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Abstract:
Membrane proteins, including G protein-coupled receptors (GPCRs), present a challenge in studying their structural properties under physiological conditions. Moreover, to better understand the activity of proteins requires examination of single molecule behaviors rather than ensemble averaged behaviors. Force–distance curve-based AFM (FD-AFM) was utilized to directly probe and localize the conformational states of a GPCR within the membrane at nanoscale resolution based on the mechanical properties of the receptor. FD-AFM was applied to rhodopsin, the light receptor and a prototypical GPCR, embedded in native rod outer segment disc membranes from photoreceptor cells of the retina in mice. Both FD-AFM and computational studies on coarse-grained models of rhodopsin revealed that the active state of the receptor has a higher Young's modulus compared to the inactive state of the receptor. Thus, the inactive and active states of rhodopsin could be differentiated based on the stiffness of the receptor. Differentiating the states based on the Young's modulus allowed for the mapping of the different states within the membrane. Quantifying the active states present in the membrane containing the constitutively active G90D rhodopsin mutant or apoprotein opsin revealed that most receptors adopt an active state. Traditionally, constitutive activity of GPCRs has been described in terms of two-state models where the receptor can achieve only a single active state. FD-AFM data are inconsistent with a two-state model but instead require models that incorporate multiple active states.

Keywords:
nanoindentation, rhodopsin, GPCR, membrane, biophysics, AFM, active, inactive, molecular dynamics, coarse graining, go-like model

Abstract:
Several membrane proteins, including G protein-coupled receptors (GPCRs), present a challenge in studying their structural and dynamical properties under physiological conditions. Moreover, to better understand the activity of proteins requires examination of single molecule behaviors rather than ensemble averaged behaviors. In this work we report the Force-distance curve-based AFM (FD-AFM) which was utilized to directly probe and localize the confor- mational states of a GPCR within an artificial membrane at nanoscale resolution. We have further validated the experimental results by molecular scale coarse-grained (CG) simulations of rhodopsin biomolecules. In the past, our CG model has been applied successfully for the study of the mechanical properties of large biological assemblies such as b-amyloid and a-synuclein fibrils. Both FD-AFM experimental results and the computational force profiles revealed that the active state of the receptor has a higher Young’s modulus compared to the inactive state of the receptor. We show how the deformation of the hydrogen bond network triggers this difference and by the statistical analysis of the native contacts we highlight the underlying mechanism. Hence, the inactive and active states of rhodopsin could be differentiated based on the stiffness of the receptor. Our work paves the route towards the molecular characterization of protein states based on the Young's modulus, which is clear indication of the mechanochemical interplay of proteins within the cell membrane.

Keywords:
Nanomechanics, GPCR. lipids, molecular dynamics, coarse graining, Go-like model, indentation, AFM, two-state model