Light Dynamics of the Retinal‐Disease‐Relevant G90D Bovine Rhodopsin Mutant

Abstract The RHO gene encodes the G‐protein‐coupled receptor (GPCR) rhodopsin. Numerous mutations associated with impaired visual cycle have been reported; the G90D mutation leads to a constitutively active mutant form of rhodopsin that causes CSNB disease. We report on the structural investigation of the retinal configuration and conformation in the binding pocket in the dark and light‐activated state by solution and MAS‐NMR spectroscopy. We found two long‐lived dark states for the G90D mutant with the 11‐cis retinal bound as Schiff base in both populations. The second minor population in the dark state is attributed to a slight shift in conformation of the covalently bound 11‐cis retinal caused by the mutation‐induced distortion on the salt bridge formation in the binding pocket. Time‐resolved UV/Vis spectroscopy was used to monitor the functional dynamics of the G90D mutant rhodopsin for all relevant time scales of the photocycle. The G90D mutant retains its conformational heterogeneity during the photocycle.


Introduction
Rhodopsin, an archetypical G-protein-coupled receptor (GPCR), belongs to the most studied G-protein coupled transmembrane receptor family.T he retinal chromophore, aderivative of vitamin A, is akey player in the photocycle of rhodopsin. It is bound to opsin [1] as Schiff base with the side chain of K296 and is stabilized by the side chain of the counter ion E113. [2] Upon illumination, the retinal cofactor undergoes a11-cis to all-trans isomerization, which induces aconformational change of rhodopsin, resulting in several intermediate states of the photocycle that can be distinguished by UV/Vis spectroscopy. [3] Thelight-activated Meta II state,inwhich the retinal is bound in the all-trans conformation, initiates the photo-transduction cascade. [4] This state is linked to the deprotonation of the Schiff base that results in an absorption maximum shift to 380 nm and is coupled to the disruption of as alt bridge with the side chain of E113 that becomes protonated indicating ap roton transfer in the hydrophobic binding pocket. [5] This initial photochemical step leads to the largest conformational changes of rhodopsin in the photocycle accompanied by opening of the Gp rotein binding site followed by the transducin activation. [4,6] In contrast to bacteriorhodopsin, the photocycle of mammalian rhodopsin involves retinal release and uptake and results in decay into opsin and free all-trans retinal. The decay of rhodopsin to opsin and free retinal can proceed through two alternative pathways with different kinetics. Relaxation via the Meta II state is characterized by adeprotonated all-trans retinal and ap rotonated counter ion E113. With ad uration of five minutes,t his process is five times faster than the relaxation via the Meta III state (25 minutes). [7] In contrast to Meta II, the Meta III state is characterized by ap rotonated retinal in all-trans-15-syn conformation with an absorption maximum of 465 nm. Since this pathway is significantly slower than the Meta II decay,the Meta III state has been proposed to act as as torage conformation of inactive rhodopsin. Approximately 40 %o f the thermal relaxation takes place via this slow kinetic pathway populating the Meta III state.I nc ontrast to this light-activated Meta II state,w hich is directly involved in signaling by interaction with visual Gp rotein, the Meta III state is inactive.T he light-induced helical rearrangements mostly affect the secondary structure elements located in the cytoplasmic region, which are crucial for the activation of the Gprotein. Furthermore,while the structure of the rhodopsin Meta II state is well studied (pdb:3pxo), [4] the Meta III state remains poorly understood and is often even not mentioned in the publications.
