![]() ![]() We constructed the reaction pathway of conformational changes between the two-end structures. In the current study, we utilized a coarse-grained (CG) model to investigate three representative membrane protein systems: the TMEM16A channel, the family C GPCRs mGlu2 receptor, and the P4-ATPase phospholipid transporter. Membrane proteins carry out vital physiological functions through conformational changes. There is a significant need to deeply understand the working mechanism of membrane proteins in detail, which is a challenging work due to the lack of available membrane structures and their large spatial scale. Approximately 60% of known drug targets are membrane proteins. Membrane proteins play crucial roles in various physiological processes, including molecule transport across membranes, cell communication, and signal transduction. Our results set the basis for the investigation of the unfolding of membrane proteins in situ, and for performing proteomics from a membrane fragment. We validated our pipeline by overexpressing 4 constructs, and this allowed us to gather structural insights of the identified proteins, revealing variable elements in the loop regions. The pipeline was tested with purified/reconstituted proteins and applied to five cell types where we classified the unfolding of their most abundant membrane proteins. We characterized and identified the embedded membrane proteins combining, within a Bayesian framework, the information of the shape of the obtained curves, with the information from Mass Spectrometry and proteomic databases. Here, we describe a pipeline for analyzing membrane proteins based on SMFS, that involves the isolation of the plasma membrane of single cells and the harvesting of force-distance curves directly from it. SMFS has been mostly used to study the unfolding of purified proteins, in solution or reconstituted in a lipid bilayer. ![]() The obtained force-distance curve encodes the unfolding pathway, and from its analysis it is possible to characterize the folded domains. Single-molecule force spectroscopy (SMFS) uses the cantilever tip of an AFM to apply a force able to unfold a single protein. ![]()
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