the construction of two more complex lipid bilayers comprising phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositide 4,5 bisphosphate (PIP2), sphingomyelin, and cholesterol. The third chapter implements the brain lipid bilayer in the development of a rotational interaction energy screening method designed to predict the most favourable orientation of a protein with respect to the cellular membrane. The functionality of the method was validated through application to two membrane proteins commonly implicated in cancer: the phosphatase and tensin homolog (PTEN), and the p110α-p85α phosphatidyl-inositol kinase (PI3Kα) complex. The fourth chapter corresponds to the main focus of this research, the behaviour of wild type PI3Kα and two of its oncogenic mutants (E545K and H1047R) with regards to membrane and substrate interaction. It was primarily found that H1047R’s increased membrane affinity allowed it to sample a catalytically competent orientation independently of Ras, unlike the wild type. Furthermore, it was also found that the position of the C terminal tail with regards to the substrate binding pocket was crucial in the achievement of a catalytically competent position against the cellular membrane. The fifth and final chapter describes a cytochrome P450 system embedded in a cellular membrane. It was primarily found that the properties of its ingress and egress tunnels depended on the presence or absence of a substrate in the active site.

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