Effects of detergents and high pressures upon the metarhodopsin I.dbr.metarhodopsin II equilibrium

Lamola, Angelo A.; Yamane, Takeshi; Zipp, Adam

doi:10.1021/bi00701a016

Cited by 122 publications

(94 citation statements)

References 18 publications

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“…Together these results reveal that the crystal structure of opsin in the region of the ionic lock reflects the active state of the receptor. We further demonstrate that Tyr223 5.58 and Ala132 3.47 in Meta II stabilize helix H5 in an active orientation. Mutation of Tyr223 5.58 to phenylalanine or mutation of Ala132 3.47 to leucine decreases the lifetime of the Meta II intermediate.…”

supporting

confidence: 55%

“…Site-directed spin-labeling studies by Hubbell and coworkers (3,4) showed that the largest change in the seven-TM-helix bundle involves an outward rotation of helix H6, consistent with an increase in volume of the receptor upon activation (5). The challenge for obtaining a high-resolution structure of the active metarhodopsin II (Meta II) intermediate has been that light activation causes the dark-state crystals of rhodopsin to dissolve (6), suggesting that the structural changes are sufficiently large to disrupt crystal packing.…”

mentioning

confidence: 90%

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Highly conserved tyrosine stabilizes the active state of rhodopsin

Goncalves

South

Ahuja

et al. 2010

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

100

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Light-induced isomerization of the 11-cis-retinal chromophore in the visual pigment rhodopsin triggers displacement of the second extracellular loop (EL2) and motion of transmembrane helices H5, H6, and H7 leading to the active intermediate metarhodopsin II (Meta II). We describe solid-state NMR measurements of rhodopsin and Meta II that target the molecular contacts in the region of the ionic lock involving these three helices. We show that a contact between Arg135 3.50 and Met257 6.40 (1)] on the intradiscal (or extracellular) side of the receptor. Absorption of light drives the 11-cis-to trans-isomerization of the retinal within a tight binding pocket. The conformational changes that occur in this process must be transmitted through the membrane-spanning portion of the bilayer to the intracellular surface in order to open up the binding site for the heterotrimeric G protein, transducin. The crystal structure of the dark, inactive state of the visual pigment rhodopsin (2) reveals a tightly packed bundle of seven transmembrane (TM) helices but offers few clues as to how the helices move upon light activation.Site-directed spin-labeling studies by Hubbell and coworkers (3,4) showed that the largest change in the seven-TM-helix bundle involves an outward rotation of helix H6, consistent with an increase in volume of the receptor upon activation (5). The challenge for obtaining a high-resolution structure of the active metarhodopsin II (Meta II) intermediate has been that light activation causes the dark-state crystals of rhodopsin to dissolve (6), suggesting that the structural changes are sufficiently large to disrupt crystal packing. Salom et al. (7) were able to determine the crystal structure to 4.15-Å resolution of a photointermediate of rhodopsin containing retinal with a deprotonated Schiff base (SB) (7). The structure did not exhibit the large helix motions characteristic of the activated receptor, suggesting that this intermediate corresponds to the Meta II substate (Meta IIa) formed prior to helix motion (8).More recently, Park et al. determined the structure of opsin (9). Opsin is formed when the Meta II intermediate decays and releases the agonist all-trans-retinal from the retinal-binding site. Opsin has low (≤1%), but detectable, basal activity in rod outer segment cell membranes (10). At pH 4, FTIR difference spectra of opsin exhibit vibrational bands characteristic of Meta II (11), suggesting that opsin adopts an active conformation. The crystals of opsin obtained at pH 6 appear to retain many features characteristic of the active state (Fig. 1). In fact, the most recent crystal structure of opsin (12) contains the bound C-terminal peptide of the Gα subunit of transducin in a conformation similar to that observed in solution NMR studies on the activated Meta II intermediate (13,14).One of the most striking features of the opsin structure is that the ionic lock involving Glu134 3.49 -Arg135 3.50 of the conserved ERY sequence on H3 and Glu247 6.30 on H6 is disrupted (Fig. 1B). The opsin structur...

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supporting

confidence: 55%

mentioning

confidence: 90%

Highly conserved tyrosine stabilizes the active state of rhodopsin

Goncalves

South

Ahuja

et al. 2010

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

100

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“…Secondary excitation of photoproducts will also be highest in those crystals. Rhodopsin is known to undergo a significant volume change on formation of the metarhodopsin II intermediate (32), which causes the Meta I 480 -Meta II equilibrium to be pressure dependent. Presumably this would have an effect on the kinetics of a photoexcited rhodopsin in a low fractionally bleached crystal where it is surrounded by unbleached molecules compared to the case of a more highly bleached crystal where it would have a higher probability to be surrounded by similarly excited rhodopsins.…”

