Distance Mapping in Proteins Using Fluorescence Spectroscopy: Tyrosine, like Tryptophan, Quenches Bimane Fluorescence in a Distance-Dependent Manner

Brunette, Amber M. Jones; Farrens, David

doi:10.1021/bi500493r

Cited by 44 publications

(52 citation statements)

References 49 publications

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“…The Ops* state was monitored by tracking the large movement of transmembrane helix 6 (TM6) that occurs during receptor activation, using the Trp-induced quenching (TrIQ) technique (17). TrIQ monitors the quenching of the small fluorescent probe bimane, which only occurs when the Trp and probe are in near contact (18).…”

Section: Methods For Simultaneously Measuring Retinal Release and The mentioning

confidence: 99%

Decay of an active GPCR: Conformational dynamics govern agonist rebinding and persistence of an active, yet empty, receptor state

Schafer

Fay

Janz

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Here, we describe two insights into the role of receptor conformational dynamics during agonist release (all-trans retinal, ATR) from the visual G protein-coupled receptor (GPCR) rhodopsin. First, we show that, after light activation, ATR can continually release and rebind to any receptor remaining in an active-like conformation. As with other GPCRs, we observe that this equilibrium can be shifted by either promoting the active-like population or increasing the agonist concentration. Second, we find that during decay of the signaling state an active-like, yet empty, receptor conformation can transiently persist after retinal release, before the receptor ultimately collapses into an inactive conformation. The latter conclusion is based on timeresolved, site-directed fluorescence labeling experiments that show a small, but reproducible, lag between the retinal leaving the protein and return of transmembrane helix 6 (TM6) to the inactive conformation, as determined from tryptophan-induced quenching studies. Accelerating Schiff base hydrolysis and subsequent ATR dissociation, either by addition of hydroxylamine or introduction of mutations, further increased the time lag between ATR release and TM6 movement. These observations show that rhodopsin can bind its agonist in equilibrium like a traditional GPCR, provide evidence that an active GPCR conformation can persist even after agonist release, and raise the possibility of targeting this key photoreceptor protein by traditional pharmaceutical-based treatments.T he superfamily of G protein-coupled receptors (GPCRs) is one of the largest targets of pharmaceutical drugs in the human genome. Classically, GPCR signaling occurs when a diffusible ligand (such as a drug) binds to the receptor and stabilizes conformations that can couple with and activate intracellular proteins. Our understanding of this process has built on the classical "ternary complex" model of receptor-ligand-G protein interaction (1), a model that, with revisions, has continued to guide our knowledge of how this critical event occurs.However, this paradigm has faced problems when applied to rhodopsin, the dim-light visual receptor. Rhodopsin is kept in an "off" state by a covalently bound inverse agonist, 11-cis retinal (11CR). Light converts the 11CR to an agonist, all-trans retinal (ATR), which enables the receptor to activate its G protein, transducin (G t ) (2, 3). The active receptor, metarhodopsin II (MII), continues signaling until the Schiff base linking ATR to the receptor is hydrolyzed, resulting in the release of ATR and the decay of MII into an inactive apoprotein, opsin (4, 5). Binding of a new 11CR to opsin reforms the dark state (DS), enabling another round of photon detection (6).Due to this unusual light-activated, covalently bound ligand, rhodopsin has usually been considered "different" from the larger superfamily of diffusible ligand-binding GPCRs. However, we recently discovered that rhodopsin behaves more like a traditional ligand-binding GPCR than previously thought (7). Our expe...

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Section: Methods For Simultaneously Measuring Retinal Release and The mentioning

confidence: 99%

Decay of an active GPCR: Conformational dynamics govern agonist rebinding and persistence of an active, yet empty, receptor state

Schafer

Fay

Janz

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…26 Recent developments in the field of site-directed fluorescence labeling (SDFL) have made it possible to overcome this problem and investigate the dynamics of proteins within the range of 7−15 Å. One such approach employs either tryptophan-induced quenching (TrIQ) or tyrosine-induced quenching (TyrIQ) to certain fluorescent probes, 27,28 processes that occur efficiently only in the distance range of 7−15 Å, presumably through a photoinduced electron transfer (PET) process. 29,30 In this study, we used the TrIQ method 27 to investigate the activation mechanism of TL lipase and certain lid variants with FAEA character.…”

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confidence: 99%

The Enzymatic Activity of Lipases Correlates with Polarity-Induced Conformational Changes: A Trp-Induced Quenching Fluorescence Study

et al. 2015

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Triacylglycerol hydrolases (EC 3.1.1.3) are thought to become activated when they encounter the water-lipid interface causing a "lid" region to move and expose the catalytic site. Here, we tested this idea by looking for lid movements in Thermomyces lanuginosus lipase (TL lipase), and in variants with a mutated lid region of esterase (Esterase) and esterase/lipase (Hybrid) character. To measure lid movements, we employed the tryptophan-induced quenching (TrIQ) fluorescence method to measure how effectively a Trp residue on the lid of these mutants (at position 87 or 89) could quench a fluorescent probe (bimane) placed at nearby site 255 on the protein. To test if lid movement is induced when the enzyme detects a lower-polarity environment (such as at the water-lipid interface), we performed these studies in solvents with different dielectric constants (ε). The results show that lid movement is highly dependent on the particular lid residue composition and solvent polarity. The data suggest that in aqueous solution (ε = 80), the Esterase lid is in an "open" conformation, whereas for the TL lipase and Hybrid, the lid remains "closed". At lower solvent polarities (ε < 46), the lid region for all of the mutants is more "open". Interestingly, these behaviors mirror the structural changes thought to take place upon activation of the enzyme at the water-lipid interface. Together, these results support the idea that lipases are more active in low-polarity solvents because the lid adopts an "open" conformation and indicate that relatively small conformational changes in the lid region play a key role in the activation mechanism of these enzymes.

