From bench to biomolecular simulation : phospholipid modulation of potassium channels

Potassium (K+) ion channels are crucial in numerous cellular processes as they hyperpolarise a cell through K+ conductance, returning a cell to its resting potential. K+ channel mutations result in multiple clinical complications such as arrhythmia, neonatal diabetes and migraines. Since 1995, the regulation of K+ channels by phospholipids has been heavily studied using a range of interdisciplinary methods such as cellular electrophysiology, structural biology and computational modelling. As a result, K+ channels are model proteins for the analysis of protein-lipid interactions. In this review, we will focus on the roles of lipids in the regulation of K+ channels, and how atomic-level structures, along with experimental techniques and molecular simulations, have helped guide our understanding of the importance of phospholipid interactions

NaViA: A Program for the Visual Analysis of Complex Mass Spectra

MOTIVATION: Native mass spectrometry is now a well-established method for the investigation of protein complexes, specifically their subunit stoichiometry and ligand binding properties. Recent advances allowing the analysis of complex mixtures lead to an increasing diversity and complexity in the spectra obtained. These spectra can be time consuming to tackle through manual assignment and challenging for automated approaches. RESULTS: Native Mass Spectrometry Visual Analyser (NaViA) is a web-based tool to augment the manual process of peak assignment. In addition to matching masses to the stoichiometry of its component subunits it allows raw data processing, assignment and annotation and permits mass spectra to be shared with their respective interpretation. AVAILABILITY: NaViA is open-source and can be accessed online under https://navia.ms. The source code and documentation can be accessed at https://github.com/d-que/navia, under the BSD 2-Clause license. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online

NaViA : a program for the visual analysis of complex mass spectra

Motivation: Native mass spectrometry is now a well-established method for the investigation of protein complexes, specifically their subunit stoichiometry and ligand binding properties. Recent advances allowing the analysis of complex mixtures lead to an increasing diversity and complexity in the spectra obtained. These spectra can be time-consuming to tackle through manual assignment and challenging for automated approaches. Results: Native Mass Spectrometry Visual Analyser is a web-based tool to augment the manual process of peak assignment. In addition to matching masses to the stoichiometry of its component subunits, it allows raw data processing, assignment and annotation and permits mass spectra to be shared with their respective interpretation. Availability and implementation: NaViA is open-source and can be accessed online under https://navia.ms. The source code and documentation can be accessed at https://github.com/d-que/navia, under the BSD 2-Clause licence. Supplementary information: Supplementary data are available at Bioinformatics online

Investigating the role of lipids for membrane protein complexes using native mass spectrometry and molecular dynamics simulations

The work described in this thesis is a combination native mass spectrometry experiments, molecular dynamics simulations and scientific software development. It consists of three results chapters which each chapter describing a distinct project with a focus on one of the aspects above. In chapter 3, I employed molecular dynamics simulations in a collaborative project on using surface-induced dissociation to probe the effect of lipid binding on the stability of oligomerisation interfaces. My simulations offer a rationale of lipid bind- ing to different locations to explain these stability differences. In addition, I contributed to experiments to probe the molecular features of these interfaces through site-directed mutagenesis to weaken these interfaces. Chapter 4 describes my investigation on lipid binding to a bacterial potassium channel, MthK. Native mass spectrometry and molecular dynamics simulations are used to characterise lipid binding after initial experiments indicated endogenous lipids being bound to MthK. Cardiolipin has been revealed to bind specifically to MthK. These results are compared to the eukaryotic homologues through further simula- tions. A part of this chapter outlines different approaches to obtain the in vivo stoichiometry. Among these approaches I employed SoLVe-MS, which leads to very complex mass spectra. The analysis of these spectra sparked the development of the program described in chapter 5. In the final results chapter, a web-based program for the analysis of native mass spectra, NaViA, is described. This program allows one to analyse native mass spectra through a visual user interface directly in a web browser. In addition, it offers auxiliary tools to facilitate the analysis as well as the ability to save and load sessions for sharing spectra including analysis or depositing them for publication. The ease of use and the instructive auxiliary tools make it a valuable addition to the suite of software available for the analysis of native mass spectrometry

Capturing a rhodopsin receptor signalling cascade across a native membrane.

G protein-coupled receptors (GPCRs) are cell-surface receptors that respond to various stimuli to induce signalling pathways across cell membranes. Recent progress has yielded atomic structures of key intermediates1,2 and roles for lipids in signalling3,4. However, capturing signalling events of a wild-type receptor in real time, across a native membrane to its downstream effectors, has remained elusive. Here we probe the archetypal class A GPCR, rhodopsin, directly from fragments of native disc membranes using mass spectrometry. We monitor real-time photoconversion of dark-adapted rhodopsin to opsin, delineating retinal isomerization and hydrolysis steps, and further showing that the reaction is significantly slower in its native membrane than in detergent micelles. Considering the lipids ejected with rhodopsin, we demonstrate that opsin can be regenerated in membranes through photoisomerized retinal-lipid conjugates, and we provide evidence for increased association of rhodopsin with unsaturated long-chain phosphatidylcholine during signalling. Capturing the secondary steps of the signalling cascade, we monitor light activation of transducin (Gt) through loss of GDP to generate an intermediate apo-trimeric G protein, and observe Gαt•GTP subunits interacting with PDE6 to hydrolyse cyclic GMP. We also show how rhodopsin-targeting compounds either stimulate or dampen signalling through rhodopsin-opsin and transducin signalling pathways. Our results not only reveal the effect of native lipids on rhodopsin signalling and regeneration but also enable us to propose a paradigm for GPCR drug discovery in native membrane environments