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 rece… Show more
“…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.…”
“…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.…”
“…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…”
ABSTRACTWhile the notion that G protein-coupled receptors (GPCRs) associate into homo- and hetero-oligomers has gained more recognition in recent years, a lack of consensus remains among researchers regarding the functional relevance of GPCR oligomerization. A relatively recent technique, Förster resonance energy transfer (FRET) spectrometry, allows for the determination of the oligomeric (or quaternary) structure of proteins in living cells via analysis of efficiency distributions of energy transferred from optically excited fluorescent tags acting as donors of energy to fluorescent tags acting as acceptors of energy and residing within the same oligomer. In this study, we significantly improved the resolution of the FRET-spectrometry approach to detect small differences between the interprotomeric distances among GPCR oligomers with subtle differences in quaternary structures. We then used this approach to study the conformational substates of oligomers of sterile 2 α-factor receptor (Ste2), a class D GPCR found in the yeast Saccharomyces cerevisiae of mating type a. Ste2 has previously been shown to form tetrameric oligomers at relatively low expression levels (between 11 and 140 molecules/μm2) in the absence of its cognate ligand, the α-factor pheromone. The significantly improved FRET spectrometry technique allowed us to detect multiple distinct quaternary conformational substates of Ste2 oligomers, and to assess how the α-factor ligand altered the proportion of such substates. The ability to determine quaternary structure substates of GPCRs provides exquisite means to elucidate functional relevance of GPCR oligomerization.
“…, rhodopsin (opsin + chromophore) (Rho) is both a light receptor and a structural protein ( 4 , 96 ). Disabling the chromophore-binding site in opsin ( 97 ) results in a constitutively active visual pigment molecule ( 98 ), but also changes the structural integrity of the unliganded opsin in the disk membranes ( 99 , 100 ), thereby leading to a gain-and-loss of function (GLF). Thus, different mutations in one gene can lead to a variety of diseases such as autosomal dominant (adRP) and autosomal recessive retinitis pigmentosa (arRP) and autosomal dominant congenital stationary blindness (CSNB) ( 101 , 102 , 103 , 104 ), all of which can be caused by mutations in the opsin gene.…”
Section: Genetic Disease-causing Changes In the Visual Cyclementioning
All that we view of the world begins with an ultrafast cis to trans photoisomerization of the retinylidene chromophore associated with the visual pigments of rod and cone photoreceptors. The continual responsiveness of these photoreceptors is then sustained by regeneration processes that convert the trans- retinoid back to an 11-cis configuration. Recent biochemical and electrophysiological analyses of the retinal G protein-coupled receptor (RGR) suggest that it could sustain the responsiveness of photoreceptor cells, particularly cones, even under bright light conditions. Thus, two mechanisms have evolved to accomplish the re-isomerization: one involving the well-studied retinoid isomerase (RPE65), and a second photoisomerase reaction mediated by the RGR. Impairments to the pathways that transform all- trans-retinal back to 11-cis-retinal are associated with mild to severe forms of retinal dystrophy. Moreover, with age there also is a decline in the rate of chromophore regeneration. Both pharmacological and genetic approaches are being used to bypass visual cycle defects and consequently mitigate blinding diseases. Rapid progress in the use of genome editing also is paving the way for the treatment of disparate retinal diseases. In this review, we provide an update on visual cycle biochemistry and then discuss visual cycle-related diseases and emerging therapeutics for these disorders. There is hope that these advances will be helpful in treating more complex diseases of the eye, including age-related macular degeneration (AMD).
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