Oligomeric distribution of membrane proteins in native-membrane environment at nanoscale spatial and single-molecule resolution.

Abstract

Membrane proteins comprise ∼1/4 the human proteome and play indispensable roles in cellular physiology. Recent studies have highlighted the role of oligomerization and clustering in membrane protein-mediated signaling and physiological responses. The protein-centricity of experimental biophysics has led to a view of membrane protein function devoid of two-dimensional cell membranes, which play pivotal roles in their regulation, structure, oligomerization, and function. Quantitation of membrane protein oligomeric states directly from native-membrane environments remains challenging. Some of the main problems are: an inability to preserve the native-membrane environment while attaining precise spatial and high molecular resolution, insufficient sensitivity to detect and analyze membrane proteins at endogenous levels of expression, and dependence on bulk analyses that lack single-molecule information. We propose a single-molecule total internal reflection fluorescence microscopy (TIRF)-based imaging assay to quantify membrane protein oligomeric states directly from native-membrane environments at nanoscale spatial (∼10 nm) and single-molecule resolution. We benchmarked and validated this experimental platform using several membrane proteins with well-established oligomeric states. We then applied this method to quantify as yet unknown oligomeric distributions of diverse membrane-proteins from E. Coli, mammalian cells, and patient-derived cell lines expressing target proteins at endogenous levels. We combined this innovative experimental approach with mutagenesis, truncations, antibody/nanobody binding, and inhibitor/drug treatments to (1) identify changes in oligomeric states and (2) map oligomeric interfaces in membrane proteins. We finally correlated our results with membrane-localized signaling to connect the physical modulation of protein oligomeric states to physiological outcomes. Our approach can be extended to identify compounds/antibodies that can modulate oligomeric distributions to ablate/alter signaling in disease-relevant membrane proteins. This general experimental pipeline ushers in a new era of studying membrane proteins in their native-membrane environments at an unprecedented spatial and molecular resolution.