Misfolded rhodopsin mutants display variable aggregation properties

The largest class of rhodopsin mutations causing autosomal dominant retinitis pigmentosa (adRP) is mutations that lead to misfolding and aggregation of the receptor. The misfolding mutants have been characterized biochemically, and categorized as either partial or complete misfolding mutants. This classification is incomplete and does not provide sufficient information to fully understand the disease pathogenesis and evaluate therapeutic strategies. A Förster resonance energy transfer (FRET) method was utilized to directly assess the aggregation properties of misfolding rhodopsin mutants within the cell. Partial (P23H and P267L) and complete (G188R, H211P, and P267R) misfolding mutants were characterized to reveal variability in aggregation properties. The complete misfolding mutants all behaved similarly, forming aggregates when expressed alone, minimally interacting with the wild-type receptor when coexpressed, and were unresponsive to treatment with the pharmacological chaperone 9-cis retinal. In contrast, variability was observed between the partial misfolding mutants. In the opsin form, the P23H mutant behaved similarly as the complete misfolding mutants. In contrast, the opsin form of the P267L mutant existed as both aggregates and oligomers when expressed alone and formed mostly oligomers with the wild-type receptor when coexpressed. The partial misfolding mutants both reacted similarly to the pharmacological chaperone 9-cis retinal, displaying improved folding and oligomerization when expressed alone but aggregating with wild-type receptor when coexpressed. The observed differences in aggregation properties and effect of 9-cis retinal predict different outcomes in disease pathophysiology and suggest that retinoid-based chaperones will be ineffective or even detrimental.     

Opsin stability and folding: the role of Cys185 and abnormal disulfide bond formation in the intradiscal domain

The structure in the extracellular, intradiscal domain of rhodopsin surrounding the Cys110–Cys187 disulfide bond has been shown to be important for correct folding of this receptor in vivo. Retinitis pigmentosa misfolding mutants of the apoprotein opsin (such as P23H) misfold, as defined by a deficiency in ability to bind 11-cis retinal and form rhodopsin. These mutants also possess an abnormal Cys185–Cys187 disulfide bond in the intradiscal domain. Here, by mutating Cys185 to alanine, we eliminate the possibility of forming this abnormal disulfide bond and investigate the effect of combining the C185A mutation with the retinitis pigmentosa mutation P23H. Both the P23H and P23H/C185A double mutant suffer from low expression and poor 11-cis retinal binding. Our data suggest that misfolding events occur that do not have an absolute requirement for abnormal Cys185–Cys187 disulfide bond formation. In the detergent-solubilised, purified state, the C185A mutation allows formation of rhodopsin at wild-type (WT) levels, but has interesting effects on protein stability. C185A rhodopsin is less thermally stable than WT, whereas C185A opsin shows the same ability to regenerate rhodopsin in detergent as WT. Purified C185A and WT opsins, however, have contrasting 11-cis retinal binding kinetics. A high proportion of C185A opsin binds 11-cis retinal with a slow rate that reflects a denatured state of opsin reverting to a fast-binding, open-pocket conformation. This slower rate is not observed in a stabilising lipid/detergent system, 1,2-dimyristoyl-sn-glycero-3-phosphocholine/Chaps, in which C185A exhibits WT (fast) retinal binding. We propose that the C185A mutation destabilises the open-pocket conformation of opsin in detergent resulting in an equilibrium between correctly folded and denatured states of the protein. This equilibrium can be driven towards the correctly folded rhodopsin state by the binding of 11-cis retinal.     

Retinitis Pigmentosa Mutants Provide Insight into the Role of the N-terminal Cap in Rhodopsin Folding,Structure, and Function

Autosomal dominant retinitis pigmentosa (ADRP) mutants (T4K, N15S, T17M, V20G, P23A/H/L, and Q28H) in the N-terminal cap of rhodopsin misfold when expressed in mammalian cells. To gain insight into the causes of misfolding and to define the contributions of specific residues to receptor stability and function, we evaluated the responses of these mutants to 11-cis-retinal pharmacological chaperone rescue or disulfide bond-mediated repair. Pharmacological rescue restored folding in all mutants, but the purified mutant pigments in all cases were thermo-unstable and exhibited abnormal photobleaching, metarhodopsin II decay, and G protein activation. As a complementary approach, we superimposed this panel of ADRP mutants onto a rhodopsin background containing a juxtaposed cysteine pair (N2C/D282C) that forms a disulfide bond. This approach restored folding in T4K, N15S, V20G, P23A, and Q28H but not T17M, P23H, or P23L. ADRP mutant pigments obtained by disulfide bond repair exhibited enhanced stability, and some also displayed markedly improved photobleaching and signal transduction properties. Our major conclusion is that the N-terminal cap stabilizes opsin during biosynthesis and contributes to the dark-state stability of rhodopsin. Comparison of these two restorative approaches revealed that the correct position of the cap relative to the extracellular loops is also required for optimal photochemistry and efficient G protein activation.     

