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300 0968 – 0004/99/$ – See front matter © 1999, Elsevier Science. All rights reserved. PII: S0968-0004(99)01432-2

detected a putative homologue (E 5 1023)of the bacterial smr gene inSaccharomyces cerevisiae (Fig. 1).Eukaryotes seem to have only mutS1-typegenes: the presence of an smr gene istherefore not surprising.

The complex phylogenetic distributionof these proteins makes it difficult todetermine whether the mutS2 family arosefrom an ancient fusion between a mutS1gene and an smr gene, or whether smrgenes and the mutS1 family are the resultof splitting of a mutS2-type gene. Theexistence of the MutS2 family and Smrproteins suggests that the MMR pathwayinvolves factors other than those alreadycharacterized, even in well knownorganisms such as E. coli. The smrsequence is present in a variety ofbacteria and also in eukaryotes, either as

an independent gene or as a domain ofmutS2 genes. This suggests that Smr playsan important role that is probably relevantto MMR through an interaction withMutS1. Elucidation of the functions ofthese new proteins will require integratedbiochemical and genetic approaches.

AcknowledgementsWe thank H. Brinkmann, H. Le

Guyader, P. López-García and threeanonymous referees for critical readingof the manuscript. D. M. is a postdoc-toral fellow of the Spanish Ministerio deEducación y Cultura.

References1 Lahue, R. S., Au, K. G. and Modrich, P. (1989)

Science 245, 160–1642 Worth, L., Clark, S., Radman, M. and Modrich,

P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91,

3238–32413 Modrich, P. (1991) Annu. Rev. Genet. 25,

229–2534 Pont-Kingdom, G. et al. (1998) J. Mol. Evol. 46,

419–4315 Fishel, R. and Wilson, T. (1997) Curr. Opin.

Genet. Dev. 7, 105–1136 Fishel, R. et al. (1993) Cell 75, 1027–10387 Leach, F. S. et al. (1993) Cell 75,

1215–12258 Eisen, J. A. (1998) Nucleic Acids Res. 26,

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539–54710 Schuler, G. D. et al. (1991) Proteins 9,

180–190

DAVID MOREIRA AND HERVÉ PHILIPPE

UPRESA Q8080-Equipe Phylogénie etEvolution Moléculaires, Bâtiment 444,Université Paris-Sud, 91405 Orsay Cedex,France.Email: david.moreira@bc4.u-psud.fr

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THE RETINA CONTAINS two types ofcells specialized for light detection: rodcells for dim-light vision and threeclasses of cone cells for color vision.The cones contain pigments absorbingin the blue (~425 nm), green (~530 nm)and red (~560 nm) regions of the spec-trum1–3, whose differential responses en-able color vision. These visual pigments

consist of an apoprotein (opsin) and an11-cis-retinal chromophore that isbound to opsin by a protonated Schiffbase (PSB) linkage4 to a specific lysineresidue (Lys296). Absorption of lighttriggers the femtosecond isomerizationof the chromophore5, producing the ac-tive signaling state that leads to hyper-polarization of the photoreceptor cellmembrane and generation of a visualnerve impulse6.

Since Isaac Newton’s famous treatiseon color vision7, a fundamental aim in vi-sion research has been the elucidationof the factors that determine the ab-sorption maximum of the opsin-bound

chromophore. Although the PSB of 11-cis-retinal absorbs at 440 nm in organicsolvents, vertebrate color pigments pro-vide environmental perturbations thattune the absorption maximum of theretinal chromophore over a very widerange – from 360 to 635 nm8. This shift inthe wavelength of maximum absorbanceis called the opsin shift9. The ~60-nmshift typically observed upon proton-ation of retinal Schiff bases10 is not suffi-cient to explain the red-shifted absorp-tion maxima of green and red visualpigments. Chromophore–protein inter-actions that might cause this opsin shiftinclude: (1) a weakening of the interac-tion between the positive charge of theretinal PSB and its negative counterionor hydrogen-bonding partner11,12; (2)placement of full or partial charges13–17

or polarizable groups18,19 close to thepolyene chain; and (3) planarization ofthe polyene chain caused by the proteinenvironment20,21.