Section: Discussionmentioning

confidence: 99%

Rhodopsin Photointermediates in Two-Dimensional Crystals at Physiological Temperatures

et al. 2006

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Bovine rhodopsin photointermediates formed in 2D rhodopsin crystal suspensions were studied by measuring the time dependent absorbance changes produced after excitation with 7 nanosecond laser pulses at 15, 25 and 35 °C. The crystalline environment favored the Meta I 480 photointermediate, with its formation from Lumi beginning faster than it does in rhodopsin membrane suspensions at 35 °C and its decay to a 380 nm absorbing species being less complete than it is in the native membrane at all temperatures. Measurements performed at pH 5.5 in 2D crystals showed that the 380 nm absorbing product of Meta I 480 decay did not display the anomalous pH dependence characteristic of classical Meta II in the native disk membrane. Crystal suspensions bleached at 35°C and quenched to 19 °C showed that a rapid equilibrium existed on the ∼1 second time scale which suggests that the unprotonated predecessor of Meta II in the native membrane environment (sometimes called MII a ), forms in 2D rhodopsin crystals, but that the non-Schiff base proton uptake completing classical Meta II formation is blocked there. Thus, the 380 nm absorbance arises from an on-pathway intermediate in GPCR activation and does not result from early Schiff base hydrolysis. Kinetic modeling of the time-resolved absorbance data of the 2D crystals was generally consistent with such a mechanism, but details of kinetic spectral changes and the fact that the residuals of exponential fits were not as good as are obtained for rhodopsin in the native membrane suggested the photoexcited samples were heterogeneous. Variable fractional bleach due to the random orientation of linearly dichroic crystals relative to the linearly polarized laser was explored as a cause of heterogeneity but was found unlikely to fully account for it. The fact that the 380 nm product of photoexcitation of rhodopsin 2D crystals is on the physiological pathway of receptor activation suggests that determination of its structure would be of interest.Rhodopsin, the visual pigment used in dim light, originally attracted attention as the majority photoreceptor protein in mammalian retinas, with study of the intermediates in its photoactivation sequence starting long before its function as a G protein-coupled receptor (GPCR) was revealed (1). In virtually all other GPCRs the study of activation intermediates is extremely difficult because the diffusional nature of chemoreception does not allow the rapid triggering required for early intermediate characterization using ensemble based methods. While progress has been made in detecting intermediates in chemoreceptor GPCRs (2-4), it is only with great effort that processes 10,000 times slower than those reported here are studied. The detailed structural information available for the inactive, dark state of rhodopsin (5,6) provides an unambiguous basis for structural modeling of the earliest photointermediates. Optical methods of detection, such as time-resolved absorbance measurements (7), provide the highest time resolution of any experimen...

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“…Related conformational changes of the binding pocket and of other, more remote parts of the apoprotein eventually lead to the active G-protein binding state, metarhodopsin II (Meta II). 3 It is in equilibrium with its precursor metarhodopsin I (Meta I), depending on temperature and pH (9,10) and on other factors such as lipids, protein environment, and pressure (11)(12)(13)(14)(15).…”

mentioning

confidence: 99%

Deactivation and Proton Transfer in Light-induced Metarhodopsin II/Metarhodopsin III Conversion

Ritter

Elgeti

Hofmann

et al. 2007

Journal of Biological Chemistry

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Vertebrate rhodopsin shares with other retinal proteins the 11-cis-retinal chromophore and the light-induced 11-cis/trans isomerization triggering its activation pathway. However, only in rhodopsin the retinylidene Schiff base bond to the apoprotein is eventually hydrolyzed, making a complex regeneration pathway necessary. Metabolic regeneration cannot be short-cut, and light absorption in the active metarhodopsin (Meta) II intermediate causes anti/syn isomerization around the retinylidene linkage rather than reversed trans/cis isomerization. A new deactivating pathway is thereby triggered, which ends in the Meta III "retinal storage" product. Using time-resolved Fourier transform infrared spectroscopy, we show that the identified steps of receptor activation, including Schiff base deprotonation, protein structural changes, and proton uptake by the apoprotein, are all reversed. However, Schiff base reprotonation is much faster than the activating deprotonation, whereas the protein structural changes are slower. The final proton release occurs with pK ≈ 4.5, similar to the pK of a free Glu residue and to the pK at which the isolated opsin apoprotein becomes active. A forced deprotonation, equivalent to the forced protonation in the activating pathway, which occurs against the unfavorable pH of the medium, is not observed. This explains properties of the final Meta III product, which displays much higher residual activity and is less stable than rhodopsin arising from regeneration with 11-cis-retinal. We propose that the anti/syn conversion can only induce a fast reorientation and distance change of the Schiff base but fails to build up the full set of dark ground state constraints, presumably involving the Glu 134 /Arg 135 cluster.The photoreceptor rhodopsin located in the retinal rods of the vertebrate eye contains the chromophore 11-cis-retinal bound by a protonated Schiff base to Lys 296 of the apoprotein (1). Light absorption triggers isomerization around the C 11 ϭC 12 double bond of the polyene chain of the chromophore (2-4), leading to the strained all-trans-form and storage of two thirds of the light energy in the chromophore-protein system (5-8). The receptor subsequently proceeds through a number of intermediates each characterized by its specific absorption spectrum in the UV-visible and mid infrared range. Related conformational changes of the binding pocket and of other, more remote parts of the apoprotein eventually lead to the active G-protein binding state, metarhodopsin II (Meta II).3 It is in equilibrium with its precursor metarhodopsin I (Meta I), depending on temperature and pH (9, 10) and on other factors such as lipids, protein environment, and pressure (11-15).The formation of the active species through the photointermediates has been described as a stepwise lowering of the stabilizing effect of the Schiff base counterion, which is a complex structure that comprises highly conserved Glu 181 and Glu 113 . In Meta I, the counterion appears to undergo a shift relative to the Schiff base, where...

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Effects of detergents and high pressures upon the metarhodopsin I.dbr.metarhodopsin II equilibrium

Cited by 122 publications

References 18 publications

Highly conserved tyrosine stabilizes the active state of rhodopsin

Highly conserved tyrosine stabilizes the active state of rhodopsin

Rhodopsin Photointermediates in Two-Dimensional Crystals at Physiological Temperatures

Deactivation and Proton Transfer in Light-induced Metarhodopsin II/Metarhodopsin III Conversion

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