Kinetic study of photoregeneration process of digitonin-solubilized squid rhodopsin

In the photoregeneration process of squid rhodopsin, an intermediate has been found at neutral pH values (phosphate buffer) with a flash light (λ > 540 nm). An intermediate R430, with the 11-cis retinal as chromophore, is produced from metarhodopsin in light and is converted to rhodopsin through the processes R430 → P380 and P380 → rhodopsin. The pH dependence of the velocity of the conversions suggests that processes R430 → P380 and P380 → rhodopsin involve a protolytic reaction and that the ionized group is a histidine residue of opsin. Kinetic parameters show that the largest conformational change in opsin occurs in the conversion of R430 → P380.     

Studies on cephalopod rhodopsin. Conformational changes in chromophore and protein during the photoregeneration process

The ultraviolet absorbance of squid and octopus rhodopsin changes reversibly at 234 nm and near 280 nm in the interconversion of rhodopsin and metarhodopsin. The absorbance change near 280 nm is ascribed to both protein and chromophore parts. Rhodopsin is photoregenerated from metarhodopsin via an intermediate, P380, on irradiation with yellow light (λ > 520 nm). The ultraviolet absorbance decreases in the change from rhodopsin to metarhodopsin and recovers in two steps; mostly in the process from metarhodopsin to P380 and to a lesser extent in the process from P380 to rhodopsin. P380 has a circular dichroism (CD) band at 380 nm and its magnitude is the same order as that of rhodopsin. Thus it is considered that the molecular structure of P380 is close to that of rhodopsin and that the chromophore is fixed to opsin as in rhodopsin. In the change from metarhodopsin to P380, the chromophore is isomerized from the all-trans to the 11-cis form, and the conformation of opsin changes to fit 11-cis retinal. In the change from P380 to rhodopsin, a small change in the conformation of the protein part and the protonation of the Schiff base, the primary retinal-opsin link, occur.     

Accessibility of the iodopsin chromophore

Iodopsin can replace its chromophore (11-cis retinal) by added 9-cis retinal, resulting in the formation of isoiodopsin. NaBH₄ bleaches iodopsin in the dark. In a relatively low concentration of digitonin, the scotopsin (the protein moiety of chicken rhodopsin) removes 11-cis retinal from isopsin in the iodopsin These facts suggests that the linkage of the chromophore to opsin in the iodopsin molecule (presumably a Schiff-base linkage) is accessible to these reagents, which is different from the situation in rhodopsin.     

Inherent Instability of the Retinitis Pigmentosa P23H Mutant Opsin

The P23H opsin mutation is the most common cause of autosomal dominant retinitis pigmentosa. Even though the pathobiology of the resulting retinal degeneration has been characterized in several animal models, its complex molecular mechanism is not well understood. Here, we expressed P23H bovine rod opsin in the nervous system of Caenorhabditis elegans. Expression was low due to enhanced protein degradation. The mutant opsin was glycosylated, but the polysaccharide size differed from that of the normal protein. Although P23H opsin aggregated in the nervous system of C. elegans, the pharmacological chaperone 9-cis-retinal stabilized it during biogenesis, producing a variant of rhodopsin called P23H isorhodopsin. In vitro, P23H isorhodopsin folded correctly, formed the appropriate disulfide bond, could be photoactivated but with reduced sensitivity, and underwent Meta II decay at a rate similar to wild type isorhodopsin. In worm neurons, P23H isorhodopsin initiated phototransduction by coupling with the endogenous G_i/o signaling cascade that induced loss of locomotion. Using pharmacological interventions affecting protein synthesis and degradation, we showed that the chromophore could be incorporated either during or after mutant protein translation. However, regeneration of P23H isorhodopsin with chromophore was significantly slower than that of wild type isorhodopsin. This effect, combined with the inherent instability of P23H rhodopsin, could lead to the structural cellular changes and photoreceptor death found in autosomal dominant retinitis pigmentosa. These results also suggest that slow regeneration of P23H rhodopsin could prevent endogenous chromophore-mediated stabilization of rhodopsin in the retina.     