Changes at any step of the photocycle can impair the visual cycle and therefore lead to numerous visual disorders. [8] Ah otspot for mutations of the wild type sequence is amino acid G90. Depending on the nature of the introduced amino acid, mutations of G90 can either lead to the most common human-inherited retinal dystrophy night blindness disease called retinitis pigmentosa (RP) [8] or congenital stationary night blindness (CSNB) [9,10] (G90V and G90D,r espectively). CSNB is an on-progressive inherited retinal disorder, which was found to be genetically and clinically heterogeneous.First symptoms of this disease are the reduction of dim and night vision, problems with the adaptation to darkness and in some cases loss of the general visual acuity.C SNB exhibits an overlapping phenotype with visual diseases such as RP, progressive rod-cone dystrophy and acquired night blindness (vitamin Adeficiency) but in contrast to these diseases,CSNB is non-progressive. [11] Currently,t here are no preventive measures for this disease,s imilar to RP.G ene therapy [12] and photoreceptor transplantations 13] are possible future cures,which are under development.
All CSNB relevant mutations are located in the retinal binding pocket and lead to constitutively active rhodopsin mutants. [14][15][16][17] Crystal structures of the constitutively active G90D mutant are only available for the ligand-free opsin conformation and the light-activated state (pdb:4 bez). [18] So far, no structural data of the G90D mutant in the dark state has been reported and ac omprehensive characterization of the retinal binding pocket could not yet be achieved. In their paper,S tandfuss and co-workers argue that structural heterogeneity due to the presence of opsin and rhodopsin prevents crystallization. Further,they propose that the ground state of the G90D mutant is destabilized due to the E113-K296 Schiff base disruption that would lead to increased rate of retinal thermal isomerization. Although the light active conformation of the G90D mutant was shown to be stabilized and structurally very similar to the wild type Meta II conformation, the conducted crystallographic refinement of the binding pocket indicated amixture of non-covalently bound retinal cis isomers.
Here,w ep resent ad etailed structural characterization employing liquid-and solid-state NMR together with timeresolved optical spectroscopy to contribute to our understanding of the disease-induced basal activity of G90D mutant rhodopsin. [15] In accordance with X-ray data performed by Standfuss and co-workers,the G90D mutant with the thermal stabilizing disulfide bond in the extracellular side (N2C/ D282C) was investigated. [18][19][20]

Results and Discussion
Influence of the Retinal Binding on the Folding Propensity of the Protein We conducted our experiments by investigating three different rhodopsin constructs:( i) the wild type (WT) construct, (ii)t he stabilized wild type with aN 2C/D282C double mutation (WT S-S )a nd the stabilized CSNB-related G90D mutant (G90D S-S ). Thed ouble mutation N2C/D282C introduces an additional disulfide bond on the extracellular side between the N-terminus and the loop E3, which increases the thermal stability of the protein without significantly affecting its activity and structure. [19,20] However,t hese mutations impede the retinal reconstitution efficiencya nd require optimization in the HEK293 expressed rhodopsin purification strategy.I nc ontrast to the wild type purification, where opsin was reconstituted with 11cis retinal for four hours before the extraction from the cellular membrane, [7] the opsin of the G90D mutant was incorporated with excess of 11-cis retinal overnight only after its solubilization in DDM detergent and binding to antibodies.S uccessful 11-cis retinal incorporation to the G90D opsin was confirmed by the characteristic absorption maximum at 490 nm [15,18] that corresponds to the dark state rhodopsin ( Figure S3).
Retinal binding has ac rucial effect on the overall structure of the protein, as monitored by chemical shift dispersion of the tryptophan side chain indole resonances by liquid-state NMR. Ther etinal-free opsin conformations of WT S-S and G90D S-S show poorly resolved signals at 10.1 ppm of 1D 1 HNMR spectrum, while the retinal-bound ground states display well resolved indole signals,i ndicating significant structural rearrangements and proper folding of the protein ( Figure 1).

