Role of the Retinal Hydrogen Bond Network in Rhodopsin Schiff Base Stability and Hydrolysis

Janz, Jay M.; Farrens, David

doi:10.1074/jbc.m408766200

Cited by 68 publications

(97 citation statements)

References 72 publications

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“…By contrast, in the lower temperature range of our measurements, the prefactor was found to have a more typical value, estimated from the three data points to be about 10 9±1 s −1 . Similar activation energies can be inferred from Arrhenius plots previously obtained by Hubbard in 1958 (∼100 kcal/mol) (10) and by Janz and Farrens in 2004 (∼103 kcal/mol) (11). However, they did not explicitly report prefactors, and the origins of the large E a and the sharp bending of the Arrhenius plot remained unexplored.…”

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confidence: 56%

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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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confidence: 56%

“…In addition, the mutant S186A where the H-bonds in the retinyl binding site are disrupted (Fig. 4A) has rates increased by one to two orders of magnitude (11,13). It is, therefore, natural to consider that thermal decay at 52.0-64.6°C, close to T m , might involve breaking or at least weakening H-bonds associated with internal water molecules.…”

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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“…The nature of these changes, subject to our sampling limitations imposed by molecular dynamics calculations, suggest a tight coupling within the rhodopsin-retinal system. 52,53 Thus, the large tilt changes in Helix 6 are well underway by 30 ns, the changes in the G-protein activation region (Helices 5 and 6 and cytoplasmic loop C3) have also started. But, the largest indicator of the nature of these shifts is the changes in sidechain-to-retinal coupling.…”

Section: Changes By End Of 30 Nsmentioning

confidence: 93%

How a small change in retinal leads to G‐protein activation: Initial events suggested by molecular dynamics calculations

Stevens

Woolf

2006

Proteins

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Rhodopsin is the prototypical G-protein coupled receptor, coupling light activation with high efficiency to signaling molecules. The dark-state X-ray structures of the protein provide a starting point for consideration of the relaxation from initial light activation to conformational changes that may lead to signaling. In this study we create an energetically unstable retinal in the light activated state and then use molecular dynamics simulations to examine the types of compensation, relaxation, and conformational changes that occur following the cis-trans light activation. The results suggest that changes occur throughout the protein, with changes in the orientation of Helices 5 and 6, a closer interaction between Ala 169 on Helix 4 and retinal, and a shift in the Schiff base counterion that also reflects changes in sidechain interactions with the retinal. Taken together, the simulation is suggestive of the types of changes that lead from local conformational change to light-activated signaling in this prototypical system.

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“…Such rapid proton exchange is enabled by an effective increase in water concentration near the retinal Schiff base. In D 2 O isotopic effect studies, slower rates of Schiff base hydrolysis were measured during Meta II decay, providing evidence that external D 2 O rather than internal water was involved in this process (30). Furthermore, exchange of the carbonyl oxygen of at-RAL released from Rho was visualized by FTIR spectroscopy (14).…”

Section: Discussionmentioning

confidence: 99%

“…The process of retinylidene hydrolysis is complex, involving multiple protonation/deprotonation and proton transfer events. It most likely occurs in dark state Rho by employing the internal water network to mediate cleavage of the Schiff base linkage (30), but due to structural constraints provided by this state of the receptor, 11-cis-retinal release is hindered, and the Schiff base reforms. Subtle structural relaxation at the retinal-binding site rather than major conformational changes upon photoactivation can be envisioned as the driving force for this protein's transitions to new thermodynamically stable subconfigurations (31).…”

Section: Discussionmentioning

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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Role of the Retinal Hydrogen Bond Network in Rhodopsin Schiff Base Stability and Hydrolysis

Cited by 68 publications

References 72 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

How a small change in retinal leads to G‐protein activation: Initial events suggested by molecular dynamics calculations

Role of Bulk Water in Hydrolysis of the Rhodopsin Chromophore

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