Retinobenzaldehydes as proper-trafficking inducers of folding-defective P23H rhodopsin mutant responsible for Retinitis Pigmentosa

The Retinitis pigmentosa (RP)-causing mutant of rhodopsin, P23H rhodopsin, is folding-defective and unable to traffic beyond the endoplasmic reticulum (ER). This ER retention, and in some cases aggregation, are proposed to result in ER-stress and eventually cell death. The endogenous rhodopsin ligand 11-cis-retinal and its isomer 9-cis-retinal have been shown to act as pharmacological chaperones, promoting proper folding and trafficking of the P23H rhodopsin. In spite of this promising effect, the development of retinals and related polyenealdehydes as pharmacological agents has been hampered by their undesirable properties, which include chemical instability, photolability, and potential retinoidal actions. Here, we report the design and synthesis of a class of more stable nonpolyene-type rhodopsin ligands, structurally distinct from, and with lower toxicity than, retinals. A structure–activity relationship study was conducted using cell-surface expression assay to quantify folding/trafficking efficiency of P23H rhodopsin.     

Activation of phosphodiesterase in frog rod outer segment by an intermediate of rhodopsin photolysis. I.

Guanosine 3′,5′-cyclic monophosphate phosphodiesterase (EC 3.1.4.1) in frog rod outer segment prepared by a sucrose stepwise density gradient method was activated by light in the presence of GTP. Rhodopsin in rod outer segment was solubilized with sucrose laurylmonoester and then purified by concavanalin A-Sepharose column. Addition of photo-bleached preparation of the purified rhodopsin to the rod outer segment, which had been prepared by 43% (w/w) sucrose floatation, caused the activation of phosphodiesterase in the dark, while each component of the photo-product eluted from the column (all-trans retinal and opsin) did not. Regenerated rhodopsin prepared from 11-cis retinal and purified opsin activated phosphosdiesterase when it was bleached. From these facts it is suggested that an intermediate or a process of photolysis of rhodopsin causes activation of phosphodiesterase.     

Activation of phosphodiesterase in frog rod outer segment by an intermediate of rhodopsin photolysis. II

Frog (Rana catesbeiana) rod outer segment membrane contains cyclic GMP phosphodiesterase (EC 3.1.4.1). Irradiation of dark-adapted rod outer segment membrane increased the enzyme activity by 5–20-fold in the presence of GTP. The phosphodiesterase in rod outer segment membrane is also activated by mixing a photo-product of 11-cis (regenerated), 9-cis or 7-cis rhodopsin which is stable at 0°C. However, neither opsin in the membrane nor all-trans retinal activates the enzyme. The phosphodiesterase in rod outer segment membrane is also activated by irradiation at ?4°C. Thus, we conclude that the phosphodiesterase is activated by a common photolysis intermediate of these rhodopsin isomers, perhaps before metarhodopsin II decays.     

Calnexin Improves the Folding Efficiency of Mutant Rhodopsin in the Presence of Pharmacological Chaperone 11-cis-Retinal

The lectin chaperone calnexin (Cnx) is important for quality control of glycoproteins, and the chances of correct folding of a protein increase the longer the protein interacts with Cnx. Mutations in glycoproteins increase their association with Cnx, and these mutant proteins are retained in the endoplasmic reticulum. However, until now, the increased interaction with Cnx was not known to increase the folding of mutant glycoproteins. Because many human diseases result from glycoprotein misfolding, a Cnx-assisted folding of mutant glycoproteins could be beneficial. Mutations of rhodopsin, the glycoprotein pigment of rod photoreceptors, cause misfolding resulting in retinitis pigmentosa. Despite the critical role of Cnx in glycoprotein folding, surprisingly little is known about its interaction with rhodopsin or whether this interaction could be modulated to increase the folding of mutant rhodopsin. Here, we demonstrate that Cnx preferentially associates with misfolded mutant opsins associated with retinitis pigmentosa. Furthermore, the overexpression of Cnx leads to an increased accumulation of misfolded P23H opsin but not the correctly folded protein. Finally, we demonstrate that increased levels of Cnx in the presence of the pharmacological chaperone 11-cis-retinal increase the folding efficiency and result in an increase in correct folding of mutant rhodopsin. These results demonstrate that misfolded rather than correctly folded rhodopsin is a substrate for Cnx and that the interaction between Cnx and mutant, misfolded rhodopsin, can be targeted to increase the yield of folded mutant protein.     

