Retinal–Protein Interactions in Halorhodopsin from Natronomonas pharaonis: Binding and Retinal Thermal Isomerization Catalysis

doi:10.1016/j.jmb.2009.09.024 J. Mol. Biol. (2009) 394, 472–484

Available online at www.sciencedirect.com

Retinal–Protein Interactions in Halorhodopsinfrom Natronomonas pharaonis: Binding and RetinalThermal Isomerization Catalysis

Tushar Kanti Maiti1, Martin Engelhard2 and Mordechai Sheves1⁎
1Department of OrganicChemistry, The WeizmannInstitute of Science,Rehovot 76100, Israel2Max-Planck-Institut fürMolekulare Physiologie,Otto Hahn Strasse 11,44227 Dortmund, Germany
Received 13 March 2009;received in revised form7 September 2009;accepted 14 September 2009Available online18 September 2009

*Corresponding author. E-mail [email protected].

Abbreviations used: bR, bacteriorhalorhodopsin; NpHR, Natronomonahalorhodopsin; HsHR, Halobacteriumhalorhodopsin; PSB, protonated Schbase; TFE, trifluoroethanol; HFIP, h

0022-2836/$ - see front matter © 2009 E

Halorhodopsin from Natronomonas pharaonis (NpHR) is a member of theretinal protein group and serves as a light-driven chloride pump in whichchloride ions are transported through the membrane following lightabsorption by the retinal chromophore. In this study, we examined twomain issues: (1) factors controlling the binding of the retinal chromophore tothe NpHR opsin and (2) the ability of the NpHR opsin to catalyze thethermal isomerization of retinal isomers. We have revealed that the recons-titution process of pharaonis HR (NpHR) pigment from its apoprotein andall-trans retinal depends on the pH, and the process has a pKa of 5.8±0.1. Itwas proposed that this pKa is associated with the pKa of the lysine residuethat binds the retinal chromophore (Lys256). The pigment formation isregulated by the concentration of sodium chloride, and the maximum yieldwas observed at 3.7 M NaCl. The low yield of pigment in a lower con-centration of NaCl (b3 M) may be due to an altered conformation adoptedby the apomembrane, which is not capable of forming the pigment.Unexpectedly and unlike the apomembrane of bacteriorhodopsin, NpHRopsin produces pigments with 11-cis retinal and 9-cis retinal owing to thethermal isomerization of these retinal isomers to all-trans retinal. The iso-merization rate depends on the pH, and it is faster at a higher pH. The pKavalue of the isomerization process is similar to the pKa of the bindingprocess of these retinals, which suggests that Lys256 is also involved in theisomerization process. The isomerization is independent of the sodiumchloride concentration. However, in the absence of sodium chloride, theapoprotein adopts such a conformation, which does not prevent the isome-rization of retinal, but it prevents a covalent bond formation with the lysineresidue. The rate and the thermodynamic parameter analysis of the retinalisomerization by NpHR apoprotein led to the conclusion that the apo-membrane catalyzes the isomerization via a triplet mechanism.

© 2009 Elsevier Ltd. All rights reserved.

Keywords: bacteriorhodopsin; halorhodopsins; pharaonis halorhodopsins;protonated Schiff base; isomerization
Edited by I. B. Holland
Introduction

Retinal proteins, a group of membrane proteins,consist of seven trans-membrane helices that cova-

ress:

hodopsin; HR,s pharaonissalinarum

iff base; SB, Schiffexafluoroisopropanol.

lsevier Ltd. All rights reserve

lently bind a retinal chromophore via a protonatedSchiff base (PSB) linkage to a lysine residue in theseventh helix of the protein. Following light absorp-tion, the retinal undergoes isomerization, which in-duces protein structural alterations, consequentlyinitiating biological functions such as vision (rho-dopsin), proton pump [bacteriorhodopsin (bR)],chloride pump [halorhodopsin (HR)], or phototaxis[sensory rhodopsin (SRI and SRII)].HR is a light-driven chloride pump that pumps

chloride ions from the extracellular to the cytoplas-mic side.1 Various analogues of HR have been iden-tified and reported, but the most studied pigments

d.

http://dx.doi.org/10.1016/j.jmb.2009.09.024

mailto:[email protected]

473Retinal–Protein Interactions in NpHR

are those from Halobacterium salinarum (HsHR) andNatronomonas pharaonis (NpHR).2,3 N. pharaonis wasisolated from soda lakes in North Africa, whose pHis 10.5–11.4,5 These organisms grow optimally at 4 MNaCl like halobacteria but at pH 9.5 rather than atpH 7. They are only distantly related to the halo-bacteria, as indicated by a significant difference inthe GC content of their DNAs.6 The finding of retinalpigment in NpHR, which functions as a chloridepump, is important for the extension of organismsthat harbor retinal proteins. Therefore, it is interest-ing to extend the studies of HRs to NpHR. In bothHRs, functionally important exceptions are theproton acceptor Asp85 and the proton donorAsp96 in bR, which are replaced by Thr and Ala,respectively. Lack of a proton acceptor creates ananion binding site and ensures that during thephotocycle, the Schiff base (SB) remains protonated,while chloride is transported through themembrane.At present, the crystal structure of NpHR remains tobe determined, whereas the structure of HsHR isknown at 1.8 Å resolution.7 However, based on thehigh level of homology between the two chloride ionpumps and their similar photocycle, it is conceivablethat both proteins have similar structures. The crys-tal structure of HsHR reveals one chloride bindingsite and a cluster of three water molecules in theretinal binding pocket.7 From earlier studies, threechloride binding sites were proposed, one of which isat the transport site and has an affinity of 2.5 mM; it islocated in the vicinity of PSB.8 This location wasfound via X-ray studies and was also previouslysuggested by Fourier transform infrared spectroscopyand resonance Raman data9,10 as well as by absorp-tion spectroscopy.11,12 The second binding site was atthe cytoplasmic release site, with an affinity of 5.7 M,whereas the third one was suggested at the extracel-lular site, with a binding constant of 200 mM.13

The photocycle of NpHR is still under debate. Onedescription of the photocycle includes the followingevents: NpHR→→K↔L↔N↔O↔NpHR′.8 In thismodel, the release of chloride ions occurs duringthe N→O transition, whereas the uptake of chlo-ride ions occurs during the O→pHR′ step. Inanother model, in analogy to the M1→M2transition in the bR photocycle, two O intermedi-ates are always in fast equilibria with L and Nintermediates, respectively, suggesting the follo-wing: NpHR→→K→L→L→ (L↔O)→ (O↔N)→NpHR′.8,14–16 In this scheme, chloride release anduptake occur during the fast L↔O and O↔Nequilibria, respectively. Following kinetic studieson NpHR, a photocycle scheme that includes aspectrally silent transition between two L interme-diates was proposed.17,18

