Reconstitution of Caveolin-1 into Artificial Lipid Membrane: Characterization by Transmission Electron Microscopy and Solid-State Nuclear Magnetic Resonance

Abstract

Caveolin-1 (CAV1), a membrane protein that is necessary for the formation and maintenance of caveolae, is a promising drug target for the therapy of various diseases, such as cancer, diabetes, and liver fibrosis. The biology and pathology of caveolae have been widely investigated; however, very little information about the structural features of full-length CAV1 is available, as well as its biophysical role in reshaping the cellular membrane. Here, we established a method, with high reliability and reproducibility, for the expression and purification of CAV1. Amyloid-like properties of CAV1 and its C-terminal peptide CAV1(168-178) suggest a structural basis for the short linear CAV1 assemblies that have been recently observed in caveolin polyhedral cages in Escherichia coli (E. coli). Reconstitution of CAV1 into artificial lipid membranes induces a caveolae-like membrane curvature. Structural characterization of CAV1 in the membrane by solid-state nuclear magnetic resonance (ssNMR) indicate that it is largely α-helical, with very little β-sheet content. Its scaffolding domain adopts a α-helical structure as identified by chemical shift analysis of threonine (Thr). Taken together, an in vitro model was developed for the CAV1 structural study, which will further provide meaningful evidences for the design and screening of bioactive compounds targeting CAV1.