Chromcrphore-Environment Interaction of Visual Pigments in Model System

SEVERAL laboratories^1–6 have recently been concerned with the mechanism of the bathochromic shift of about 120 nm which results when 11-cis retinal (λ _max 380 nm) combines with the protein opsin to form rhodopsin (λ_max 498 nm). A red shift of up to 186 nm is involved in the formation of iodopsin from 11-cis retinal and cone opsin^7,8. The active site of bovine rhodopsin consists of the 11-cis retinylidene chromophore attached to a primary amine group of the protein forming a Schiff-base linkage of the type shown in Fig. 1, Ia. On the basis of the chemical reactions of rhodopsin and its derivatives it has been suggested that an interaction between a protonated form of the chromophore (structure of the type Ib) and a lipophilic environment contributes¹¹ to the red shift.     

Systematic profiling of temperature- and retinal-sensitive rhodopsin variants by deep mutational scanning

Membrane protein variants with diminished conformational stability often exhibit enhanced cellular expression at reduced growth temperatures. The expression of “temperature-sensitive” variants is also typically sensitive to corrector molecules that bind and stabilize the native conformation. There are many examples of temperature-sensitive rhodopsin variants, the misfolding of which is associated with the molecular basis of retinitis pigmentosa. In this work, we employ deep mutational scanning to compare the effects of reduced growth temperature and 9-cis-retinal, an investigational corrector, on the plasma membrane expression of 700 rhodopsin variants in HEK293T cells. We find that the change in expression at reduced growth temperatures correlates with the response to 9-cis-retinal among variants bearing mutations within a hydrophobic transmembrane domain (TM2). The most sensitive variants appear to disrupt a native helical kink within this transmembrane domain. By comparison, mutants that alter the structure of a polar transmembrane domain (TM7) exhibit weaker responses to temperature and retinal that are poorly correlated. Statistical analyses suggest that this observed insensitivity cannot be attributed to a single variable, but likely arises from the composite effects of mutations on the energetics of membrane integration, the stability of the native conformation, and the integrity of the retinal-binding pocket. Finally, we show that the characteristics of purified temperature- and retinal-sensitive variants suggest that the proteostatic effects of retinal may be manifested during translation and cotranslational folding. Together, our findings highlight several biophysical constraints that appear to influence the sensitivity of genetic variants to temperature and small-molecule correctors.     

Thermal Stability of Rhodopsin and Progression of Retinitis Pigmentosa: COMPARISON OF S186W AND D190N RHODOPSIN MUTANTS

Over 100 point mutations in the rhodopsin gene have been associated with retinitis pigmentosa (RP), a family of inherited visual disorders. Among these, we focused on characterizing the S186W mutation. We compared the thermal properties of the S186W mutant with another RP-causing mutant, D190N, and with WT rhodopsin. To assess thermal stability, we measured the rate of two thermal reactions contributing to the thermal decay of rhodopsin as follows: thermal isomerization of 11-cis-retinal and hydrolysis of the protonated Schiff base linkage between the 11-cis-retinal chromophore and opsin protein. We used UV-visible spectroscopy and HPLC to examine the kinetics of these reactions at 37 and 55 °C for WT and mutant rhodopsin purified from HEK293 cells. Compared with WT rhodopsin and the D190N mutant, the S186W mutation dramatically increases the rates of both thermal isomerization and dark state hydrolysis of the Schiff base by 1–2 orders of magnitude. The results suggest that the S186W mutant thermally destabilizes rhodopsin by disrupting a hydrogen bond network at the receptor''s active site. The decrease in the thermal stability of dark state rhodopsin is likely to be associated with higher levels of dark noise that undermine the sensitivity of rhodopsin, potentially accounting for night blindness in the early stages of RP. Further studies of the thermal stability of additional pathogenic rhodopsin mutations in conjunction with clinical studies are expected to provide insight into the molecular mechanism of RP and test the correlation between rhodopsin''s thermal stability and RP progression in patients.     

