Reconstitution and real-time quantification of membrane remodeling by single proteins and protein complexes.

ABSTRACT: Cellular membrane processes, from signal transduction to membrane fusion and fission, depend on acute membrane deformations produced by small and short-lived protein complexes working in conditions far from equilibrium. Real-time monitoring and quantitative assessment of such deformations are challenging; hence, mechanistic analyses of the protein action are commonly based on ensemble averaging, which masks important mechanistic details of the action. In this protocol, we describe how to reconstruct and quantify membrane remodeling by individual proteins and small protein complexes in vitro, using an ultra-short (80- to 400-nm) lipid nanotube (usNT) template. We use the luminal conductance of the usNT as the real-time reporter of the protein interaction(s) with the usNT. We explain how to make and calibrate the usNT template to achieve subnanometer precision in the geometrical assessment of the molecular footprints on the nanotube membrane. We next demonstrate how membrane deformations driven by purified proteins implicated in cellular membrane remodeling can be analyzed at a single-molecule level. The preparation of one usNT takes ~1 h, and the shortest procedure yielding the basic geometrical parameters of a small protein complex takes 10 h.

SUBMITTER: Bashkirov PV 

PROVIDER: S-EPMC10839814 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature