Structural waters define a functional channel mediating activation of the GPCR, rhodopsin

Angel, Thomas E.; Gupta, Sayan; Jastrzębska, Beata; Palczewski, Krzysztof; Chance, Mark R.

doi:10.1073/pnas.0901074106

Cited by 185 publications

(205 citation statements)

References 53 publications

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“…4A), participating in an extended network of ∼100 H-bonds (2,17). This network is thought to stabilize rhodopsin (2,18,19) and suppress thermal isomerization, conferring rhodopsin with extraordinary thermal stability to lower the dark noise and enhance photosensitivity. This hypothesis is supported by the observation that the rates of thermal reactions are slowed down threefold in deuterated water, consistent with a rate-determining step involving water interactions (20).…”

Section: Resultsmentioning

confidence: 99%

Unusual kinetics of thermal decay of dim-light photoreceptors in vertebrate vision

Guo

Sekharan

Liu

et al. 2014

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

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We present measurements of rate constants for thermal-induced reactions of the 11-cis retinyl chromophore in vertebrate visual pigment rhodopsin, a process that produces noise and limits the sensitivity of vision in dim light. At temperatures of 52.0-64.6°C, the rate constants fit well to an Arrhenius straight line with, however, an unexpectedly large activation energy of 114 ± 8 kcal/mol, which is much larger than the 60-kcal/mol photoactivation energy at 500 nm. Moreover, we obtain an unprecedentedly large prefactor of 10 72±5 s −1 , which is roughly 60 orders of magnitude larger than typical frequencies of molecular motions! At lower temperatures, the measured Arrhenius parameters become more normal: E a = 22 ± 2 kcal/mol and A pref = 10 9±1 s −1 in the range of 37.0-44.5°C. We present a theoretical framework and supporting calculations that attribute this unusual temperature-dependent kinetics of rhodopsin to a lowering of the reaction barrier at higher temperatures due to entropy-driven partial breakup of the rigid hydrogen-bonding network that hinders the reaction at lower temperatures.non-Arrhenius | dim-light vision | transition state theory | isomerization rate R hodopsin is a vertebrate dim-light photoreceptor. Molecular studies of rhodopsin in recent decades have largely focused on its photochemistry and photoactivation (1-3). However, complete understanding of rhodopsin's function requires characterization of its thermal properties because thermal isomerization of the 11-cis retinyl chromophore can trigger the same physiological response as photo-isomerization, generating false visual signals as dark noise that jeopardizes photosensitivity (4-6). To enhance dim-light vision, rhodopsin has evolved to acquire remarkable thermal stability with a half-life of 420 y as determined by electrophysiological experiments using the outer segments of rod cells at 36°C (4). However, the molecular mechanism for the thermal stability has remained unclear. Here, we have addressed this by exploring the temperature dependence of thermal decay rate constants k(T) associated with isomerization of the retinyl chromophore and hydrolysis of the chromophore protonated Schiff-base (PSB) linkage, for temperatures ranging from the physiological temperature of 37.0°C to 64.6°C. In the upper range of temperatures from 52.0°C to 64.6°C, we find that the rate constants determined by UV-visible spectroscopy follow a linear Arrhenius model, k(T) = A pref exp(−E a /k B T), where k B is the Boltzmann constant (Fig. 1A). The slope, however, is very steep, giving an elevated activation energy, E a = 114 ± 8 kcal/mol. Surprisingly, this value is much higher than the photoactivation energy at visible wavelengths (60 kcal/mol at 500 nm). E a is also much higher than the reaction enthalpy change (32-35 kcal/mol) (7, 8) (Fig. 1B). In the lower temperature range of our rate constant measurements, 37.0-44.5°C, the slope of the Arrhenius plot decreases abruptly, as shown in Fig. 1A. Fitting a straight line through the low temperature points prod...