Crystallographic analysis of the primary photochemical reaction of squid rhodopsin

Visual signal transduction is initiated by the photoisomerization of 11-cis retinal upon rhodopsin ligation. Unlike vertebrate rhodopsin, which interacts with Gt-type G-protein to stimulate the cyclic GMP signaling pathway, invertebrate rhodopsin interacts with Gq-type G-protein to stimulate a signaling pathway that is based on inositol 1,4,5-triphosphate. Since the inositol 1,4,5-triphosphate signaling pathway is utilized by mammalian nonvisual pigments and a large number of G-protein-coupled receptors, it is important to elucidate how the activation mechanism of invertebrate rhodopsin differs from that of vertebrate rhodopsin. Previous crystallographic studies of squid and bovine rhodopsins have shown that there is a profound difference in the structures of the retinal-binding pockets of these photoreceptors. Here, we report the crystal structures of all-trans bathorhodopsin (Batho; the first photoreaction intermediate) and the artificial 9-cis isorhodopsin (Iso) of squid rhodopsin. Upon the formation of Batho, the central moiety of the retinal was observed to move largely towards the cytoplasmic side, while the Schiff base and the ionone ring underwent limited movements (i.e., the all-trans retinal in Batho took on a right-handed screwed configuration). Conversely, the 9-cis retinal in Iso took on a planar configuration. Our results suggest that the light energy absorbed by squid rhodopsin is mostly converted into the distortion energy of the retinal polyene chain and surrounding residues.     

Photochemical reaction of 9-cis-retro-γ-rhodopsin at low temperatures

9-cis-Retro-γ;rhodopsin (λ_max = 420 nm) was prepared from 9-cis-retro-γ-retinal and cattle opsin. After cooling to liquid nitrogen temperature (77 K), the pigment was irradiated with light at 380 nm. The spectrum shifted to the longer wavelengths, owing to formation of a batho product. This fact indicates that the conjugated double bond system from C-5 to C-8 of the chromophoric retinal in rhodopsin was not necessary for formation of bathorhodopsin. Reirradiation of the batho product with light at wavelengths longer than 520 nm yielded a mixture composed of presumably 9- or 11-cis forms of retro-γ-rhodopsin. These three isomers are interconvertible by light at liquid nitrogen temperature. Thus the retro-γ-rhodopsin system is similar in photochemical reaction at 77 K to cattle rhodopsin system. Each system has its own batho product. Based on these results, it was infered that the formation of bathorhodopsin is due to photoisomerization of the chromophoric retinal of rhodopsin and is not due to translocation of a proton on the ring or on the side chain from C-6 to C-8 of the chromophoric retinal to the Schiff-base nitrogen.     

The thermal stability of rhodopsin and opsin

Rhodopsin, the red photosensitive pigment of rod vision, is composed of a specific cis isomer of retinene, neo-b (11-cis), joined as chromophore to a colorless protein, opsin. We have investigated the thermal denaturation of cattle rhodopsin and opsin in aqueous digitonin solution, and in isolated rod outer limbs. Both rhodopsin and opsin are more stable in rods than in solution. In solution as well as in rods, moreover, rhodopsin is considerably more stable than opsin. The chromophore therefore protects opsin against denaturation. This is true whether rhodopsin is extracted from dark-adapted retinas, or synthesized in vitro from neo-b retinene and opsin. Excess neo-b retinene does not protect rhodopsin against denaturation. The protection involves the specific relationship between the chromophore and opsin. Similar, though somewhat less, protection is afforded opsin by the stereoisomeric iso-a (9-cis) chromophore in isorhodopsin. The Arrhenius activation energies (E_a) and entropies of activation (ΔS‡) are much greater for thermal denaturation of rhodopsin and isorhodopsin than of opsin. Furthermore, these values differ considerably for rhodopsins from different species —frog, squid, cattle—presumably due to species differences in the opsins. Heat or light bleaches rhodopsin by different mechanisms, yielding different products. Light stereoisomerizes the retinene chromophore; heat denatures the opsin. Photochemical bleaching therefore yields all-trans retinene and native opsin; thermal bleaching, neo-b retinene and denatured opsin.     