Liquid-State NMR Experiments
Five tryptophan residues in the rhodopsin sequence were selectively 15 Ni sotope labeled using as tably transfected cell line in HEK293 cells and used as reporter signals in liquidstate NMR experiments.R esidues W126 3.41 ,W 161 4.50 and W265 6.48 are highly conserved among GPCRs.T hey are located in the trans-membrane region and are involved in light-induced conformational changes. [1] Thet ryptophan signals are sensitive to light-induced conformational changes of the protein, resulting in chemical shift perturbations (CSPs) of the light active conformation. [7] Monitoring NMR signals in 2D SOFAST-HMQC 1 H, 15 NN MR experiments for the different constructs thus provides ad irect readout for potential conformational changes.Nodifference in the indole resonances between WT and WT S-S could be detected, indicating no effect of the N2C/D282C mutation on the ground state ( Figure S4). However,t he stabilizing effect of the disulfide bridge was clearly observed on the illuminated rhodopsin conformation that, in contrast to WT,d id not aggregate and remained stable ( Figure 2A).
Thel ight-induced photocycle of the not-stabilized wild type is completed within 25 minutes,r esulting in opsin and free all-trans retinal. [7] Retinal release from the binding pocket is irreversible and leads to sample aggregation of opsin, accompanied with the decrease and vanishing of all tryptophan signal intensities.D ue to the low sample concentration, all 2D spectra of WT S-S and G90D S-S were recorded within several hours,which is beyond the photocycle regime. This is in contrast to previously published spectra by Stehle et al.,where the high sample concentration allowed us to run experiments for shorter times and signals for the light state could be recorded. [7] Thetryptophan resonances of WT S-S in the light state are in agreement with the chemical shift assignment of WT in the Meta II state. [7] Three residues located in the transmembrane region (W265 6.48 ,W 161 4.50 and W126 3.41 )a re sensitive to the light-induced conformational changes and show characteristic Meta II CSPs,w hile two other tryptophan amino acids from the extracellular domain (W35 1.30 and W175 4.65 )d on ot undergo any chemical shift perturbations and are resistant to the protein structural rearrangements.
Remarkably,t he G90D S-S mutant shows significant CSPs of the W161 4.50 and W265 6.48 signals and, more importantly,an additional signal at 11.65 ppm (labelled WX in Figure 2B). Taking into account that rhodopsin has five tryptophan residues,t he additional sixth tryptophan signal indicates the presence of two long-lived states of the protein. We cloned and expressed the following triple mutants G90D S-S (W161F) and G90D S-S (W265F) to try to assign the additional sixth signal. This W-Fm utation strategy could be successfully applied to assign the 5t ryptophan reporter signals in WT rhodopsin. [21] In fact, the protein can be cloned and expressed ( Figure S1). However,i nt he G90D S-S mutant series,t he additionally introduced mutation leads to lower affinities of retinal to the triple mutant protein, and as ar esult, reconstitution and purification of these mutants was not possible.
Interestingly,u nlike WT S-S ,t he G90D S-S NMR signals of tryptophan residues do not change upon light activation ( Figure 2B). Even W265, which is located in the binding pocket and is highly sensitive to the retinal isomerization, shifts downfield (vs.upfield shift of WT) much less compared to the WT S-S .
Thea dditional WX signal at 11.65 ppm also does not undergo significant CSPs in the dark and light states,showing an almost identical chemical shift compared to the light induced downfield shift of the W161 from WT S-S in Meta II state.W 161 4.50 is located in the middle of H4, which is not directly involved in the light-induced conformational changes. But due to the direct connection to H3, which undergoes large structural rearrangements,t his residue is co-affected by the light exposure and is involved indirectly in the Meta II formation. This suggests the Meta II-origin of the additional sixth signal from the G90D S-S mutant, which supports the hypothesis of ap re-active conformation of the G90D S-S mutant in the dark state.
Based on its location, G90D would be expected to lead to adisruption of the ligand binding pocket, which in turn could result in different long-lived ground state populations.   15 NNMR spectra of rhodopsin constructs in the dark and light states. All spectra were recorded at 600 MHz and 298 K. The dark state is colored in blue and the light state is shown in red. Light state experiments started after complete illumination, that was monitored by changes in the absorption maximum at 500 nm and 490 nm for WT S-S and G90D S-S respectively,were performed under light exposure. The experiments have been conducted for 8h.A)Stabilized wild type (WT S-S ). B) Stabilized G90D mutant (G90D S-S ). Arrows indicate CSPs of W161 and W265. The additional tryptophan signal in the G90D S-S mutant is labelled WX, tentative assignments are discussed in the main text.