Resonance Raman vibrational spectroscopyThe cloning and expression of visual

pigments and mutant pigments2,3,22 iden-tified Glu113 as the primary counterionof the retinal PSB chromophore23–25 aswell as amino acids that are associatedwith the green-to-blue and green-to-redopsin shifts13,26–28. However, compari-sons of structural data on the chro-mophore in the relevant color pigmentsare critical to understanding the opsin-shift mechanism. By combining site-directed mutagenesis with resonanceRaman vibrational spectroscopy, we cannow compare the vibrational structures

How color visual pigments aretuned

Gerd G. Kochendoerfer, Steven W. Lin,Thomas P. Sakmar and Richard A. Mathies

The absorption maximum of the retinal chromophore in color visual pig-ments is tuned by interactions with the protein (opsin) to which it is bound.Recent advances in the expression of rhodopsin-like transmembrane re-ceptors and in spectroscopic techniques have allowed us to measure res-onance Raman vibrational spectra of the retinal chromophore in recombi-nant visual pigments to examine the molecular basis of this spectraltuning. The dominant physical mechanism responsible for the opsin shiftin color vision is the interaction of dipolar amino acid residues with theground- and excited-state charge distributions of the chromophore.

G. G. Kochendoerfer and R. A. Mathies areat the Dept of Chemistry, University ofCalifornia, Berkeley, CA 94720, USA; and S. W. Lin and T. P. Sakmar are at the HowardHughes Medical Institute, RockefellerUniversity, New York, NY 10021, USA.Email: rich@zinc.cchem.berkeley.edu

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of the chromophore in differ-ent visual pigments directlyand elucidate the physicalmechanism underlying theopsin shift. Towards this end,we have obtained resonanceRaman vibrational spectra ofvisual pigments absorbing inthe blue, green and red by ex-ploiting preresonance and res-onance Raman microprobetechniques26,29–32. Comparisonof these spectra shows thatthe interaction of dipolarresidues with the chro-mophore ground- and excited-state charge distributions isthe single most importantphysical interaction that de-termines the opsin shift.

Figure 1 presents a compari-son of the Raman spectra ofthe 11-cis-retinal PSB chro-mophore in methanol (lmax= 440 nm), in a blue-rhodopsinanalog (lmax = 438 nm)(Ref. 26)*, in the human greenpigment (lmax = 530 nm) and inthe human red pigment (lmax= 560 nm). The assignments ofthe retinal vibrational modesare summarized in Fig. 1, andBox 1 presents a brief tutorialon resonance Raman spec-troscopy and the concepts ofvibrational structure determi-nation. A more detailed pres-entation of these assignmentsand of the properties of vari-ous pigment mutants is foundelsewhere26,32,33. The first strik-ing observation is that the hy-drogen out-of-plane (HOOP)wagging, C–C stretch and C–Hrocking, C=C stretching andC=NH Schiff-base modes of the11-cis-retinal PSB in the bluepigment analog are nearlyidentical to those of the iso-lated PSB chromophore inmethanol†. The similarity ofthe skeletal mode frequenciesindicates that there are nostrong location-specific per-turbations of the chromo-phore imposed by the protein

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Figure 1Resonance Raman vibrational spectra of the 11-cis-retinal protonated Schiff-base (PSB) chromophore inmethanol, in the blue-rhodopsin pigment analog*, in the human green cone pigment and in the human redcone pigment. The traces are broken at ~1600 cm–1 to facilitate presentation of expanded spectra of theSchiff-base region. These expanded insets also present portions of spectra recorded in D2O buffers. Thegreen and the red pigment data are reproduced, with permission, from Ref. 32. Raman spectra of the 11-cis-retinal PSB and of the blue pigment analog were obtained by focusing a 50-mW, 720-nm beam from aLexel 479 Ti:sapphire laser pumped by the all-lines output from an Ar+ laser (Spectra-Physics 2020) into~3 ml of pigment solution in a 300 mm I.D. circular capillary cooled to ~0°C (Ref. 32). The green pigmentsamples were excited similarly with 30 mW of 795 nm light. The Raman microprobe apparatus used toobtain the low-temperature red-pigment Raman spectra was described previously29–31.

*The blue absorbing (lmax = 438 nm) rhodopsin analog described in Ref. 26 was used instead of the blue cone pigment because it is easily prepared and more suit-able for Raman studies while exhibiting ~80% of the expected opsin shift. The blue-rhodopsin analog is a mutant chimera consisting of bovine rhodopsin with nineamino acid replacements (M86L/G90S/A117G/E122L/A124T/W265Y/A292S/A295S/A299C). Chromophore replacement studies with retinal analogs demon-strated that the chromophore is specifically bound to this mutant opsin. Furthermore, it is our experience that high-quality Raman spectra can only be obtained frompigments that have regenerated to form a stable native and homogeneous structure.†The lower intensity of the C-14–C-15 stretching mode at 1187 cm21 in the blue-rhodopsin pigment is due to a subtraction artifact. All reported pigment spectra areof the pigment minus a bleached background. As the spectrum of all-trans-retinal in the metarhodopsin I bleaching intermediate (see Ref. 38) exhibits a strong lineat 1187 cm21, this subtraction will unavoidably reduce the 1187 cm21 band intensity. The shoulder at 960 cm21 in the red pigment spectrum originates from a smallpopulation of the 9-cis isomer that is present in the photostationary state (see Ref. 32).

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pocket that alter the frequencies or in-tensities of specific vibrational modes.In particular, the similarity of the Schiff-base vibrational properties shows thatthe Schiff-base group in the blue pig-ment analog experiences a dielectricand hydrogen-bonding environmentthat is equivalent to that of the chro-mophore in methanolic solution. Theidentity of the HC-11=C-12H HOOP fre-quencies and intensities at 970 cm–1 in-dicates that the skeletal twists in the C-10–C-13 region of the chromophoreare also very similar33. These obser-vations reveal that the absorption maxi-mum of the chromophore in the bluepigment analog is not determined bystrong local perturbations of the chro-mophore structure by the protein,

because no such vibrational alterationsare observed. Instead, the protein envi-ronment solvates the positively chargedSchiff-base group in a similar manner tomethanol. These dielectric interactionsstabilize the Schiff-base complex ion,prevent delocalization of the charge onthe retinal chromophore and blue shiftthe absorption maximum.

Comparison of the spectrum of theblue pigment analog with that of the green pigment reveals several im-portant differences associated withamino acid residue side-chain alter-ations. Although the ‘fingerprint’ modes(1200–1300 cm21) and the HOOP modes(970 cm21) of the two pigments arenearly identical, there is a dramatic fre-quency reduction of the C=C mode

(1559 to 1531 cm21) in the green pig-ment. This is expected because the al-tered chromophore–protein interac-tions produce a more delocalizedelectronic structure that has less bondalternation. In addition, there is a signifi-cant shift in the Schiff-base mode (1660to 1641 cm21). The shift induced bydeuteration (see Box 1) is also reducedfrom ~28 cm21 in the blue pigment ana-log to 221 cm21 in the green pigment. Thefrequency of the Schiff-base mode andthe magnitude of the shift induced bydeuteration correlate with the strengthof the hydrogen bonding and electro-static interaction between the Schiff-base imine cation and its counterion11.Thus, formation of the green pigment involves a significant reduction of the

Box 1. Raman spectroscopy

Raman spectroscopy is an inelastic light-scattering process where the energy loss of the scattered photons (expressed in wavenumbers) is proportional to the molecule’s vibrational frequencies. By choosing laser excitation that lies within the visible electronic absorption band of theretinal chromophore in rhodopsin, scattering from the chromophore is strongly enhanced compared to the surrounding protein and buffer modes.Some of the observed vibrational modes, such as the Schiff-base stretch and the hydrogen out-of-plane or HOOP modes, are localized on a particular part of the chromophore and can thus be used as a probe of local structure. Other modes, such as the illustrated ethylenic stretch, aredelocalized and involve motion of many atoms throughout the molecule. Vibrational modes in the fingerprint region are mixtures of C–C stretchingand C–H rocking motions whose pattern of frequencies and intensities is very sensitive to the configuration and comformation of the chro-mophore. The N–H proton on the Schiff-base group can be exchanged by placing the pigment in D2O-containing buffer; the characteristic isotopicshifts that arise provide an additional probe of Schiff-base vibrational structure. (Online: see Fig. I)

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dielectric interaction betweenthe Schiff-base group and itsprotein environment. Further-more, the lack of other pertur-bations in the vibrationalstructure tells us that thischange in Schiff-base environ-ment is the primary structuraldifference between the chro-mophore in the blue pigmentand the chromophore in thegreen pigment.

Comparison of the green-pigment spectrum with thered-pigment spectrum revealsyet another pattern. The red-pigment ‘fingerprint’ modesare found at frequencies thatare nearly the same as thoseof the green and blue pig-ments. The alteration in inten-sity of the C–C modes in thered pigment is a consequenceof the altered resonance-enhancement conditions. TheHOOP mode in all these pig-ments is found between 970and 973 cm21, which indicatesthat the C-10–C-13 region ex-periences a similar skeletaltwist (Ref. 33; see † on p. 301)The ethylenic mode has shifteddown to 1526 cm21, which is consistentwith the red-shifted absorption and delocalized electronic structure. Banddeconvolution demonstrates that theC=NH mode in the red pigment is at1644 cm21. The Schiff-base frequency ofthis pigment in D2O buffers was not de-termined. The slightly elevated fre-quency of the C=NH mode comparedwith that of the green pigment is prob-ably due to the fact that the spectrum ofthe red pigment was taken at 77 K. Thereduced temperature induces a 3-cm21

frequency increase in the Schiff-basemode of the green pigment32. Assuminga similar shift for the red pigment, weconclude that the Schiff-base vibrationalstructures of the green and red pig-ments are identical. Thus, althoughthere are clear indications from the ab-sorption spectrum and from theethylenic mode frequency that the elec-tronic structure is more delocalized,there is no evidence of any specific alterations in the skeletal or Schiff-basevibrational structure that are caused bystrong local interactions. The interactionsthat generate the difference between theabsorption maxima of the green and red pigments must involve some de-localized perturbation of the molecules’electronic structure (see below).

Origin of the opsin shiftBy considering the sequences of these

pigments and molecular models of theirstructures, we have produced a newmodel for the origin of the opsin shiftthat explains these structural obser-vations. Figure 2 presents a structuralmodel of the green visual pigment. The11-cis-retinal chromophore in the greenpigment is surrounded mainly by non-polar residues‡. Replacement of thesenon-polar residues (Gly90, Ala292 andAla295) by more polar groups (Ser90,Ser292 and Ser295) on transmembranehelices 2 and 7 in the blue pigment ana-log and in the human blue pigment ex-poses the chromophore’s Schiff-basegroup to a much more polar, methanol-like environment. This is illustrated moreclearly in Fig. 3, where the nine residuealterations that convert rhodopsin to theblue pigment analog are indicated26.These polar residues might also re-arrange or stabilize water molecules that

are thought to be present in the vicinityof the Schiff base34. Finally, the fact thatadditional helix 3 mutations (Ala117Glyand Glu122Leu) in rhodopsin are necess-ary for the fully blue-shifted pigment ab-sorption suggests that a slight movementof the counterion towards the Schiff basealso helps to stabilize the ground-statecharge distribution26. This synergistic di-electric stabilization leads to reducedelectron delocalization and the blue-shifted absorption.

Conversion of the human green visualpigment into a red-absorbing pigmentrequires seven amino acid replace-ments13. In rhodopsin, the replacementof three conserved non-polar residues(Ala164, Phe261 and Ala269) by hy-droxyl amino acids produces shifts of75, 400 and 550 cm21, respectively; thissuggests that these residues play an im-portant role in tuning the color fromgreen to red27. The fact that no specificlocal perturbations of the vibrationalstructure are observed in the red pig-ment is consistent with the distance ofthese residues from the chromophore(Fig. 2). This suggests that these dipolarresidues interact electrostatically withthe chromophore charge distribution inthe ground and excited states andthereby shift the absorption maximum.