The chloride ion can be replaced by other anions,as revealed by titration with different sodium saltsmonitored by absorption spectroscopy.11,18 Thebinding constant of NpHR at pH 6.0 was found tobe 10 mM for azide19 and 11 mM for nitrate.20 If thechloride ion is replaced by anions such as Br−, BrO3

−,I−, NO3

−, and N3−, the PSB does not deprotonate

[except NpHR(N3−)11] during the photocycle. In the

presence of N3−, the chloride pump is converted to a

proton pump similarly to bR and an M-like interme-diate is observed.19 The M-like intermediate is alsoobserved if the chloride ion is replaced by HCOO−,NO2

−, and OCN− anions whose pKa≥3.22.21 It hasbeen previously shown that the reconstitution of thepurple complex from bR apomembrane and retinaloccurs via at least two spectroscopically distinctintermediates22 according to the scheme:

retinalþ bR opsinY400 nm chromophoreY430=460 nm chromophoreYpurple complex

The purple complex can be formed by bindingeither all-trans retinal or 13-cis isomer, whereas 11-cis and 9-cis isomers do not form the purple complex.A planar ring-chain conformation was suggested tobe a prerequisite for pigment formation. All-transretinal and 13-cis retinal fulfill this requirement,whereas 9-cis and 11-cis retinal do not.22,23 At hightemperatures, ∼60 °C, 11-cis retinal produces apigment following mixing with bR opsin in thedark, when 11-cis retinal produces all-trans retinalvia thermal isomerization.The photoisomerization of retinal isomers, as well

as its PSB, has been extensively studied, but thethermal isomerization process of these chromo-phores has been less extensively explored. Retinalthermal isomerization is a very important process inthe photocycle of archaeal rhodopsins such as bR,HRs, and SRs. Following photo-excitation, the all-trans retinal produces 13-cis retinal, which thermallyreisomerizes to all-trans retinal within several milli-seconds. This thermal isomerization is obviouslycatalyzed by the protein, but the exact mechanismhas not been established.24,25 In this respect, it isimportant to extend the thermal isomerizationprocess studies to NpHR. To understand the retinalthermal isomerization in the pigments, it is worth-while to investigate this process in solution (withoutthe protein). It is well known that thermal cis–transisomerization is effectively catalyzed by a variety ofacids,26 amines,27,28 and several nucleophiles.29 Theiodine-catalyzed isomerization of retinals has alsobeen described.30,31 In addition, a limited number ofenzymatic cis–trans isomerization reactions havebeen examined.32 It was suggested that the chargedelocalization along the polyene, especially in theretinal PSB, reduces the energy barrier for the doublebond isomerization and increases the rate of thethermal isomerization.33,34

The functional reconstitution and chromophore–protein interaction of HR (HsHR) have been stu-died,35,36 whereas the reconstitution of NpHR pig-ment with the retinal chromophore has beenexplored less. Moreover, it is very difficult to obtainartificial pigments (derived from synthetic retinalanalogs) of HsHR since the apomembrane is veryunstable and the pigment is only obtained undercertain special conditions.35,36 The photochemicaland transport activities are restored by the reconsti-tution pigment of HsHR, whereas in some retinal

Fig. 1. Absorption difference spectra of the retinal (1.5equivalent) binding process to the apoprotein of NpHR atvarying pH values at 3.2 M NaCl. The process was carriedout at room temperature in the dark for ∼18 h. Bindingspectra are shown at pH 4.2 (i), pH 4.62 (ii), pH 4.8 (iii),pH 5.18 (iv), pH 5.56 (v), pH 5.80 (vi), pH 6.03 (vii),pH 6.50 (viii), pH 6.69 (ix), and pH 6.80 (x).

474 Retinal–Protein Interactions in NpHR

analogues, the reconstitution does not restore thechloride pumping activity.35 The asparagine andarginine residues are the key residues for the ioni-zable amino acid that interacts with the retinal at theactive site. In NpHR, the PSB hydrolyzes at elevatedtemperatures and the rate of hydrolysis is acceleratedfollowing light illumination.37 Upon cooling to 25 °C,80% of the pigment was recovered. However, therecovered pigment differed from the native pigment,which suggests that the apomembrane adopts amodified conformation that rebinds the retinal andproduces a new conformation of NpHR pigment.37

In the present report, we studied the bindinginteraction of retinal isomers with the NpHR opsin.Importantly,we established that the retinal binding toNpHR opsin is regulated by the pH and by the chlo-ride ion concentration. A most interesting feature ofNpHR opsin is its ability to catalyze the retinalthermal isomerization. This isomerization is indepen-dent of the sodium chloride concentration. Wepropose that the protonation state of the retinalbinding site lysine (Lys256) regulates the pigmentformation as well as the retinal isomerization. Thethermodynamic analysis revealed that the isomeriza-tion process strongly resembles the iodine-catalyzedisomerization process of the retinal in solution, andthus, it is conceivable that the mechanism underlyingthe retinal isomerization at the protein binding siteproceeds via a triplet mechanism.

Results and Discussion

Dependence of all-trans retinal binding on pHand on the NaCl concentration

Reconstitution of the NpHR opsin with 1.5 equi-valent all-trans retinal generates a 578-nm pigmentat pH 7.0 and in the presence of 3.2 M sodiumchloride. This reconstitution strongly depends onthe pH of the solution. At a high pH (pH ∼8.0), a578-nm pigment was formed but at a low pH(pH ∼4.2), almost no pigment formation wasdetected. Instead, at low pH, an ∼430-nm absorbingspecies was monitored (Fig. 1). To verify that lack ofpigment formation at a low pH was not due toprotein degradation, we first carried out the bindingprocess at pH 5.0. At this pH, retinal binding gene-rates a 430-nm species along with a small amount ofthe 578-nm pigment. Upon increasing the pH topH 7.0 (by addition of sodium hydroxide), a pig-ment was produced concomitantly with a decreaseof the 430-nm species (Fig. 2a). In order to establishthat both procedures (reconstitution at pH 7 andpH 5 followed by increasing the pH to 7) led tosimilar amounts of pigment, we carried out bleach-ing experiments with hydroxylamine. Similaramounts of pigment (Fig. 2b and c) indicated thatat low pH, the protein did not undergo degradation.It is not entirely clear why protonation of a proteinresidue prevents the binding process. One canexpect that since the binding process is slow,