Retinal binding to K296 in opsin takes place via aS chiff base,w hich causes ac haracteristic chemical shift change for the 15 N-Lys resonance from around 40 to 183 ppm (pSBprotonated Schiff base,v alue re-referenced to liquid ammonium). Upon Schiff base deprotonation, af urther shift by 127 ppm is expected. [22] The2 D 15 N-13 C-TEDOR (Transferred-Echo DOuble Resonance) spectrum of 15 N-G90D S-S with 13 C 2 -retinal shows characteristic crosspeaks between a 15 Np SB resonance at 179.5 ppm and both retinal carbons C14 (125.0 ppm) and C15 (167.7 ppm) providing clear evidence for the Schiff base formation and therefore,f or the retinal being covalently bound to K296 (Figure 3). ThepSB chemical shift is similar to that observed for the wild type and no resonance for adeprotonated SB species is observed ( Figure S8). Downfield to the main pSB signal as houlder around 188.5 ppm can be detected, which could arise from al owly populated second conformation. This second conformation shows additional crosspeaks with C14 and C15 (Figure 3). Theresonance of the retinal carbon C14 also shows am inor conformation at 125.0 ppm (shoulder) for which, however,n oa dditional crosspeak with the pSB could be detected within the signalto-noise limitations of this experiment.
In order to test whether the observed conformational heterogeneity can also be detected for other retinal carbons, the WT S-S and the G90D S-S mutant was reconstituted with 13 C 3 (C12,13,20)-retinal. Here,p ositions C12, C13 and C20 were chosen as the most light-sensitive retinal carbons,which are in close proximity to the Schiff base.
Ac omparison between double quantum filtered (DQF) 13 C-spectra of WT S-S and G90D S-S is shown in Figure 4A,B. Thec hemical shift assignment was deduced from the 2D double quantum-single quantum (DQ-SQ) correlation experiment ( Figure 4B,C) and is consistent with Patel et al. [23] However,i nc ontrast to C13, whose chemical shift (170.4 ppm) remains similar for both constructs,C 12 and C20 of G90D S-S show significant downfield CSPs of 0.5 and 0.7 ppm with respect to WT S-S .

Light State Solid-State NMR Experiments
Further characterization of the retinal conformational changes induced by illumination and analysis of the impact of the G90D mutation on the binding pocket geometry was performed in situ in the MAS rotor under cryogenic conditions at 100 Kfollowed by thermal relaxation. Illuminating the sample at 100 Kf or two hours with blue light allowed trapping of the early photoproduct bathorhodopsin, [24] while its subsequent warming to room temperature led to the light active Meta II state conformation. [23] Similar to the dark state,t he batho and Meta II states show characteristic protonated Schiff base signals at 179.5 ppm. Forb oth intermediates,t he pSB signal profiles are broader than for the dark state,w ith an additional shoulder shifted 3ppm upfield ( Figure 5A). However,t he low signal-to-noise ratio does not allow unambiguous assignment of these shapes to the second conformational population. Thesignal in the protonated SB region (179.5 ppm) and the absence of any signal in the deprotonated SB area ( Figure S8) indicates that the Meta II conformation of the G90D S-S mutant is protonated and not deprotonated as in WT rhodopsin.
Fort he analysis of the conformational changes of the retinal under light exposure the retinal carbons C14 and C15 were chosen as reporters.The DQF spectrum of cryo-trapped G90D S-S bathorhodopsin reveals an ew C14 signal appearing at ac hemical shift of 119.8 ppm, which is consistent with the WT batho state. [24] Thedetectable minor signal at 123 ppm is assigned to ar esidual dark state population which remained due to an incomplete illumination process.C arbon C14 in light adapted Meta II state of the G90D S-S mutant shows conformational heterogeneity with residual signals corresponding to the dark state (123 ppm) and bathorhodopsin (119.8 ppm;F igure 5B,C).
