| Summary: | <p>Integral membrane proteins (IMPs) and their interactions form the basis underlying many processes essential for life of all living organisms. Despite their pivotal importance, they are challenging to study due to the presence of a bilayer-embedded transmembrane domain requiring astute strategies to achieve solubilisation in aqueous environments. Here we present mass photometry as a novel technique to investigate IMPs and their interactions in a variety of membrane mimetic systems, uncovering the interactions underlying the bacterial outer membrane architecture. We then apply mass photometry to viral membrane glycoproteins, the HIV-1 envelope spike and SARS-CoV-2 spike, and their interactions with a soluble inhibiting protein and host cell receptor, respectively. In both cases, we describe how the oligomeric nature of the involved proteins facilitates intermolecular crosslinking, facilitating enhanced inhibition.</p>
<p>We begin by establishing the applicability of mass photometry to measure a variety of membrane mimetic systems, including detergents, by determining the molecular weight of different IMPs in detergents, amphipols, lipid nanodiscs,
and native nanodiscs. We further show the heterogeneity stemming from native membrane protein extraction with native nanodiscs, as well as the utility of mass photometry in predicting IMP functionality in different lipid nanodisc assemblies. We apply these findings to characterise the architecture and interactions between the IMP of the Gram-negative bacterial outer membrane, providing important insights into the outer membrane island formation and contributions to antibiotic resistance.</p>
<p>Next, we focus on the interactions of the HIV-1 envelope spike glycoprotein with its natural inhibitor, banana lectin BanLec. Through careful mass photometry measurements, we determine the dynamics of this multivalent interaction, resulting in the formation of highly heterogeneous species through intermolecular crosslinking. This approach is then applied to investigate the mechanism behind SARS-CoV-2 infection. We measure the interactions between the viral spike and its human host cell receptor, ACE2, showing the importance of its dimeric nature for achieving its full neutralisation potential through spike crosslinking.</p>
<p>This work introduced mass photometry as an important tool for membrane protein research with broad applications in protein-protein interactions as well as in characterisation of complex heterogeneous reactions important in viral infections.</p>
|
|---|