equilibrium will eventually result in completebinding. However, it is possible that irreversiblepigment formation may “freeze” the unprotonatedprotein fraction and will prevent further pigmentformation. Clarification of this issue requires furtherfuture studies.A plot of pH versus the amount of pigment gene-

rated produced a sigmoidal curve (Fig. 3) fromwhich a pKa of 5.8±0.1 was calculated. It is rea-sonable to assume that this pKa is associated withLys252 (the lysine in the retinal binding site).Protonation of this residue would prevent SBformation. Although the pKa of a lysine residue isusually around 10, it should significantly decrease inthe environment of the protein to allow SB formationat physiological pH values. In bR, the binding reac-tion needed to form the pigment proceeds even at apH as low as 3.0. One way to tune the lysine groupfor such a reaction would be to partially deprotonatethe amino group by the presence of another appro-priately positioned group in its vicinity, which willserve as the proton acceptor. In bacterio-opsin,Asp85 might play a major role, accepting a protonfrom the amino group of Lys216. Asp212, which ispresent in the Lys216 vicinity, cannot completelysubstitute Asp85 as the proton acceptor. Similarly,Asp212 does not serve as a proton acceptor in D85Nmutant photocycle, which does not form an M-likeintermediate (characterized by a deprotonated reti-nal SB). NpHR lacks the equivalent of Asp85 residue,and therefore, the pKa of the lysine residue in NpHRopsin is higher than that in bacterio-opsin but still issignificantly lower than the usual lysine pKa.Similarly to the bR D85N mutant, in NpHR,Asp227 (the equivalent of Asp212 in NpHR) cannotsubstitute Asp85 as a proton acceptor. The relativelylow pKa may be attributed to a relatively nonpolar

Fig. 2. (a) The absorption difference spectra of retinalbinding (1.5 equivalent) to NpHR opsin at 3.2 M NaCl inthe dark for ∼18 h at pH 7.0 (i) and at pH 5.0 (ii). After∼18 h binding at pH 5.0, the pH of the solution waschanged to pH 7.0 using NaOH and was incubated foranother∼18 h (iii). (b) The reconstituted pigment at pH 7.0was subjected to a hydroxylamine reaction (1 M) and wasirradiated for 6 h using a 540-nm cutoff filter. The spectraafter 30 s (i), 5 min (ii), 30 min (iii), 2 h (iv), and 6 h (v) areshown. (c) The reconstituted pigment after changing thepH from pH 5.0 to pH 7.0 was subjected to hydroxylaminereaction (1 M) and irradiated for 6 h using a 540-nm cutofffilter. The spectra after 30 s (i), 5 min (ii), 30 min (iii), 2 h(iv), and 6 h (v) are shown.


environment in the Lys252 protein vicinity. In thisrespect, we noted that low lysine pKa was alsodetected in classical studies on acetoacetate decar-boxylase by Kokesh and Westheimer.38 We notedthat the pH effect on the spectroscopy and photo-cycle of HRs was previously observed and that,below pH 5, the photocycle yield is very low.39

The NpHR opsin did not generate a pigmentfollowing incubation with all-trans retinal in theabsence of sodium chloride. The binding of all-trans

Fig. 3. Effect of pH on the yield of the pigmentformation following all-trans retinal binding. Black, red,blue, and green lines represent the binding at 3.2 M NaCl,3 M NaBr, 3 M NaNO3, and 3 M NaN3, respectively.

retinal with NpHR opsin in various sodium chlorideconcentrations at pH 7.5 produced a bell-shapedcurve in which the best yield of pigment generationwas obtained at 3.76 M sodium chloride concentra-tion. Above this concentration, the yield of pigmentformation decreased considerably (Fig. 4). Thereason underlying this effect is not clear, but it isprobably associated with a high concentration ofchloride anion inducing a protein conformation thatcannot form the pigment. At a low salt concentration,part of the opsin is inactive in generating a pigment,and this is probably due to conformational altera-tions. To support this interpretation, we carried outthe binding experiment at 1 M salt concentration foran extended period of time (overnight at pH 7.0) todetermine whether more pigment will be formed.However, this extended incubation time did notincrease the yield. Furthermore, the elevation of saltconcentration to 3.2 M did not generate additionalpigment. Therefore, it can be concluded that a highsalt concentration (N3 M) is needed for apoproteinstabilization and that low salt concentrations irre-versibly lead to a protein conformation that cannotform the pigment. The binding of chloride to NpHRcan be detected by a blue shift of the absorptionmaximum11 characterized by a dissociation constantof 1.7 mM, but the stability of the pigment is notaffected, and the change in the absorption isreversible. Moreover, exposing NpHR pigment tolow salt concentration followed by bleaching at 3.2Msodium chloride yielded NpHR opsin that generateda pigment following incubationwith all-trans retinal.This demonstrates that the retinal chromophoreplays a role in stabilizing NpHR, and its opsin issignificantly less stable. The effects of chloride anionson L33 HR membrane were previously reported. Itwas observed that addition of chloride affected thephotocycle kinetics and efficiency, and at high pHvalues, it induced spectroscopic changes as theabsorption increased at 590 nm and decreased at410 nm.39

Fig. 4. Effect of sodium chloride concentration on theyield of pigment following all-trans retinal (1.5 equivalent)binding at pH 7.5.

Fig. 6. (a) Effect of pH on the binding yield of 11-cisretinal at 3.2 M NaCl (black) and at 3 M NaBr (blue). (b)Effect of pH on the isomerization of 11-cis retinal at 3.2 MNaCl (black) and without NaCl (blue).


