Eric D. B. Foley

Determining the identity, quantity and interactions of proteins is fundamental to studies of biomolecular function and (dis)regulation in disease. Here, we combine mass photometry with supported lipid bilayers (SLBs) to specifically recruit proteins of interest from complex mixtures such as cell lysate and demonstrate sensitive detection and mass measurement of single biomolecules and their complexes without prior purification. Extending to heterologously expressed proteins, we provide rapid information on oligomerisation and concentration in lysate. Introduction of polyethylene glycol in the SLBs passivates against a variety of cell lysates and serum, allows specific recruitment of proteins from complex mixtures and mass-specific detection and affinity measurement at sub-nM concentrations. Our approach preserves the mass resolution and accuracy of mass photometry, thereby enabling visualisation and quantification of oligomeric states, interactions and complexes directly from cell lysates.

The quantification of membrane-associated biomolecular interactions is crucial to our understanding of various cellular processes. State-of-the-art single-molecule approaches rely largely on the addition of fluorescent labels, which complicates the quantification of the involved stoichiometries and dynamics because of low temporal resolution and the inherent limitations associated with labeling efficiency, photoblinking and photobleaching. Here, we demonstrate dynamic mass photometry, a method for label-free imaging, tracking and mass measurement of individual membrane-associated proteins diffusing on supported lipid bilayers. Application of this method to the membrane remodeling GTPase, dynamin-1, reveals heterogeneous mixtures of dimer-based oligomers, oligomer-dependent mobilities, membrane affinities and (dis)association of individual complexes. These capabilities, together with assay-based advances for studying integral membrane proteins, will enable the elucidation of biomolecular mechanisms in and on lipid bilayers.

We introduce dynamic mass photometry, a method for label-free imaging, tracking and mass measurement of membrane-associated proteins. Our method enables quantitative studies of their mobility, membrane affinity and interactions at the single molecule level. Application to the membrane remodelling GTPase dynamin1 reveals heterogeneous mixtures of oligomers suggesting that the fundamental building block for oligomerisation is a dimer, challenging current tetramer-centric models. Dynamic mass photometry has the ability to transform our approach to studying biomolecular mechanisms in and on lipid bilayers.

Interactions between biomolecules control the processes of life in health and their malfunction in disease, making their characterization and quantification essential. Immobilization‐ and label‐free analytical techniques are desirable because of their simplicity and minimal invasiveness, but they struggle with quantifying tight interactions. Here, we show that mass photometry can accurately count, distinguish by molecular mass, and thereby reveal the relative abundances of different unlabelled biomolecules and their complexes in mixtures at the single‐molecule level. These measurements determine binding affinities over four orders of magnitude at equilibrium for both simple and complex stoichiometries within minutes, as well as the associated kinetics. These results introduce mass photometry as a rapid, simple and label‐free method for studying sub‐micromolar binding affinities, with potential for extension towards a universal approach for characterizing complex biomolecular interactions.

Interactions between biomolecules control the processes of life in health and their malfunction in disease, making their characterization and quantification essential. Immobilization‐ and label‐free analytical techniques are desirable because of their simplicity and minimal invasiveness, but they struggle with quantifying tight interactions. Here, we show that mass photometry can accurately count, distinguish by molecular mass, and thereby reveal the relative abundances of different unlabelled biomolecules and their complexes in mixtures at the single‐molecule level. These measurements determine binding affinities over four orders of magnitude at equilibrium for both simple and complex stoichiometries within minutes, as well as the associated kinetics. These results introduce mass photometry as a rapid, simple and label‐free method for studying sub‐micromolar binding affinities, with potential for extension towards a universal approach for characterizing complex biomolecular interactions.

The first in-depth, systematic study of the photoswitching properties of Donor–Acceptor Stenhouse Adducts (DASAs) is reported. Barbituric acid derived DASAs functionalised with 14 different amines ranging from dimethylamine to 4-methoxy-N-methylaniline were structurally characterised in solution using ¹H and ¹³C NMR spectroscopy and, in eight cases, in the solid state by single crystal X-ray diffraction. The distribution of coloured and colourless isomers in the dark, their photostationary states under irradiation, apparent thermal half-lives, and fatigue resistance are systematically compared. A simple kinetic model is used to characterise photoswitching behaviour and reveals that minor structural modifications can significantly improve the photoswitching properties of DASA photochromes. These modifications result in excellent photoswitching properties for ‘1^st generation’ DASAs in chloroform, including exceptional fatigue resistance, opening the door for these photochromic molecules to find widespread applications.