Label-free tracking and mass measurement of single protein complexes on lipid bilayers

Thesis

Protein-protein interactions are essential for biological processes, but can display a level of polydispersity that is difficult to study with bulk measurements. Recently, mass photometry, a label-free single molecule imaging technique, has been used to quantify protein-protein interactions by measuring the mass of single proteins. So far, however, measurements are limited to solution-based systems and nM concentrations. Here, we aim to expand the applications of mass photometry to proteins on lipid bilayers, and to increase the concentration range of solution-based measurements.

We implement a surface passivation protocol that reduces non-specific binding by up to four orders of magnitude, and apply further surface function- alisation strategies aimed at accessing measurements at μM concentrations. Next, we develop a mass photometry-based method for tracking individual protein complexes on lipid bilayers, thereby demonstrating quantification of oligomer-dependent kinetics and mobilities. We then extend this method to interactions of the HIV-1 envelope glycoprotein (Env) with broadly neutral- ising antibodies. Using our assay, we resolve polydisperse anitbody-induced cross-linking and quantify the corresponding binding affinities. We show that lipid bilayers give access to mass photometry measurements at μM protein concentrations, and demonstrate specific pull-down of target proteins from complex mixtures. Finally, we discuss aspects of the data analysis for these assays with a specific focus on systematic errors in the diffusion and mass measurements, and outline strategies for their correction.

Authors: Foley, EDB

• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: single molecule pull-down; optical microscopy; surface passivation; single particle tracking; lipid bilayers; mass photometry lipid; HIV
• Subjects: Microscopy; HIV antibodies; Bilayer lipid membranes; Biophysics