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“…7C, integrated fluorescence intensities (F 0 and F w ) of the mutant G16I and G23I were shown respectively. For the RH2 model, F 0 /F w equaled 1 after adding W-G16I in mBBr-G16I, no fluorescence quenching occurs, whereas for the RH1 model, F 0 /F w equaled 1.3 after adding W-G23I in mBBr-G23I, indicating that the C␣-C␣ distance is ϳ11 Å by calculation (46). Because the applicable C␣-C␣ distance range is 5-15 Å for tryptophan/mBBr FRET, these results were significantly consistent with the results of CG simulations (Fig.…”

Section: Two Packing Patterns Of Cd36 Tm1 Homodimer Confirmed By Fretmentioning

confidence: 96%

“…Two packing models switch in CD36 TM1 homodimer phan-induced quenching FRET method (45)(46)(47) to probe the N-terminal distances of both RH1(TM1-G23I) and RH2 (TM1-G16I) models. This method provides a straightforward and sensitive way to address questions of conformational dynamics in proteins by mBBr labeling.…”

Section: Two Packing Patterns Of Cd36 Tm1 Homodimer Confirmed By Fretmentioning

confidence: 99%

Critical residues and motifs for homodimerization of the first transmembrane domain of the plasma membrane glycoprotein CD36

Wei

Sun

Zuo

et al. 2017

Journal of Biological Chemistry

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The plasma transmembrane (TM) glycoprotein CD36 is critically involved in many essential signaling processes, especially the binding/uptake of long-chain fatty acids and oxidized lowdensity lipoproteins. The association of CD36 potentially activates cytosolic protein tyrosine kinases that are thought to associate with the C-terminal cytoplasmic tail of CD36. To understand the mechanisms by which CD36 mediates ligand binding and signal transduction, we have characterized the homo-oligomeric interaction of CD36 TM domains in membrane environments and with molecular dynamics (MD) simulations. Analysis of pyreneand coumarin-labeled TM1 peptides in SDS by FRET confirmed the homodimerization of the CD36 TM1 peptide. Homodimerization assays of CD36 TM domains with the TOXCAT technique showed that its first TM (TM1) domain, but not the second TM (TM2) domain, could homodimerize in a cell membrane. Smallresidue, site-specific mutation scanning revealed that the CD36 TM1 dimerization is mediated by the conserved small residues Gly CD36 is a transmembrane glycoprotein receptor with many diverse functions, playing a critical role in innate immunity, thrombosis, atherosclerosis, cellular adhesion, and lipid transport (1-5). Widely distributed in a variety of cells, CD36 expression is prominent on platelets and capillary epithelial cells where it is thought to take part in transmembrane signaling and in regulating vessel angiogenesis (6, 7). As a scavenger receptor, CD36 is involved in the binding and uptake of oxidized lowdensity lipoprotein by macrophages and the formation of foam cells during arterial atherogenesis (8 -11). As the receptor for thrombospondin 1 (TSP-1) 3 on endothelial cells, CD36 is reported to mediate the anti-angiogenic effect of TSP-1 (12, 13). CD36 also serves as a selective and non-redundant receptor or sensor of microbial diacylglycerides that signal via the TLR2/6 heterodimer (2).CD36 is the canonical member of class B scavenger receptor family. In this protein family, all the members conform to a two-TM membrane topology, with the first and the second TM domains flanking a large extracellular loop that is highly N-glycosylated (see Fig. 1A). Similar to CD36, many members of the class B scavenger receptor family share a common functional feature in recognizing and binding hydrophobic molecules such as fatty acids and pheromones (14,15). The extracellular domain of CD36 is characterized by a hydrophobic stretch (residues 184 -204) that may interact with the plasma membrane (16). The binding sites of CD36 for fatty acids, modified LDL, the growth hormone releasing peptide, and hexarelin have been mapped to residues 155-183 (17), whereas that for TSP-1 was shown to be residues 93-120 (18). Both intracellular domains of CD36 are relatively short but important for its efficient expression in the plasma membrane (19). Moreover, there are two cysteine residues in each intracellular domain, and their palmitoylation is thought to be an important factor in positioning CD36 into caveolae and lipid rafts (20 -2...

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Distance Mapping in Proteins Using Fluorescence Spectroscopy: Tyrosine, like Tryptophan, Quenches Bimane Fluorescence in a Distance-Dependent Manner

Cited by 44 publications

References 49 publications

Decay of an active GPCR: Conformational dynamics govern agonist rebinding and persistence of an active, yet empty, receptor state

Decay of an active GPCR: Conformational dynamics govern agonist rebinding and persistence of an active, yet empty, receptor state

The Enzymatic Activity of Lipases Correlates with Polarity-Induced Conformational Changes: A Trp-Induced Quenching Fluorescence Study

Critical residues and motifs for homodimerization of the first transmembrane domain of the plasma membrane glycoprotein CD36

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