It has been noted that certain monovalent anionsare transported by NpHR. Therefore, pigment for-mation was studied independent of the type ofanion. Sodium bromide produced a pigment at 3 Mconcentration with λmax=576 nm. As was observedfor NaCl, the pigment formation was also pHdependent (pKa=5.8±0.1). Other anions testedgave lower yields, with nitrate higher than azide(Fig. 5). The pKa values were found to be 5.7±0.1(3 M NaNO3) and 6.5±0.1 (NaN3). The latter pKa isrelatively far from the pKa of NaN3 in water (4.7),and therefore, it is conceivable that the protonatedform of NaN3 is low and that the measured pKa (6.5)is not distorted. This picture will be different,however, if the protein significantly raises the pKa.Previously, it was reported that the NpHR mem-brane transports nitrate ions as effectively as chlo-ride and that the absorption kinetic measurementshowed that the photocycle of NpHR(NO3

−) is verysimilar to that of NpHR(Cl−).20 The equilibriumconstants (half maximal binding) for chloride andnitrate at pH 6.0 are 1 and 16mM, respectively.11 Wenoted that the high concentration of the variousanions needed for the reconstitution process is incontrast with the low equilibrium constants for thebinding of the various anions.11 Obviously, theanions' effect on the retinal binding process is notassociated with the binding to specific sites. Azideions may alter the structure of the apomembraneupon binding, causing the active-site lysine pKa toincrease. In addition, azide anion may not stabilizethe apomembrane as effectively as chloride does,resulting in a lower yield of pigment formation. Thelow yield of pigment formation in the presence ofnitrate ion can similarly be explained.

Binding of 11-cis retinal

It was reported that bacterio-opsin does not pro-duce pigment following incubation with 11-cisretinal. It produces only 430-nm species (following

Fig. 5. Absorption difference spectra of all-trans retinalbinding (1.5 equivalent) to the apoprotein of NpHR atpH 7.5 under salt-free conditions (i), at 3 M NaN3 (ii), at3 M NaNO3 (iii), and at 3.2 M NaCl (iv).

occupation of the opsin retinal binding site) probablysince the non-planarity of the 11-cis retinal isomerperturbs the covalent bond formation between theretinal and the binding site lysine residue.23 How-ever, at a higher temperature (∼60 °C), 11-cis retinalthermally isomerizes to all-trans retinal and pro-duces a pigment. We carried out a binding experi-ment between NpHR opsin and 1.5 equivalent 11-cisretinal at pH 7.5 at 25 °C in the presence of 3.2 Msodium chloride in the dark for ∼18 h. Unexpec-tedly, 11-cis retinal generated a pigment at roomtemperature with a half time of about 80 min. Thebinding characteristic was similar to that of all-transretinal at different pH values (Fig. 6a), yielding a pKaof 6.0±0.1 for the binding. A similar binding processwas observed for 11-cis retinal in the presence of 3 MNaBr, yielding a pKa of 6.1±0.1.At low pH, a 430-nm species was produced,

similar to the behavior of all-trans retinal. Theformation of 430-nm species supports the assump-tion that the 11-cis retinal chromophore occupies theretinal binding site but does not form the retinal–protein covalent bond. Incubation of 1.5 equivalentof 11-cis retinal with NpHR opsin for 20 min atpH 7.5 in the presence of 3.2 M sodium chloridefollowed by retinal extraction and HPLC analysisclearly indicated that no trace of 11-cis retinal waspresent in the mixture. The pigment fraction formedwithin this time period is ∼8–10%. This observationclearly indicates that the isomerization of 11-cisretinal occurs prior to the pigment formation. Theabove explanation has also been substantiated fromthe analysis of the rate. The isomerization rate of 11-cis retinal at pH 7.5 in the presence of 3.2 M NaCl at25 °C is 2.02×10− 3 s− 1. The isomerization is fast (t1/2at 25.5 °C∼5 min), whereas the binding reaction isslow (t1/2∼80 min). Approximately 4–5% iso-merization occurs in a pH 7.5 buffer, even after48-h incubation in the dark at 25 °C.The activation energy for 11-cis retinal isomeriza-

tion to all-trans isomer in solution is quite high, and

Table 1. Rate constants (k) for retinal isomerization at different temperatures11-cis retinal Temperature (K) k1 (sec

−1)

NpHR opsin, pH 7.5, 3.2 M NaCl 290 6.50×10−4

294 9.90×10−4

298.5 2.02×10−3

308 7.23×10−3

NpHR opsin, pH 5.0, 3.2 M NaCl 299 2.60×10−4

304 5.10×10−4

309.5 1.30×10−3

314.5 2.27×10−3

NpHR opsin, salt- free, pH 7.5 291 7.30×10−4

298 1.79×10−3

303 3.93×10−3

bR opsin, pH 7.0, 0.1 M NaCl 303 3.00×10−5

308 6.00×10−5

314.5 1.30×10−4

D85N bR, pH 7.0, 0.1 M NaCl 303 8.00×10−5

308 1.40×10−4

311 2.30×10−4

314 3.50×10−4

Dioxane (retinal concentration=28 μM) 358 3.00×10−5

363 4.00×10−5

368 7.00×10−5

371 1.10×10−4

Dioxane-I2 (retinal=28 μM, I2=30 μM) 319 2.90×10−4

324 9.90×10−4

329 1.59×10−3

334 2.03×10−3

TFE 329 7.00×10−5

334 9.00×10−5

339 1.30×10−4

10% HFIP 280 7.13×10−4

284 7.53×10−4

289 1.03×10−3

298 2.2×10−3

n-Heptanea (retinal=15–20 μM) 353 1.02×10−5

356 1.41×10−5

360.6 2.18×10−5

365.2 3.58×10−5

n-Heptane-I2a (I2=7.02 μM), retinal=15–20 μM 303.5 1.21×10−4

307.5 1.77×10−4

312.5 3.47×10−4

317.3 6.23×10−4

321.2 1.39×10−3

9-cis retinal Temperature (K) k1 (sec−1) k2 (sec

−1)

Dioxane-I2 (30 μM I2) 323 2.80×10−4

328 9.70×10−4

333 2.12×10−3

338 2.85×10−3

343 3.57×10−3

348 6.04×10−3

Dioxane-I2 (164 μM I2) 333 1.79×10−3 1.3×10−4

343 5.22×10−3 2.6×10−4

353 6.87×10−3 5.6×10−4

363 1.05×10−2 1.3×10−3

NpHR opsin, salt-free, pH 7.5 298 2.65×10−3 5.80×10−4

303 3.72×10−3 9.50×10−4

308 4.48×10−3 1.13×10−3

313 5.30×10−3 2.16×10−3

13-cis retinal Temperature (K) k1 (sec−1)

Dioxane-I2 (30 μM I2) 303 6.50×10−4

308 8.40×10−4

313 1.16×10−3

318 3.48×10−3

NpHR opsin, pH 7.5, salt- free 298 1.08×10−3

303 1.51×10−3

308 2.55×10−3

313 4.32×10−3

a Data obtained from Ref. 30.