TheM eta II formation is confirmed by an ew signal resonating at 132.6 ppm, which is in agreement with downfield shifted wild type Meta II C14 resonance. [23] Ac loser examination of C14 DQF spectra shows that even in bathorhodopsin al ow intensity Meta II signal around 120 ppm is visible.T his was not observed for the WT [25] and might indicate the presence of the light active state in earlier stages of the photocycle,w hich could possibly result from the preactive conformation in the dark state.F urthermore,t he aldehyde carbon C15 of non-covalently bound retinal would resonate around 190 ppm. [26] Theabsence of any signal in this range provides clear evidence that only bound retinal is detected in the dark state.T he weak spectral feature around The 2D spectrum is overlaid in the F2-dimension with a 13 CD QF spectrum and in the 1D dimension with a 15 N-CP spectrum. The chemical shifts of C14 and C15 could be transferred from Patel et al. [23] See text for further details.

Angewandte Chemie
Research Articles presence of an additional minor retinal species.

Time-Resolved Optical Spectroscopy
We further investigated the effect of the G90D mutation on the rhodopsin photocycle by the kinetic experiments.T he time-resolved absorption measurements comprise kinetics from picoseconds up to two hours and provide information on the early retinal isomerization processes up to the evolution of the light active Meta II and Meta III intermediate states.
Kinetic experiments detected af our-fold slower bathorhodopsin state formation of the stabilized wild type.F lash photolysis also reports astabilizing effect of the N2C/D282C mutation. Thus,the decay of the intermediate states,Meta II and Meta III, is significantly delayed, the covalently bound retinal is stabilized and its hydrolysis is prolonged ( Figure 6).  TheG 90D S-S mutant shows as trongly altered photocycle compared to WT [7] and WT S-S ,resulting in aunique signature pattern presented in al ifetime density map (LDM) profile. [27,28] In contrast to the wild type,t he photorhodopsin conformation was detectable for the G90D S-S mutant, indicating the delayed retinal isomerization ( Figure S9). Then ext transition to the bathorhodopsin occurred faster and relaxed more rapidly,r esulting in al onger lived batho state.U nlike WT S-S ,G 90D S-S mutant shows aw eak absorption decay at 0.4 minutes at approximately 490 nm, [29] which is assigned to the photo-intermediate state lumirhodopsin (signature G). This decay is accompanied by an absorption increase at 390 nm, which is due to the formation of ab lue shifted intermediate (signature H) that appears before the active intermediate states are formed. Furthermore,i ntermediate Meta III formation and Meta II decay of the G90D S-S mutant are in the same temporal regime as for the WT S-S ,while Meta III decay is 20 minutes faster. Finally,the Meta II state of the G90D S-S mutant was found in the spectrally slightly blueshifted protonated form (signature C*) confirming the results from solid-state NMR.
Taken together,t he N2C/D282C mutations stabilize the light active conformation of rhodopsin delaying the relaxation of its Meta states.T he photocycle of the G90D S-S mutant is unique,showing aprotonated Meta II state and an untypical retinal behavior:r etinal isomerization is delayed, while the early appearance of ablue shifted component might indicate structural distortion or even transient deprotonation before the active Meta-intermediates are formed.

