Membrane proteins enter the fold

Abstract

Membrane proteins have historically been recalcitrant to biophysical folding studies. However, recent adaptations of methods from the soluble protein folding field have found success in their applications to transmembrane proteins composed of both α-helical and β-barrel conformations. Avoiding aggregation is critical for the success of these experiments. Altogether these studies are leading to discoveries of folding trajectories, foundational stabilizing forces and better-defined endpoints that enable more accurate interpretation of thermodynamic data. Increased information on membrane protein folding in the cell shows that the emerging biophysical principles are largely recapitulated even in the complex biological environment.

Figure 1. Membrane protein folding and stability flow chart.

(a) The relevant thermodynamic equilibria describing membrane protein stability and the experimental approaches used to measure each free energy are shown. $\Delta G_{U_W,F}^{\circ }$ describes the coupled folding and insertion of an unfolded, water-soluble membrane protein into the bilayer and is calculated from chemical denaturation titrations of β-barrels (PDB: 1QD5). This approach has been used to investigate side-chain transfer free energies [–5,8,13,53] and folding transition states [3]. $\Delta G_{U_M,F}^{\circ }$ describes the association/folding of helices in a membrane unfolded state and has been measured using both steric trapping [20,23] and single-molecule force spectroscopy [29,30]. $\Delta G_{Olig}^{\circ }$ describes the oligomerization of membrane proteins and is currently measured using single-molecule fluorescence photobleaching (PDB: 3Q17) [25,26]. (b) The growing knowledge of the thermodynamic parameters that define membrane protein folding and structure have led to the successful design of functional membrane proteins (PDBs: 6TMS (left) and 6MCT (right)) [27,35]. (c) In vitro-derived parameters of membrane protein stability (Panel A) have also been applied to membrane protein folding. Model systems for investigating folding in vivo include CFTR (PDB: 5UAK), PMP22 [54], and rhodopsin (PDB: 1L9H). The residues for each protein that have been discussed here are shown with a space-filling representation and are colored red. For rhodopsin, the entire TM7 helix has been investigated using deep mutational scanning [43]. For each system, the general trend is that stability is correlated with the surface expression of each membrane protein.