Kubicek, J. | bioRxiv Science

bioRxiv2025

Cryo-EM structure of a cell-free synthesized full-length human β1-adrenergic receptor in complex with Gs

The third intracellular loop (ICL3) of the {beta}1-adrenergic receptor ({beta}1AR) plays a critical role in regulating G protein coupling, yet the structural basis has remained unclear due to truncations of ICL3 in all available structures of the {beta}1AR in complex with Gs or a G protein mimetic nanobody. To address this, we used cell-free cotranslational insertion of full-length human {beta}1AR into nanodiscs and determined its cryo-EM structure in complex with Gs. In this structure, ICL3 extends transmembrane helix 5, resulting in enhanced interactions with Gs and in a slight rotation of the engaged G protein. This repositioning enables new polar interactions between Gs, ICL2 and helix 8, while ICL1 and helix 8 form additional contacts with G{beta}. These structural insights, supported by mutational analysis, demonstrate that ICL3 enhances G protein activation and downstream cAMP signaling by promoting more extensive interactions between the receptor and the heterotrimeric G protein.

biochemistry↗

bioRxiv2023

The bigger picture: global analysis of solubilization performance of classical detergents versus new synthetic polymers utilizing shotgun proteomics

Integral membrane proteins are critical for many cellular functions. Roughly 25% of all human genes code for membrane proteins, and about 70% of all approved drugs target them. Despite their importance, laborious and harsh purification conditions often hinder their characterization. Traditionally, they are removed from the membrane using detergents, thereby taking the proteins out of their native environment, affecting their function. Recently, a variety of synthetic polymers have been introduced, which can extract membrane proteins together with their native lipids into a so-called native nanodisc. However, they usually show lesser solubilization capacity than detergents, and their general applicability for membrane protein biochemistry is poorly understood. Here, we used Hek293 cell membrane extracts and LC-MS-based proteomics to compare the ability of nanodisc-forming polymers against state-of-the- art detergents to solubilize the membrane proteome. Our data demonstrates the general ability of synthetic co-polymers to extract membrane proteins, rivaling the efficacy of commonly used detergents. Interestingly, each class of solubilization agent presents specific solubilization profiles. We found no correlation between efficiency and number of transmembrane domains, isoelectric point, or GRAVY score for any compound. Our data shows that these polymers are a versatile alternative to detergents for the biochemical and structural study of membrane proteins, functional proteomics, or as components of native lysis/solubilization buffers. Our work here represents the first attempt at a proteome-scale comparison of the efficacy of nanodisc-forming polymers. These data should serve as starting reference for researchers looking to purify membrane proteins in near native conditions.

biochemistry↗