Differentiating between Inactive and Active States of Rhodopsin by Atomic Force Microscopy in Native Membranes

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 coarsegrained 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 *

Quantitative determination of mechanical stability in the novel coronavirus spike protein

“…To further demonstrate the mechanical stability of the RBD, we employed a validated structure-based modelling approach. 15,28,29,31,32 Our computational structure-based study (see Fig. 3) shows that the RBD is a mechanically stable component in the spike (see next section) and also identifies the structural motif (see Fig.…”

In-cell detection of conformational sub-states of a GPCR quaternary structure: Modulation of sub-state probability by cognate ligand binding

“…Early hypotheses regarding possible GPCR tertiary structures depicted the receptor as a simple switch which existed in either an inactive or active state. However, a growing body of evidence suggests that activation of GPCRs is not a binary operation, but rather that the tertiary structure conformation can exist in a series of intermediate states 50 , with a range of activity levels possible across the distribution of conformations as shown by fluorescence-based techniques 30,73 , FNMR 32 , AFM 74 , electron microscopy 75 , X-ray crystallographic studies 38 , and theoretical simulations using crystal structures 2,33,[76][77] . From crystal structure studies of individual GPCRs, it has been observed that the intracellular region of TM1, TM3, TM5, TM6, and TM7 move outward from one another when ligand is bound 2 , with TM6…”

Pathways and disease-causing alterations in visual chromophore production for vertebrate vision

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