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‡An exception to this trend is the presence of Glu865 Å from the protonated Schiff base. However, theabsence of strong perturbations of the C=N–Dstretching mode in the green and red pigments provides structural evidence that the electrostaticinteraction between Glu86 and the chromophore is strongly shielded, possibly by the presence ofseveral water molecules in the binding site.

Figure 2Structural model of the human green-cone pigment viewed from the cytoplasmic side of the membrane.The 11-cis-retinal chromophore and its Glu113 counterion are shown in green. The green-pigmentresidues whose alteration is important for the color shift from the green-to-blue pigment are shown inblue; green-pigment residues whose alteration is important for the color shift from the green-to-red pig-ment are shown in red. The model was built in Insight, and the protein structure was minimized inDiscover. Transmembrane helices I–VII are indicated.

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Retinals and retinal PSBs experience adramatic change in charge distributionupon electronic excitation. Stark shift(electric field perturbation) measure-ments have shown that this change indipole moment is as large as 10–15 debye(1 debye = 3.336 3 10230 C m) and that

there is a shift of net positive charge to-wards the ionone ring upon excitation35,36.In the structural model in Fig. 4, the di-polar residues Ser164, Tyr261 and Thr269are ideally positioned to stabilize this excited-state charge distribution and produce a red shift. Semiempirical

calculations predict that there is an ab-sorption red shift of 1150 cm21 if the pro-tein dipoles are placed in the orientationshown in Fig. 4, establishing our model asa fully competent mechanism for explain-ing the ~1000 cm21 absorption shift between green and red visual pigments32.

ConclusionsThe vibrational data pre-

sented here, together with se-quence alignment and molecu-lar modeling, allow us toadvance the understanding ofcolor-pigment tuning beyondprevious explanations that arebased on differential point-charge perturbations9. Giventhat the chromophore–pro-tein-binding-site complex iselectrically neutral35, chargedamino acid side-chains otherthan the Schiff-base counter-ion cannot play an importantrole in the opsin-shift mecha-nism. Instead, direct dipolarelectrostatic interactions withthe ground-state chromo-phore charge distributiondominate the green-to-blueshift in the pigment absorp-tion maximum11,12,28 in synergywith movement of the counter-ion26. Longer-range, dipolar interactions between polarprotein hydroxy dipoles, andthe change in electric dipole

Figure 3Structural model of the regionaround the chromophore in theblue visual pigment indicatingthe chromophore–protein inter-actions that are responsible forthe absorption difference be-tween green and blue visualpigments. The polyene colorcoding represents the calcu-lated ground-state-charge distri-bution difference between thegreen and blue pigments fromsemiempirical INDO (intermedi-ate neglect of differential over-lap) electronic structure calcu-lations32. The introduction ofthe polar amino acid residuesin the vicinity of the Schiff-basegroup, in synergy with a slightmovement of the Glu113 coun-terion, creates an electrostaticpotential that stabilizes thepositive charge near the proto-nated Schiff-base group in theblue pigment, thereby loweringthe ground-state energy andshifting the absorption fromgreen to blue.

Figure 4Structural model of the region around the chromophore in the red visual pigment, indicating the chro-mophore–protein interactions that are responsible for the absorption difference between green andred visual pigments. The orientation of the hydroxyl groups was constrained by INDO calculations mod-eling the green–red opsin shift32. The color coding of the polyene chain represents the INDO-calculatedcharge distribution difference between the ground and first-excited states of the chromophore in thered pigment. The three hydroxyl-bearing amino acid residues in the red pigment interact preferentiallywith the excited-state charge distribution to lower the excited-state energy and shift the absorptionfrom green to red.

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moment upon electronic excitation areresponsible for the shift of the absorp-tion maximum from green to red pig-ments. Nature thus exploits the dielec-tric interaction of polar protein residueswith the asymmetric and highly polariz-able charge distribution of the retinalprosthetic group36,37 to give us the vividsensation of color.

AcknowledgementsWe thank Daniel Oprian and Lubert

Stryer for constructive comments onthis manuscript, and the NIH (EY 02051)and the Allene Reuss Memorial Trust forsupport.

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Pete Jeffs is a freelancer working in Paris, France.