Conformation and Stability of α-Helical Membrane Proteins. 1. Influence of Salts on Conformational Equilibria between Active and Inactive States of Rhodopsin

Little is known about the molecular mechanism of Schiff base hydrolysis in rhodopsin. We report here our investigation into this process focusing on the role of amino acids involved in a hydrogen bond network around the retinal Schiff base. We find conservative mutations in this network (T94I, E113Q, S186A, E181Q, Y192F, and Y268F) increase the activation energy (E a ) and abolish the concave Arrhenius plot normally seen for Schiff base hydrolysis in dark state rhodopsin. Interestingly, two mutants (T94I and E113Q) show dramatically faster rates of Schiff base hydrolysis in dark state rhodopsin, yet slower hydrolysis rates in the active MII form. We find deuterium affects the hydrolysis process in wild-type rhodopsin, exhibiting a specific isotope effect of ϳ2.5, and proton inventory studies indicate that multiple proton transfer events occur during the process of Schiff base hydrolysis for both dark state and MII forms. Taken together, our study demonstrates the importance of the retinal hydrogen bond network both in maintaining Schiff base integrity in dark state rhodopsin, as well as in catalyzing the hydrolysis and release of retinal from the MII form. Finally, we note that the dramatic alteration of Schiff base stability caused by mutation T94I may play a causative role in congenital night blindness as has been suggested by the Oprian and Garriga laboratories.Rhodopsin, the dim light photoreceptor of rod cells, is arguably the best characterized member of the class A superfamily of GPCR (1-7). A transmembrane receptor, it has evolved into an efficient photoreceptor by covalently binding its chromophore, 11-cis-retinal, to lysine 296 via a protonated Schiff base linkage within the helical bundle (8, 9). Dim light vision begins when the 11-cis-retinal chromophore absorbs a photon and isomerizes to the all-trans-retinal form. This change in retinal initiates a series of photo-intermediates and conformational changes in the protein, resulting in the formation of metarhodopsin II (MII), 1 the active conformation of rhodopsin that is able to bind and activate the G-protein transducin. The MII photoproduct is in dynamic equilibrium with its predecessor MI, and this MI/MII pool is thought to decay through two processes (10). The MII product may be directly hydrolyzed and release all-trans-retinal from the binding pocket, or the MI pool may undergo an addition thermal isomerization along the chromophore CϭN double bond (all-trans 15-syn) giving rise to the MIII storage product (10). This MIII intermediate also decays to opsin and all-trans-retinal (albeit at a slower rate) either through the MI/MII pool or possibly direct retinal Schiff base hydrolysis of the MIII intermediate. Rhodopsin deactivation ultimately requires hydrolysis of the all-trans-retinal Schiff base linkage and release of retinal from the binding pocket. Recycling the receptor and returning it to a photosensitive conformational state completes the recovery process (11). The retinoid cycle accomplishes this task by converting the released all-trans-...

Section: Rational For Choice Of Mutants-supporting

confidence: 89%

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

Janz¹,

Farrens

2004

“…Recent studies (43,71,72) have begun to address carefully the thermodynamics of rhodopsin protein stability. In the present work, our conclusions about rhodopsin thermal stability are primarily based on monitoring the stability of the retinal Schiff base linkage, which we measure as the loss of absorbance at 500 nm.…”

Section: Discussionmentioning

confidence: 99%

“…Thermal stability of the mutants was determined by first measuring the samples from 650 to 250 nm at 1-min intervals at a given temperature. Thermal decay rates were then measured by monitoring the decrease of the 500 nm absorbing dark state species from these measurements over time (41)(42)(43). Base-line drift was corrected for by normalizing all spectra to an absorbance of 0 at 650 nm.…”

Section: Thermal Bleaching Of Rhodopsin Samplesmentioning

confidence: 99%

Stability of Dark State Rhodopsin Is Mediated by a Conserved Ion Pair in Intradiscal Loop E-2

Janz

Fay

Farrens

2003

The rhodopsin crystal structure reveals that intradiscal loop E-2 covers the 11-cis-retinal, creating a "retinal plug." Recently, we noticed the ends of loop E-2 are linked by an ion pair between residues Arg-177 and Asp-190, near the highly conserved disulfide bond. This ion pair appears biologically significant; it is conserved in almost all vertebrate opsins and may occur in other G-protein-coupled receptors. We report here that the Arg-177/Asp-190 ion pair is critical for the folding and stability of dark state rhodopsin. We find ion pair mutants that regenerate with retinal are functionally and spectrally wild-type-like yet thermally unstable in their dark state because of rapid hydrolysis of the retinal Schiff base linkage. Surprisingly, Arrhenius analysis indicates that the activation energies for the hydrolysis process are similar between the ion pair mutants and wild-type rhodopsin. Furthermore, the ion pair mutants do not show increased reactivity toward hydroxylamine, suggesting that their instability is not caused by an increased exposure to bulk solvent. Our results indicate that the loop E-2 ion pair is important for rhodopsin stability and thus suggest that retinitis pigmentosa observed in patients with Asp-190 mutations may in part be the result of thermally unstable rhodopsin proteins.

“…In rhodopsin, the existence of multiple conformers is evident from absorbance changes (37). Activation occurs by transition through intermediate conformations (38), with the equilibrium between these forms showing a characteristic pH sensitivity (39). The existence of multiple receptor conformers is also evident in single molecule spectroscopy studies of the ␤ adrenergic receptor (40).…”

mentioning

confidence: 94%

Conserved Helix 7 Tyrosine Acts as a Multistate Conformational Switch in the 5HT2C Receptor

Prioleau¹,

Visiers²,

Ebersole³

et al. 2002

114

Studies in many rhodopsin-like G-protein-coupled receptors are providing a general scheme of the structural processes underlying receptor activation. Microdomains in several receptors have been identified that appear to function as activation switches. However, evidence is emerging that these receptor proteins exist in multiple conformational states. To study the molecular control of this switching process, we investigated the function of a microdomain involving the conserved helix 7 tyrosine in the serotonin 5HT2C receptor. This tyrosine of the NPXXY motif was substituted for all naturally occurring amino acids. Three distinct constitutively active receptor phenotypes were found: moderate, high, and "locked-on" constitutive activity. In contrast to the activity of the other receptor mutants, the high basal signaling of the locked-on Y7.53N mutant was neither increased by agonists nor decreased by inverse agonists. The Y7.53F mutant was uncoupled. Computational modeling based on the rhodopsin crystal structure suggested that Y7.53 interacts with the conserved aromatic ring at position 7.60 in the recently identified helix 8 domain. This provided a basis for seeking revertant mutations to correct the defective function of the Y7.53F receptor. When the Y7.53F receptor was mutated at position 7.60, the wild-type phenotype was restored. These results suggest that Y7.53 and Y7.60 contribute to a common functional microdomain connecting helices 7 and 8 that influences the switching of the 5HT2C receptor among multiple active and inactive conformations.