Fig. 7. Time course of 11-cis retinal isomerization byNpHR opsin. (a) Isomerization of 11-cis retinal by NpHRopsin at 3.2 M NaCl, pH 7.5, at 17 °C (black), 21 °C (red),25.5 °C (green), and 35 °C (blue). (b) Isomerization of 11-cisretinal by NpHR opsin at 3.2 M NaCl, pH 5, at 26 °C(black), 31 °C (red), 36.5 °C (green), and 41 °C (blue). (c)Isomerization of 11-cis retinal by NpHR opsin withoutNaCl, pH 7.5, at 18°C (black), 25 °C (red), and 30 °C(green).


the isomerization requires relatively high tempera-tures. Therefore, apparently, the thermal isomeri-zation process that occurs following incubation of11-cis retinal with the apomembrane is catalyzed bythe apoprotein. To support the possibility that the11-cis retinal isomerization process requires theretinal to occupy the retinal binding site, we incu-bated 11-cis retinal with NpHR membrane at roomtemperature for ∼18 h. The isomerization of 11-cisretinal to all-trans retinal was followed by chromo-phore extraction and HPLC analysis. We clearlyobserved that no isomerization of 11-cis retinal hadoccurred (data not shown). This suggests that retinalisomerization occurs only if the retinal chromophoreoccupies the retinal binding site of the apoprotein.Thus, we can conclude that upon binding of 11-cisretinal to the apoprotein, the retinal first isomerizesto all-trans retinal before reacting with the ɛ-aminogroup of Lys252 to form the PSB linkage.To shed further light on the isomerization

process, we checked whether the isomerization of11-cis retinal in the apoprotein is pH dependent.Importantly, we observed that the isomerizationprocess is faster at pH 7.5 (2.02×10−3 s−1) than atpH 5.0 (2.6×10− 4 s− 1) at 25.5 °C in the presence ofNaCl (Table 1). The pKa of this reaction was 5.8±0.1(Fig. 6b), which is identical with the pKa detected inthe formation of the retinylidene SB. This suggeststhat the same NpHR opsin residue is involved inboth 11-cis retinal isomerization and pigmentformation. As outlined above, this residue mightbe assigned to Lys252, which is characterized by arelatively low pKa. Apparently, protonation of thisactive-site lysine inhibits both reactions: isomeri-zation and pigment formation.

Catalysis of 11-cis retinal isomerization

The NpHR opsin-catalyzed isomerization of 11-cisto all-trans retinal in the presence of 3.2 M sodiumchloride at pH 7.5 and pH 5 was monitored atdifferent temperatures by HPLC analysis (Fig. 7 andTable 1). The ratio of the isomerization rate at pH 7.5and pH 5.0 at 25 °C is ∼10. Under salt-free condi-tions, the isomerization rate did not change sub-stantially. The pKa of the isomerization of 11-cisretinal in the absence of NaCl is 6.0±0.1 (Fig. 6b).The ratio between the rate of isomerization in thepresence and absence of sodium chloride at 25 °C(pH 7.5) is ∼1.12. This result suggests that althoughsodium chloride affects the apoprotein conforma-tion, it interferes with retinal–protein covalent bondformation but not with catalysis of the thermal iso-merization process. The new conformation adoptedby the protein due to low salt concentration preventsformation of the retinal–protein covalent bond but iscapable of 11-cis isomerization catalysis.To shed light on possible parameters that affect the

rate of 11-cis retinal isomerization, we have studiedthe isomerization process of the free chromophore insolution without the protein. The thermodynamicparameters of 11-cis retinal isomerizationwere deter-mined from the temperature dependence of the iso-

merization rate under different conditions, includingdifferent solvents and iodine effects. An Arrheniusplot was generated by plotting lnk versus 1/T (Fig. 8)according to Eq. (2). Activation energy (Ea) andfrequency factor (A) were calculated from the slopeand intercept of the graph. Activation enthalpy(ΔH‡) and activation entropy (ΔS‡) of this isome-rization reaction were calculated from the slope andintercept of ln(k/T) versus 1/T plot (Eyring plot)(Fig. 8) according to Eq. (3). All the rate constants andparameters are listed in Tables 1 and 2, respectively.Solvents characterized by hydrogen-bonding capa-bility such as trifluoroethanol (TFE) or hexafluoroi-sopropanol (HFIP) (10% in methylene chloride)exhibit energy of activation (Ea), frequency factor(A), and entropy (ΔS‡) values of 13.7 kcal/mol,8.7×104, and −38.4 e.u., respectively, for TFE and10.7 kcal/mol, 1.65×105, and −36.59 e.u., respec-tively, for HFIP. The activation energy is much lowerthan that detected for polar or nonpolar solvents. Inthe presence of neat HFIP, the reaction is so fast thatit was not possible to measure the kinetic parametersat 25 °C by HPLC or UV–Vis spectroscopy. In neatHFIP, the activation energy may further decreaseand accelerate the reaction. The hydroxyl group ofTFE or HFIP is hydrogen bonded with the retinylcarbonyl group; it induces a partial positive chargeon the carbonyl carbon atom, which delocalizesalong the retinal polyene. The positive charge delo-calization lowers the bond order, thereby loweringthe isomerization activation energy. Thus, in thepresence of TFE or HFIP, the isomerization probablyoccurs through an ionic intermediate. An increasedcharge delocalization along the retinal chromophoreand retinal PSB, owing to interaction with fluorinat-ed alcohols, was previously suggested to explain the13C NMR results and the red-shifted absorption that

Fig. 8. Arrhenius plots for the isomerization of 11-cis retinal by NpHR opsin at 3.2 M NaCl, pH 7.5 (a), pH 5.0 (c), andwithout NaCl at pH 7.5 (e). Eyring plots for the isomerization of 11-cis retinal by NpHR opsin at 3.2 M NaCl, pH 7.5 (b),pH 5.0 (d), and without NaCl at pH 7.5 (f).


was induced by fluorinated alcohols.33,40 These stu-dies in solution are relevant for the process in theprotein since the retinal once occupying the binding

Table 2. Thermodynamic parameters of retinal isomerization

Ea (cal/mol) A (sec−1)

11-cis retinal1-Propanola 22,400 9.0×107

n-Heptanea 26,200 1.0×1011

n-Heptane+I2a 24,200 2.0×1013

Dioxane 26,230 2.08×1011

Dioxane-I2 (30 μM) 26,848 9.32×1014

TFE 13,704 8.73×104

10% HFIP in DCM 10,784 1.65×105

NpHR opsin, pH 7.5 24,255 1.17×1015

NpHR opsin, pH 5.0 26,764 8.80×1015

bR opsin, pH 7.0 24,092 7.25×1012

NpHR opsin, salt-free, pH 7.5 24,374 1.14×1014

D85N-bR, pH 7.5 25,540 2.03×1014

9-cis retinalDioxane-I2 (164 μM) 11,888 1.36×105

15,174 9.64×105

Dioxane-I2 (30 μM ) 18,992 5.1×109

NpHR opsin, salt-free, pH 7.5 8417 4.13×103

15,244 8.75×107

13-cis retinalNpHR opsin, salt-free 16,853 2.30×109

Dioxane-I2 (30 μM) 16,873 8.39×108

a Data obtained from Ref. 30.

site can form hydrogen bonds, for example, withbound water present in the lysine residue vicinity.The isomerization process analysis indicated that the

ΔH‡ (cal/mol) ΔG‡ (cal/mol) ΔS‡ (e.u.)

21,700 29,300 −21.4025,500 29,100 −10.0023,600 23,400 +0.7025,507 28,610 −8.4026,198 23,655 +7.7913,041 25,777 −38.4010,209 20,790 −36.6023,597 21,100 +8.2126,154 22,317 +12.5123,478 24,015 −1.7423,806 21,175 +8.9124,927 23,424 +4.87

11,198 24,182 −37.3414,486 26,113 −33.4518,321 23,846 −16.357810 21,255 −44.00

14,630 22,037 −24.00

16,246 21,622 −17.7316,255 22,488 −19.92

Fig. 9. (a) Effect of pH on the binding of 9-cis retinal toNpHR opsin at 3.2 M NaCl. (b) Effect of pH on the isome-rization of 9-cis retinal to NpHR opsin without NaCl.


11-cis retinal isomerization in polar/nonpolar andhydrogen-bonding solvents is associated with nega-tive entropy of activation. It was previously reportedthat the thermal isomerization of 11-cis retinal innonpolar solvents such as n-heptane occurs favor-ably through a hypothetical triplet-state mecha-nism, as indicated by the low value of thefrequency factors, whereas in polar solvents suchas 1-propanol, it occurs through a predominantlysinglet-state mechanism characterized by a highfrequency factor value.30 In both cases, the transi-tion state is considered to be biradical, althoughthere is no unequivocal proof that such a stateexists. The iodine-catalyzed isomerization mecha-nism involved the addition of an iodine radical tothe C11 atom, and it generates a monoradical that isdelocalized over the carbonyl group.30,31 The iodineprobably reacts with the triplet state, and therefore,the reaction proceeds through the triplet state andconsequently the iodine-catalyzed reaction acceler-ates the reaction mainly by increasing the A value,which lowers the entropy barrier without affectingthe Ea.

30 Accordingly, our results indicated that theiodine-catalyzed isomerization of 11-cis retinal isassociated with a higher entropy of activation in allsolvents.In the case of the NpHR opsin-catalyzed isomeri-

zation, the activation energy and enthalpy at pH 7.5are 24.2 and 23.6 kcal/mol, respectively, and atpH 5.0, the values are 26.7 and 26.2 kcal/mol, res-pectively (Table 2). At high pH, the activation energyand activation enthalpy are lower than those innonpolar mediums such as n-heptane, but at lowpH, the activation energy and activation enthalpyare similar to those of n-heptane. It was observedthat the frequency factor at low pH (8.8×1015) isslightly higher than that at high pH (1.17×1015). Theentropy of isomerization at pH 7.5 and 5.0 is +8.2and +12.5 e.u., respectively. The thermodynamicparameters of the isomerization process were notaffected by the removal of sodium chloride at pH 7.5.To shed further light on the isomerization mecha-

nism, we measured the isomerization kinetics fol-lowing incubation of 11-cis retinal with bR opsin in0.1 M sodium chloride at pH 7.0. Interestingly, weobserved that unlike the NpHR opsin, the isome-rization process in the apomembrane of bR was veryslow (t1/2=386 min at 30 °C). The activation energy(Ea), frequency factor (A), enthalpy (ΔH‡), freeenergy (ΔG‡), and entropy (ΔS‡) values are24 kcal/mol, 7.3×1012, 23.5 kcal/mol, 24 kcal/mol,and −1.74 e.u., respectively (Table 2). To study thereasons that underlie the different behavior betweenNpHR opsin and bacterio-opsin, we have examinedbR mutant D85N. Since NpHR lacks a residuecorresponding to the Asp85 residue of bR, wemeasured the isomerization of 11-cis retinal follow-ing incubation with the D85N mutant of bR opsin.The rate is faster than that in bR but still muchslower than that in NpHR opsin (t1/2=137 min at30 °C). The thermodynamic parameters Ea, A, ΔH‡,ΔG‡, and ΔS‡ are 25.5 kcal/mol, 2.0 × 1013,24.9 kcal/mol, 23.4 kcal/mol, and +4.9 e.u., respec-

tively. We found that all parameters lie between thebR and NpHR values. The parameters extracted forall opsin-catalyzed reactions are best mimicked bythe iodine-catalyzed reaction in n-heptane/dioxanesolvent, which is much better than polar orhydrogen-bonded solvents. Considering all para-meters of NpHR, bR, and D85N bR, it is conceiv-able that isomerization in the NpHR opsin occursthrough a triplet mechanism as observed in theiodine-catalyzed reaction. The retinal binding siteoffers the hydrophobic environment to the retinalpolyene, which may promote the radical generationand triplet mechanism. The effect of D85N muta-tion on bR behavior supports the possibility that theabsence of this residue enhances the catalysis of 11-cis retinal and may point to one difference betweenthe catalysis of bR and NpHR. Further studiesshould clarify whether specific protein residuessuch as tryptophanes promote such a mechanismfor the thermal isomerization reaction. As proposedabove, the protonation state of the active lysine inNpHR (Lys256) plays an important role in expedi-ting the isomerization process.

Binding of 9-cis retinal

It was previously reported that 9-cis retinal doesnot produce pigment with bR opsin in the dark. Itoccupies the retinal binding site without changingits absorption spectrum and without forming theretinal–protein covalent bond.23 However, over-night incubation of 9-cis retinal with NpHR opsin(1.5: 1) at pH 7.0 in the presence of 3.2 M sodiumchloride produced a pigment (λmax=578 nm) resembling that obtained from all-trans retinal. Incubationof 9-cis retinal with NpHR opsin at a low pH did notgenerate a 578-nm pigment but produced instead a430-nm absorbing species. Similarly to all-transretinal and 11-cis retinal, pigment formation from9-cis retinal with the NpHR opsin was stronglyregulated by the pH. The pKa of the binding processof 9-cis retinal was 6.6±0.1 (Fig. 9a), which is higherthan the all-trans retinal and 11-cis retinal binding

Fig. 10. (a) Effect of pHon the binding of 13-cis retinal toNpHR opsin at 3.2 M NaCl. (b) Effect of pH on the iso-merization of 13-cis retinalwithNpHRopsinwithoutNaCl.


pKa. Incubation of 9-cis retinal with the NpHR opsinfor 4 h at pH 7.5 in the presence of 3.2 M NaCl,followed by retinal extraction and analysis, revealedthat almost 80% of 9-cis retinal was converted to all-trans retinal, 9–13 dicis, and 13-cis retinal. Therefore,it appears that the apomembrane of NpHR alsocatalyzes the isomerization of 9-cis retinal similar tothat observed for 11-cis retinal. However, the iso-merization process is of the same order of magni-tude as the pigment reconstitution by all-transretinal (t1/2=90 min versus t1/2=80 min). Therefore,it is very difficult to monitor the rate of 9-cisisomerization in the presence of 3.2 M sodiumchloride. To circumvent this difficulty, we carriedout the isomerization experiments under salt-freeconditions, where reconstitution of pigment doesnot occur. In this instance, the pKa of isomerizationwas 6.6±0.1 (Fig. 9b). Assuming that the pKa ofbinding/isomerization processes represents the pKaof the active lysine, it is conceivable that differentretinal isomers can differentially affect the microen-vironment of the binding site, thereby altering thepKa of active-site lysine.

Catalysis of 9-cis retinal isomerization

In solution, 9-cis retinal does not isomerize evenafter the solvent (dimethyl sulfoxide) is warmed to120 °C for∼7 h. However, it was possible to catalyzethe isomerization of 9-cis retinal in dioxane solventby addition of iodine and to achieve isomerization ata lower temperature. Rando and Chang showed thatat low concentrations of iodine, 9-cis retinal pro-duced only the kinetically controlled product 9, 13-dicis retinal isomer in n-heptane solvent, whereas at ahigher concentration of iodine, all-trans retinal, 13-cisretinal, along with 9, 13-dicis retinal were pro-duced.31 We analyzed 9-cis retinal isomerization ata lower concentration (30 μM) and a higher concen-tration of iodine in solution (dioxane). It wasobserved (by HPLC analysis) that almost 26% of 9-cis retinalwas converted to 9, 13-dicis isomer and thata very small amount of all-trans retinal (4%) wasconverted after heating at 50–70 °C (∼7 h to 20 min,respectively) using a low concentration of iodine.The rate of isomerization of 9-cis retinal to 9, 13-dicisretinal is 2.12×10− 3 s− 1 at 60 °C (Table 1). Theactivation energy and activation entropy were∼19 kcal/mol and −16 e.u., respectively. At a higherconcentration of iodine (164 μM), a biphasic type ofkinetics was observed with two rate constants(1.73×10− 3 s− 1 and 1.3×10− 4 s− 1). The fast rateconstant represents the formation of 9, 13-dicisretinal, which is a kinetically controlled product,and the slow rate represents the formation of all-transretinal. The isomerization around the 13-bond isfaster sincemore positive charge is delocalized alongthe 13-double bond, which reduces the energy ofactivation and facilitates its isomerization. All therate constants and parameters are listed in Tables 1and 2, respectively.Note that the NpHR opsin-catalyzed isomeriza-

tion of 9-cis retinal also exhibited a similar biphasic

type of kinetics, as observed in an iodine-catalyzedreaction in dioxane solution. The rate constants forNpHR opsin-catalyzed isomerization at pH 7.5 at25 °C are 2.65×10− 3 s− 1 and 5.80×10− 4 s− 1

(Table 1). The rate constants and the thermodynamicparameters (especially for the first phase) are similarto those for iodine-catalyzed reactions. The secondphase is less similar, but we noted that the exactnumbers depend on the iodine concentration. Allthese observations may support a triplet mechanismfor the 9-cis retinal isomerization process operatingin the NpHR opsin, as was proposed above for 11-cisretinal.

Binding and isomerization of 13-cis retinal

The binding process of 13-cis retinal was similar tothat of all-trans retinal in the presence of 3.2 M NaClat pH7.5. ThepKa of the binding processwas 5.6±0.1,which is somewhat lower than that of all-trans retinal(Fig. 10a). One cannot accuratelymeasure the kineticsof isomerization of 13-cis retinal in 3.2 M sodiumchloride at pH 7.5 because ∼50% of pigment formswithin 1 h. Therefore, we monitored the isomeri-zation kinetics of 13-cis retinal in the presence ofNpHRopsin at pH 7.5without salt.We observed thatafter 70% conversion of 13-cis retinal to all-transisomer, an equilibrium between the two isomers wasestablished. The rates of isomerization at differenttemperatures are presented in Table 1. The isome-rization of 13-cis retinal is also pH dependent, similarto 9-cis and 11-cis retinal, and it has a pKa of 5.7±0.1(Fig. 10b). To evaluate the catalysis effect of the pro-tein, we monitored the isomerization rate in dioxanein the presence of iodine (Table 1). The thermo-dynamic parameters were also estimated similarly,and it was found that the activation energy,frequency factors, enthalpy, free energy, and entropyof NpHR opsin-catalyzed isomerization correlatewell with iodine-catalyzed isomerization (Table 2).These observations indicate that a similar mecha-nism underlies both NpHR opsin isomerization andiodine-catalyzed isomerization. Therefore, appa-


rently, in the protein, all the retinal isomers share asimilar mechanism of thermal double bond isome-rization that favors a triplet mechanism.

