Single-molecule force spectroscopy of G-protein-coupled receptors

Abstract

The applicability of single-molecule force spectroscopy (SMFS) to characterize membrane proteins in vitro is developing rapidly and opening a wide range of fascinating possibilities to study how intra- and intermolecular interactions determine their structural stability and functional state. In particular, understanding how molecular interactions contribute to the functional state of G-protein-coupled receptors (GPCRs) is of importance because they mediate most of our physiological responses and act as therapeutic targets for a broad spectrum of diseases. In our review we focus on SMFS to characterize GPCRs embedded in their physiologically relevant membranes and exposed to physiologically relevant conditions. SMFS uses a molecularly sharp stylus to grasp the terminal end of a GPCR and to quickly unfold the receptor while recording interaction forces. The positional accuracy of SMFS localizes these interactions to structural segments of the GPCR whereas the sensitivity of SMFS enables their stabilizing interaction forces to be quantified. To further investigate the kinetic, energetic and mechanical properties of the structural segments, dynamic SMFS (DFS) probes their stability over a wide range of loading rates. These parameters provide insight into the energy landscape that provides information on the structural and functional properties of the GPCRs. Selected highlights exemplify the application of SMFS to characterize inter- and intramolecular interactions, which change the properties of GPCRs in relation to their functional state (e.g., ligand binding), diseased state (e.g., mutation), or lipid environment such as cholesterol.

Imaging G protein–coupled receptors while quantifying their ligand-binding free-energy landscape

“…Because SMFS spectra recorded upon unfolding of PAR1 closely resembled those previously recorded for the human β 2 -adrenergic receptor (β 2 AR) 12 , a class A GPCR showing high structural homology to PAR1 (ref. 13), we concluded that PAR1 folded correctly into the proteoliposomes 14 . For FD-based AFM we imaged PAR1 proteoliposomes adsorbed onto freshly cleaved mica in buffer solution at 37 °C ( Fig.…”

Seeing and sensing single G protein-coupled receptors by atomic force microscopy

“…AFM-based SMFS characterizes the mechanical and kinetic stability of the structural intermediates of GPCRs at the resolution of single α-helices [ 34 ] or a few amino acids [ 35 • ]. In a nutshell, the tip of the AFM cantilever acts as a ‘sticky finger’ to pick up single membrane receptors from either terminal end.…”

Force spectroscopy reveals the presence of structurally modified dimers in transthyretin amyloid annular oligomers

“…Differences in the extent with which unfolding intermediates are observed in aoTTR in comparison to nTTR are suggestive of considerable folding differences, and also of stabilization of unfolding intermediates of the aoTTR dimer by its neighboring environment. To illustrate this last point, we may take the example of 2 different classes of α‐helical proteins: while membrane‐embedded G‐protein coupled receptors tend to unfold via the stepwise unraveling of their individual transmembrane α‐helices, soluble α‐helical bundles of spectrin unfold in a single event . Likewise, mechanical unfolding of β‐barrel membrane proteins such as OmpG reveals multiple unfolding intermediates while the water‐soluble β‐barrel GFP does not .…”

“…The double-stack arrangement of aoTTR likely contributes to stabilize unfolding intermediates. D, Diagram of the assembly of annular oligomers with monomers oriented in a head-to-head and tail-to-tail configuration forming strong and weakly interacting interfaces of α-helical proteins: while membrane-embedded G-protein coupled receptors tend to unfold via the stepwise unraveling of their individual transmembrane α-helices, 49 soluble α-helical bundles of spectrin unfold in a single event. 50 Likewise, mechanical unfolding of β-barrel membrane proteins such as OmpG reveals multiple unfolding intermediates while the water-soluble β-barrel GFP does not.…”