As such, mass photometry could be ideally suited to address the shortcomings of existing fluorescence-based techniques for in vitro applications preciso studying IMPs and MAPs
State-of-the-art single-molecule approaches rely largely on the additif 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, verso method for label-free imaging, tracking and mass measurement of individual membrane-associated proteins diffusing on supported lipid bilayers. Application of this method preciso 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 durante and on lipid bilayers.
The quantification of membrane-associated biomolecular interactions is crucial sicuro our understanding of various cellular processes
Integral membrane proteins (IMPs) and membrane-associated proteins (MAPs) are essential for per number of cellular processes such as signaling and vesicular trafficking, and this makes them important therapeutic targets 1,2 . Their function often relies on homo- and hetero-oligomerization 3,4 , and this complexity, combined with the need for lipid bilayers, makes it particularly challenging to accurately characterize the stoichiometries and kinetics of the biomolecular interactions underlying IMP and MAP function and regulation. Advances per celibe-molecule fluorescence-based microscopy methods 5,6 have enabled in vivo and durante vitro investigations of IMP interactions, such as dimerization of G-protein-coupled receptors 7,8 and basso-clustering 9 , and MAP interactions, such as the coordination of Min proteins during bacterial cell division 10 , and the mechanism of amyloid-? come per vedere chi si ama sul xpress senza pagare plaque formation on cell membranes, which is associated with Alzheimer’s disease 11 . The main challenges esatto fluorescence-based methods, however, arise from quantitative uncertainties caused by incomplete labeling of the sample, photochemical and photophysical effects such as photoblinking, photobleaching and quenching, and the distinct labeling required to detect multiple species simultaneously. These limitations have made it challenging to accurately quantify processes such as membrane (un)binding of MAPs and the dynamics and stoichiometries of protein–protein interactions for both MAPs and IMPs. Although numerous approaches aimed at molecular subunit counting exist 12,13,14 , the analysis and interpretation of the resulting oligomeric distributions is complicated and the number of heterogeneous species that can be detected simultaneously remains limited. Given the critical functional importance of homo- and hetero-oligomeric interactions for membrane-associated processes, there is an urgent need for per quantitative and dynamic approach that is athletique of complementing the information accessible from existing methods.
Mass photometry is per label-free method that detects celibe biomolecules con solution and measures their mass with an overall mass accuracy and resolution of 2% and 20 kDa, respectively 15 . These capabilities enable the quantification of protein–protein interactions sopra solution with sufficient sensitivity preciso accurately determine stoichiometry and rate of reactions 16 . Existing implementations of mass photometry rely on the stationary binding of individual molecules to verso surface, usually per glass coverslip. By averaging images taken before a binding event and subtracting them from averaged images taken after verso binding event, the signal due sicuro glass surface roughness is removed and the shot noise is lowered sufficiently sicuro detect individual molecules binding esatto the surface 17,18,19 . When molecules remain trasportabile after binding onesto the surface, however, the resulting signals are a convolution of the positions of the molecules over the averaged time frame, which makes their detection and quantification difficult. Here, by implementing a new background processing methodology, we spettacolo that the capabilities of mass photometry can be extended onesto durante vitro studies of individual protein complexes diffusing on supported lipid bilayers (SLBs).