The heat-shock response co-inducer arimoclomol protects against retinal degeneration in rhodopsin retinitis pigmentosa

Retinitis pigmentosa (RP) is a group of inherited diseases that cause blindness due to the progressive death of rod and cone photoreceptors in the retina. There are currently no effective treatments for RP. Inherited mutations in rhodopsin, the light-sensing protein of rod photoreceptor cells, are the most common cause of autosomal-dominant RP. The majority of mutations in rhodopsin, including the common P23H substitution, lead to protein misfolding, which is a feature in many neurodegenerative disorders. Previous studies have shown that upregulating molecular chaperone expression can delay disease progression in models of neurodegeneration. Here, we have explored the potential of the heat-shock protein co-inducer arimoclomol to ameliorate rhodopsin RP. In a cell model of P23H rod opsin RP, arimoclomol reduced P23H rod opsin aggregation and improved viability of mutant rhodopsin-expressing cells. In P23H rhodopsin transgenic rat models, pharmacological potentiation of the stress response with arimoclomol improved electroretinogram responses and prolonged photoreceptor survival, as assessed by measuring outer nuclear layer thickness in the retina. Furthermore, treated animal retinae showed improved photoreceptor outer segment structure and reduced rhodopsin aggregation compared with vehicle-treated controls. The heat-shock response (HSR) was activated in P23H retinae, and this was enhanced with arimoclomol treatment. Furthermore, the unfolded protein response (UPR), which is induced in P23H transgenic rats, was also enhanced in the retinae of arimoclomol-treated animals, suggesting that arimoclomol can potentiate the UPR as well as the HSR. These data suggest that pharmacological enhancement of cellular stress responses may be a potential treatment for rhodopsin RP and that arimoclomol could benefit diseases where ER stress is a factor.     

A Ligand Channel through the G Protein Coupled Receptor Opsin

The G protein coupled receptor rhodopsin contains a pocket within its seven-transmembrane helix (TM) structure, which bears the inactivating 11-cis-retinal bound by a protonated Schiff-base to Lys296 in TM7. Light-induced 11-cis-/all-trans-isomerization leads to the Schiff-base deprotonated active Meta II intermediate. With Meta II decay, the Schiff-base bond is hydrolyzed, all-trans-retinal is released from the pocket, and the apoprotein opsin reloaded with new 11-cis-retinal. The crystal structure of opsin in its active Ops* conformation provides the basis for computational modeling of retinal release and uptake. The ligand-free 7TM bundle of opsin opens into the hydrophobic membrane layer through openings A (between TM1 and 7), and B (between TM5 and 6), respectively. Using skeleton search and molecular docking, we find a continuous channel through the protein that connects these two openings and comprises in its central part the retinal binding pocket. The channel traverses the receptor over a distance of ca. 70 Å and is between 11.6 and 3.2 Å wide. Both openings are lined with aromatic residues, while the central part is highly polar. Four constrictions within the channel are so narrow that they must stretch to allow passage of the retinal β-ionone-ring. Constrictions are at openings A and B, respectively, and at Trp265 and Lys296 within the retinal pocket. The lysine enforces a 90° elbow-like kink in the channel which limits retinal passage. With a favorable Lys side chain conformation, 11-cis-retinal can take the turn, whereas passage of the all-trans isomer would require more global conformational changes. We discuss possible scenarios for the uptake of 11-cis- and release of all-trans-retinal. If the uptake gate of 11-cis-retinal is assigned to opening B, all-trans is likely to leave through the same gate. The unidirectional passage proposed previously requires uptake of 11-cis-retinal through A and release of photolyzed all-trans-retinal through B.     

Human Blue Cone Opsin Regeneration Involves Secondary Retinal Binding with Analog Specificity

Human color vision is mediated by the red, green, and blue cone visual pigments. Cone opsins are G-protein-coupled receptors consisting of an opsin apoprotein covalently linked to the 11-cis-retinal chromophore. All visual pigments share a common evolutionary origin, and red and green cone opsins exhibit a higher homology, whereas blue cone opsin shows more resemblance to the dim light receptor rhodopsin. Here we show that chromophore regeneration in photoactivated blue cone opsin exhibits intermediate transient conformations and a secondary retinoid binding event with slower binding kinetics. We also detected a fine-tuning of the conformational change in the photoactivated blue cone opsin binding site that alters the retinal isomer binding specificity. Furthermore, the molecular models of active and inactive blue cone opsins show specific molecular interactions in the retinal binding site that are not present in other opsins. These findings highlight the differential conformational versatility of human cone opsin pigments in the chromophore regeneration process, particularly compared to rhodopsin, and point to relevant functional, unexpected roles other than spectral tuning for the cone visual pigments.