Conformational Heterogeneity
Both from liquid-state and solid-state NMR spectroscopy, we find evidence for conformational mixture of two longlived states of the CSNB-related G90D mutant in the dark state comprising asecond minor population of the Schiff base and attached retinal. Structural heterogeneity consisting of amixture of opsin and rhodopsin conformations of the G90D mutant in the dark state was proposed by Singhal et al. as ar eason for not obtaining crystals in the dark state.I n addition to that we suppose that the heterogeneity is caused by as econd minor populated protein conformation with incorporated 11-cis retinal. Retinal conformational heterogeneity was observed only on the C14 carbon and is referred to the slightly different steric position of 11-cis retinal in the binding pocket. Furthermore,weidentify the crucial effect of the retinal binding on the overall protein structure.Unlike for the stabilized wild type WT S-S ,liquid-state NMR results of the G90D mutant indicated no significant structural changes of the tryptophan signals between dark and light active conformations,which is consistent with previous results obtained by Fourier-transform infrared (FTIR), [30] electron paramagnetic resonance (EPR), [5] and dynamic single-molecule force spectroscopy (SMFS) [31] experiments.M oreover,u nlike the wild type,t he Meta II conformation of the G90D mutant exists in the protonated Schiff base form fully consistent with the FTIR analysis performed by Zvyaga et al. [30] Changes in the Structure and Photocycle of the G90D Mutant Ther hodopsin photocycle is triggered by light-induced chromophore isomerization. In the dark inactive state,11-cis Figure 6. Kinetic analysis of three constructs. Top: Lifetime density maps of the transienta bsorption data. Positive (red) amplitudes account for decay of absorption, negative (blue) amplitudes account for rise of absorption. Signature Ar epresents free retinal absorption and Bs tands for the ground-state bleaching.B oth belong to the long-lived non-decaying components, which are similar for each sample and mark the end of the photodynamics. Shorter lifetime components Cand Ddescribe Meta II decay and Meta III formation,respectively.Eis assigned to the longer lifetime Meta III decay and Fcorresponds to the free retinal formation, [28] Gd escribes the photo-intermediate state lumirhodopsin, Hi st he partial retinal release,a nd C* is the protonated Meta II decay.B ottom:Schematic representation of the analysis of the lifetimes. Estimated lifetimes reported in the literature but not measured on these constructs are shown in grey.
retinal is covalently bound to K296 via ap rotonated Schiff base.A ni mportant stabilizing role plays the negatively charged E113 residue,w hich acts as ac ounter ion for the protonated Schiff base in the dark state.1 1-cis to all-trans isomerization induces aseries of intermediate photoproducts, resulting in an active Meta II conformation, which in turn is characterized by deprotonated Schiff base and all-trans covalently attached retinal. Thep roton transfer from the all-trans Schiff base bound retinal to the negatively charged counter ion E113 is akey process in the activation switch and retinal release.A na lternative relaxation pathway of the Meta Istate involves Meta III conformation, which is considered as as torage conformation of inactive rhodopsin and comprises protonated retinal in its all-trans-15-syn conformation. Both intermediates,Meta II and Meta III, subsequently decay to opsin and free all-trans retinal. Thec onstitutively active G90D mutation is located in the retinal binding pocket in close proximity to the residues K296 and E113. The charged D90 side chain interacts with the Schiff base K296, perturbing the salt bridge connection between E113 and K296. [15] However,the exact effect of the induced mutation on the retinal conformation had remained unclear. TheG 90D mutation induced distortion in the binding pocket starts in the retinal free opsin conformation. Thec loser steric position of D90 compared to WT counter ion E113 allows the WT unlike salt bridge formation between the Schiff base and the negatively charged D90 side chain, leading to the stabilization of the G90D opsin state.T his reflects on the impeded retinal binding efficiency of the G90D mutant. However, in contrast to the RP associated constitutively active mutations,C SNB related mutants are still able to incorporate retinal, retaining the increased basal activity in the ground state.M utation of glycine to aspartic acid at position 90 leads to acompeting salt bridge formation between the negatively charged E113 and G90, reflecting in the structural heterogeneity of the retinal binding pocket. According to our data, we suggest the Schiff base between 11-cis retinal and K296 to be stabilized by both counter ions,E 113 and D90.