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Section: Resultsmentioning

confidence: 99%

Unusual kinetics of thermal decay of dim-light photoreceptors in vertebrate vision

Guo

Sekharan

Liu

et al. 2014

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

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“…XF-MS generates hydroxyl radicals by activating bulk as well as protein-bound water, which can react directly with proximal side-chains (13,30). In contrast to bulk water, protein-bound water (identified by X-ray crystallography) shows a longer residence time in solution and facilitates a significantly higher yield of hydroxyl radical modifications at reactive residues (23,31). XF-MS has been used to probe the positions of internal water molecules, changes in the H-bonding networks involving bound water, and the dynamic role of internal water in hydrophobic cavities inside protein cores (23,26,(31)(32)(33).…”

Section: Hdx-msmentioning

confidence: 99%

“…In contrast to bulk water, protein-bound water (identified by X-ray crystallography) shows a longer residence time in solution and facilitates a significantly higher yield of hydroxyl radical modifications at reactive residues (23,31). XF-MS has been used to probe the positions of internal water molecules, changes in the H-bonding networks involving bound water, and the dynamic role of internal water in hydrophobic cavities inside protein cores (23,26,(31)(32)(33). In addition to carotenoid translocation (9), our data demonstrate two other structural changes upon photoactivation of the OCP: unfolding and release of the N-terminal helix from the CTD surface and complete dissociation of the NTD and CTD.…”

Section: Hdx-msmentioning

confidence: 99%

Local and global structural drivers for the photoactivation of the orange carotenoid protein

Gupta

Guttman

Leverenz

et al. 2015

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

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Photoprotective mechanisms are of fundamental importance for the survival of photosynthetic organisms. In cyanobacteria, the orange carotenoid protein (OCP), when activated by intense blue light, binds to the light-harvesting antenna and triggers the dissipation of excess captured light energy. Using a combination of small angle X-ray scattering (SAXS), X-ray hydroxyl radical footprinting, circular dichroism, and H/D exchange mass spectrometry, we identified both the local and global structural changes in the OCP upon photoactivation. SAXS and H/D exchange data showed that global tertiary structural changes, including complete domain dissociation, occur upon photoactivation, but with alteration of secondary structure confined to only the N terminus of the OCP. Microsecond radiolytic labeling identified rearrangement of the H-bonding network associated with conserved residues and structural water molecules. Collectively, these data provide experimental evidence for an ensemble of local and global structural changes, upon activation of the OCP, that are essential for photoprotection.orange carotenoid protein | photoprotection | X-ray footprinting | hydrogen deuterium exchange | SAXS P hotosynthetic organisms have evolved a protective mechanism known as nonphotochemical quenching (NPQ) to dissipate excess energy, thereby preventing oxidative damage under high light conditions (1). In plants and algae, NPQ involves pHinduced conformation changes in membrane-embedded protein complexes and enzymatic interconversion of carotenoids (2, 3). Cyanobacteria, in contrast, use a relatively simple NPQ mechanism governed by the water soluble orange carotenoid protein (OCP). The OCP is composed of an all α-helical N-terminal domain (NTD) consisting of two discontinuous four-helix bundles and a mixed α/β C-terminal domain (CTD), which is a member of the widely distributed nuclear transport factor 2-like superfamily (Fig. S1A) (4, 5). There are two regions of interaction between the NTD and CTD (4, 5): the major interface, which buries 1,722 Å of surface area, and the interaction between the N-terminal alpha-helix (αA) and the CTD (minor interface) (Fig. S1A). A single noncovalently bound keto-carotenoid [e.g., echinenone (ECN)] spans both domains in the structure of the resting (inactive) form of the protein (OCP O ).The NTD and CTD of the OCP have discrete functions. The isolated NTD acts as an effector domain that binds to the antenna whereas the CTD has been proposed to play a sensory/regulatory role in controlling the OCP's photoprotective function (6). Exposure to blue light converts OCP O to the active (red) form, OCP R (7). OCP R is involved in protein-protein interactions with the phycobilisome (PB) (5) and the fluorescence recovery protein (FRP), which converts OCP R back to OCP O (8). The OCP R form is therefore central to the photoprotective mechanism, and determining the exact structural changes that accompany its formation are critical for a complete mechanistic understanding of the reversible quenching process in cy...