Conclusions

The binding of retinal is strongly pH dependent. Itis conceivable that the protonation state of thebinding site lysine (Lys252) affects the protein–retinal covalent bond formation process since thisprocess requires a nucleophilic attack of the non-protonated lysine amino group on the retinal car-bonyl moiety. The pKa of the active-site lysine is low,reflecting a relatively hydrophobic environment.The microenvironment is affected by the nature ofthe retinal isomer, and therefore, different isomershave different pKa values for binding.The binding of retinal with NpHR opsin is regula-

ted by the NaCl concentration. The maximum yieldis observed in 3.7 M salt concentration. In theabsence of salt, the opsin does not generate pigmentprobably because the protein adopts a conformationthat cannot form the pigment. The NpHR opsingenerates pigment in the presence of sodium bro-mide similarly, but in the presence of azide andnitrate ions, the structure of the apomembrane maybecome altered upon binding, causing the active-sitelysine pKa to increase. Furthermore, these anionsmay not stabilize the apomembrane as effectively aschloride does, thus inducing a lower yield of pig-ment formation.Strikingly, the NpHR opsin efficiently catalyzes

the isomerization of 11-cis retinal to all-trans retinalwhile the retinal chromophore occupies its NpHRopsin binding site. The isomerization of the retinalisomers is pH dependent and the pKa of the isome-rization process is comparable to the pKa of theretinal binding. It is suggested that the same NpHRopsin residue, namely, Lys252, is involved in thebinding and isomerization process.The thermodynamic parameters of the thermal

double bond isomerization process in the proteinresemble those of the iodine-catalyzed reaction innonpolar solvents. It is well established that iodineradicals interact with the triplet state and initiate aradical mechanism for the thermal isomerization ofa double bond. Therefore, we suggest that the pro-tein provides a hydrophobic environment that signi-ficantly favors a triplet mechanism rather than anionic one. Future studies should be aimed at iden-tifying the specific chromophore–protein interac-tions that favor this mechanism.

Materials and Methods

All-trans retinal, 9-cis retinal, 13-cis retinal, 11-cis retinal,HFIP, and TFE were obtained from Sigma. The purity ofretinal isomers was checked by HPLC before the bindingexperiment. All absorption spectra were recorded with anAgilent 4583 diode array spectrophotometer (AgilentTechnologies, Palo Alto, CA).

NpHR sample preparation

Cell membrane containing cloned NpHR was preparedas reported earlier.14 Briefly, NpHR containing membranesuspension was prepared from H. salinarum strain L33, inwhich the N. pharaonis hop structural gene and thenovobiocin resistance gene for selection were introduced.This resulted in a greatly enhanced production of NpHR. Itwas purified using the same method as bR, with theexception that the lysed cells were centrifuged at30,000 rpm for 1 h and washed with 0.3 M NaCl.

Preparation of apomembrane

Apomembrane was prepared according to a publishedmethod.41 In brief, 1 ml of 1.25 OD/ml NpHR membranewasmixedwith 1ml of 2MNH2OHsolution at pH8.0. Thesolutionwas then irradiatedwith a 540-nm cutoff filter andirradiationwas continued until the samplewas completelybleached. The sample was then dialyzed against a 4-MNaCl solution to remove excess hydroxylamine.

Binding experiments

The NpHR opsin was mixed with 1.5 equivalent ofretinal at varying pH values in the presence of 3.2 M NaClsolution for ∼18 h in the dark. The amounts of pigmentgenerated at different pH values were plotted against thepH of the solution. The pKa of binding was determinedusing the following equation:

F xð Þ = 1

1 + 10n pKa�xð Þ ð1Þ

where n is the number of residues involved in binding andx=pH; pKa is the midpoint of the observed curve.

The effect of pH on isomerization of retinal inthe presence of NpHR opsin

TheNpHR opsinwasmixedwith 1.5 equivalent of 11-cisretinal in the presence of 3.2 M NaCl at varying pH valuesof the solution for 30 min in the dark. In the case of 9-cisretinal and 13-cis retinal, the incubation times were ∼4 hand 45 min, respectively. The retinals were extracted withn-hexane and analyzed by HPLC as reported earlier.42 Allthe reactions and the extraction of retinals were carried outin red light. The percentage of retinals that isomerize wasplotted against the pH of the solution. The pKa ofisomerization was calculated from the abovementionedequation.

The effect of temperature on isomerization of retinalin the presence of NpHR opsin

The NpHR opsin was mixed with 1.5 equivalent of 11-cis retinal in the presence of 3.2 M NaCl at pH 7.5 and atpH 5.0 in the dark at different temperatures (17, 21, 25.5,and 35 °C at pH 7.5 and 26, 31, 36.5, and 41.5 °C atpH 5.0). Aliquots of 100 μl were taken from the reactionmixture as the reaction proceeded and the retinals wereextracted and analyzed by HPLC. The percentage ofretinal that isomerizes was plotted against time andfitted with a first-order kinetic equation. From thatcurve, the rate of isomerization was calculated for aparticular temperature.


The logarithm of the rate of isomerization (ln k) isplotted against 1/T according to the equation:

lnk = � Ea

RT+ lnA ð2Þ

where Ea is the activation energy and A is the frequencyfactor. The activation energy and frequency factor arecalculated from the slope and intercept of this plot.The enthalpy (ΔH‡) and entropy (ΔS‡) values are also

calculated from the plot ln(k/T) versus the plot 1/Taccording to the Eyring equation:

lnkT= � DHz

RT+ ln

KB

h+DSzR

ð3Þ

where KB is Boltzmann's constant and h is Planck'sconstant.We also carried out the 9-cis retinal, 11-cis retinal, 13-cis

retinal, and all-trans retinal isomerization in the absence ofsodium chloride at varying temperatures, and thethermodynamic parameters were estimated from thetemperature dependence of the rate constants.

Iodine-catalyzed isomerization of 9-cis, 11-cis, 13-cis,and all-trans retinals

The iodine-catalyzed isomerization of 9-cis, 11-cis,13-cis, and all-trans retinal was studied in dioxanesolvent. The concentration of retinals was in the rangeof 28–30 μM, and the iodine concentration was 30 μM.The thermodynamic parameters were estimated fromthe rate constant's temperature dependence on theiodine-catalyzed isomerization.

Acknowledgements

This work was supported by grants from theKimmelman Center for Biomolecular Structure andAssembly and the United States–Israel BinationalScience Foundation. M.S. holds the Katzir-MakineniProfessorial Chair in Chemistry.

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Retinal–Protein Interactions in Halorhodopsin from Natronomonas pharaonis: Binding and Retinal Thermal Isomerization Catalysis