Light-induced proton transfer from positively charged protonated retinal Schiff base to the negatively charged counter ion E113 is apart of the rhodopsin activation switch. [5] In WT,i tr esults in an increased distance between the Schiff base and the side chain carboxyl group of E113 in the light active Meta II state. [4] According to the crystal structure of the light adapted Meta II state of G90D mutant, [18] the distance between K296 and E113 of G90D mutant is similar to the Meta II WT,while D90 is still in close proximate to form the salt bridge and prevent the retinal from its further hydrolysis. Taken together,s imilar to the WT,u pon illumination retinal isomerizes to the all-trans conformation, which is still bound via aSchiff base to the protein. However, the G90D mutation disrupts the hydrogen network, leaving the Meta II state protonated and, therefore,s tabilized. In contrast to the wild type,w here the retinal isomerization is essential for the corresponding intermediate states and is directly related to the activation of the transduction process,the G90D mutation disrupt this structural cascade withdrawing the antagonistic function of the retinal.

Origin of the Constitutive Activation
Constitutively active rhodopsin mutations can be classified into two groups.Inthe first group,changes in the retinal binding pocket and accompanying disturbance of retinal binding and/or retinal release (E113Q, [32,33] K296, [34] A292E, [14] G90D [15,18] and T94I [16,35] )a re proposed. In the second group,t he mutated amino acids are located close to the cytoplasmic side and influences the transducin binding site,w hich is responsible for the Gp rotein activation (M257Y [36] ). An increased basal activity is observed for both diseases (RP and CSNB). However,i ta ppears unlikely that this increased activity is the only reason for their distinct phenotypes.D ifferent mutations of the Schiff base K296 [34] and of its counter ion E113Q [32] lead to ac onstitutive active state of rhodopsin (RP), while four other constitutive active single point mutations A292E, [14] G90D, [15] T94I [16] and quiet recently discovered A295V mutation [17] are known to cause CSNB.Furthermore,the nature of the mutated amino acid at aspecific position can define the pathology,thus G90V leads to RP while G90D causes CSNB. [9,10] Three theories were proposed to explain the increased basal activity of the G90D mutant:anactive opsin conformation was proposed to be able to activate Gprotein transducin in the absence of light, [15,37] an increased thermal isomerization of the retinal in the absence of light [38] and pre-active conformation in the dark state.A lready previously,t he first two models remained ambiguous,l eading to conflicts with several reported studies. [30,39,40] Here,wepresent several arguments that strongly support the pre-active conformation of the G90D mutant.
With liquid state NMR experiments,w ef ind ac rucial influence of 11-cis retinal binding on the folding properties of the G90D mutant. DNP enhanced solid-state NMR experiments reported only 11-cis retinal bound to the G90D protein in the dark state,excluding the effect of spontaneous isomerization in the ground state.T he second retinal population observed in the dark state does not originate from the light active form and is attributed to the slightly different steric position of the 11-cis retinal. Absence of spontaneous isomerization is supported by the ultrafast absorption spectroscopy that showed the delayed light-induced retinal isomerization of the early photoproducts of the G90D mutant compared to the WT.
Theunambiguous evidence for apre-active conformation is provided by liquid-state NMR experiments.T he high similarity between the spectra of G90D mutant, recorded under dark and light conditions,i si ndicative for the third model of the increased basal activity.Furthermore,structural heterogeneity observed by both, liquid-and solid-state NMR, is consistent with the model proposed by Dizhoor et al. [39] Conclusion Taking together,our results revealed important structural information regarding the CSNB related G90D mutant in the dark and light Meta II state.F or the first time the retinal conformation is characterized for three protein states:d ark state G90D rhodopsin, G90D bathorhodopsin and G90D Meta II state.
First, we demonstrate retinal being bound to the protein via aS chiff base in all detected states.T he conformational heterogeneity of the binding pocket of the G90D mutant in the dark state is originated from the slightly different steric position of the covalently bound 11-cis retinal. Furthermore, structural heterogeneity is also reflected on the global protein conformation, which remains similar for both, dark and light state of the G90D mutant. These data in combination with aunique photocycle of the G90D mutant provide evidence for the pre-active ground state theory as an explanation of the increased basal activity of the mutant and add an important piece of information for the detailed understanding of the molecular mechanism of night blindness disease.