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“…To form the Schiff base, Lys 296 must be deprotonated, the carbonyl group must be polarized, and water must be accommodated within the chromophore-binding site (1). Hydroxyl radical footprinting revealed local conformational changes in the Rho structure following its photoactivation, presumably mediated by the dynamics of both ordered water molecules and the protein (7,8). Moreover, protein footprinting combined with rapid H 2 18 O mixing methodology and deuterium-hydrogen exchange on C 2 His residues indicated that these tightly bound internal waters do not exchange with bulk solvent in ground state Rho, Meta II, or opsin (7,8).…”

mentioning

confidence: 99%

“…Hydroxyl radical footprinting revealed local conformational changes in the Rho structure following its photoactivation, presumably mediated by the dynamics of both ordered water molecules and the protein (7,8). Moreover, protein footprinting combined with rapid H 2 18 O mixing methodology and deuterium-hydrogen exchange on C 2 His residues indicated that these tightly bound internal waters do not exchange with bulk solvent in ground state Rho, Meta II, or opsin (7,8). Thus, ordered waters function as noncovalent cofactors that actively participate in transmitting the activation signal from the retinylidene-binding pocket to the cytoplasmic face of Rho, where binding of transducin occurs.…”

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confidence: 99%

Role of Bulk Water in Hydrolysis of the Rhodopsin Chromophore

Jastrzębska

Palczewski

Golczak

2011

Journal of Biological Chemistry

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Rhodopsin (Rho) is a prototypical G protein-coupled receptor that changes from an inactive conformational state to a G protein-activating state as a consequence of its retinal chromophore isomerization, 11-cis-retinal 3 all-trans-retinal. The photoisomerized chromophore covalently linked to Lys 296 by a Schiff base is subsequently hydrolyzed, but little is known about this reaction. Recent research indicates a significant role for tightly bound transmembrane water molecules in the Rho activation process. Atomic structures of Rho and hydroxyl radical footprinting reveal ordered waters within Rho transmembrane helices that are located close to highly conserved and functionally important receptor residues, forming a hydrogen bond network. Using 18 O-labeled H 2 O, we now report that water from bulk solvent, but not tightly bound water, is involved in the hydrolytic release of chromophore upon Rho activation by light. Moreover, small molecules (and presumably, water) enter the Rho structure from the cytoplasmic side of the membrane. Thus, this work indicates two distinct origins of water vital for Rho function.Rhodopsin (Rho), 3 the visual pigment in retinal rod photoreceptors, is a G protein-coupled receptor responsible for dim light vision (1, 2). Rho is composed of its apoprotein, opsin, and a retinylidene chromophore in an 11-cis-conformation covalently linked by a Schiff base to Lys 296 of the opsin (Fig. 1A). Photoactivation of Rho is initiated by isomerization of the retinylidene ligand to its all-trans-configuration (3), allowing the photoreceptor after deprotonation of the Schiff base to adopt the active Meta II state required for G protein (transducin) activation. The atomic structure of Rho led to the identification of crystallographically ordered water molecules adjacent to functionally important conserved protein residues (Fig. 1A) (4), supporting the conclusion that these waters are essential not just for structural stabilization of the receptor but also for the activation process. One of these tightly bound waters positioned close to the chromophore-binding pocket has been postulated to play a key role in the counterion switch between ground state (dark; Glu 113 ) and activated states (Glu 181 ) of Rho (5, 6). Moreover, Glu 113 and Glu 181 are also likely involved in the hydrolytic process via the carbinol ammonium ion and in a regeneration reaction between 11-cis-retinal and opsin. To form the Schiff base, Lys 296 must be deprotonated, the carbonyl group must be polarized, and water must be accommodated within the chromophore-binding site (1). Hydroxyl radical footprinting revealed local conformational changes in the Rho structure following its photoactivation, presumably mediated by the dynamics of both ordered water molecules and the protein (7,8). Moreover, protein footprinting combined with rapid H 2 18 O mixing methodology and deuterium-hydrogen exchange on C 2 His residues indicated that these tightly bound internal waters do not exchange with bulk solvent in ground state Rho, Meta II, or opsin...

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Structural waters define a functional channel mediating activation of the GPCR, rhodopsin

Cited by 185 publications

References 53 publications

Unusual kinetics of thermal decay of dim-light photoreceptors in vertebrate vision

Unusual kinetics of thermal decay of dim-light photoreceptors in vertebrate vision

Local and global structural drivers for the photoactivation of the orange carotenoid protein

Role of Bulk Water in Hydrolysis of the Rhodopsin Chromophore

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