REVIEWS TIBS 24 – AUGUST 1999 detected a putative homologue (E ϭ 10Ϫ3) an independent gene or as a domain of 3238–3241 of the bacterial smr gene in mutS2 genes. This suggests that Smr plays 3 Modrich, P. (1991) Annu. Rev. Genet. 25, an important role that is probably relevant 229–253 Saccharomyces cerevisiae (Fig. 1). 4 Pont-Kingdom, G. et al. (1998) J. Mol. Evol. 46, Eukaryotes seem to have only mutS1-type to MMR through an interaction with 419–431 genes: the presence of an smr gene is MutS1. Elucidation of the functions of 5 Fishel, R. and Wilson, T. (1997) Curr. Opin. therefore not surprising. these new proteins will require integrated Genet. Dev. 7, 105–113 The complex phylogenetic distribution biochemical and genetic approaches. 6 Fishel, R. et al. (1993) Cell 75, 1027–1038 of these proteins makes it difficult to 7 Leach, F. S. et al. (1993) Cell 75, 1215–1225 determine whether the mutS2 family arose Acknowledgements 8 Eisen, J. A. (1998) Nucleic Acids Res. 26, from an ancient fusion between a mutS1 We thank H. Brinkmann, H. Le 4291–4300 gene and an smr gene, or whether smr Guyader, P. López-García and three 9 Tomb, J. F. et al. (1997) Nature 388, genes and the mutS1 family are the result 539–547 anonymous referees for critical reading 10 Schuler, G. D. et al. (1991) Proteins 9, of splitting of a mutS2-type gene. The of the manuscript. D. M. is a postdoc- existence of the MutS2 family and Smr 180–190 toral fellow of the Spanish Ministerio de proteins suggests that the MMR pathway Educación y Cultura. involves factors other than those already DAVID MOREIRA AND HERVÉ PHILIPPE characterized, even in well known organisms such as E. coli. The smr References UPRESA Q8080-Equipe Phylogénie et 1 Lahue, R. S., Au, K. G. and Modrich, P. (1989) Evolution Moléculaires, Bâtiment 444, sequence is present in a variety of Science 245, 160–164 bacteria and also in eukaryotes, either as 2 Worth, L., Clark, S., Radman, M. and Modrich, Université Paris-Sud, 91405 Orsay Cedex, P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, France. Email: [email protected]

chromophore. Although the PSB of 11- cis-retinal absorbs at 440 nm in organic How color visual pigments are solvents, vertebrate color pigments pro- vide environmental perturbations that tune the absorption maximum of the tuned retinal chromophore over a very wide range – from 360 to 635 nm8. This shift in the wavelength of maximum absorbance is called the opsin shift9. The ~60-nm Gerd G. Kochendoerfer, Steven W. Lin, shift typically observed upon proton- ation of retinal Schiff bases10 is not suffi- Thomas P. Sakmar and Richard A. Mathies cient to explain the red-shifted absorp- tion maxima of green and red visual The absorption maximum of the retinal chromophore in color visual pig- pigments. Chromophore–protein inter- ments is tuned by interactions with the protein (opsin) to which it is bound. actions that might cause this opsin shift Recent advances in the expression of rhodopsin-like transmembrane re- include: (1) a weakening of the interac- ceptors and in spectroscopic techniques have allowed us to measure res- tion between the positive charge of the onance Raman vibrational spectra of the retinal chromophore in recombi- retinal PSB and its negative counterion nant visual pigments to examine the molecular basis of this spectral or hydrogen-bonding partner11,12; (2) tuning. The dominant physical mechanism responsible for the opsin shift placement of full or partial charges13–17 18,19 in color vision is the interaction of dipolar amino acid residues with the or polarizable groups close to the ground- and excited-state charge distributions of the chromophore. polyene chain; and (3) planarization of the polyene chain caused by the protein environment20,21. THE RETINA CONTAINS two types of consist of an apoprotein (opsin) and an cells specialized for light detection: rod 11-cis-retinal chromophore that is Resonance Raman vibrational spectroscopy cells for dim-light vision and three bound to opsin by a protonated Schiff The cloning and expression of visual classes of cone cells for color vision. base (PSB) linkage4 to a specific lysine pigments and mutant pigments2,3,22 iden- The cones contain pigments absorbing residue (Lys296). Absorption of light tified Glu113 as the primary counterion in the blue (~425 nm), green (~530 nm) triggers the femtosecond isomerization of the retinal PSB chromophore23–25 as and red (~560 nm) regions of the spec- of the chromophore5, producing the ac- well as amino acids that are associated trum1–3, whose differential responses en- tive signaling state that leads to hyper- with the green-to-blue and green-to-red able color vision. These visual pigments polarization of the photoreceptor cell opsin shifts13,26–28. However, compari- membrane and generation of a visual sons of structural data on the chro- G. G. Kochendoerfer and R. A. Mathies are nerve impulse6. mophore in the relevant color pigments at the Dept of Chemistry, University of Since Isaac Newton’s famous treatise are critical to understanding the opsin- California, Berkeley, CA 94720, USA; and 7 S. W. Lin and T. P. Sakmar are at the Howard on color vision , a fundamental aim in vi- shift mechanism. By combining site- Hughes Medical Institute, Rockefeller sion research has been the elucidation directed mutagenesis with resonance University, New York, NY 10021, USA. of the factors that determine the ab- Raman vibrational spectroscopy, we can Email: [email protected] sorption maximum of the opsin-bound now compare the vibrational structures 300 0968 – 0004/99/$ – See front matter © 1999, Elsevier Science. All rights reserved. PII: S0968-0004(99)01432-2 TIBS 24 – AUGUST 1999 REVIEWS of the chromophore in differ- ent visual pigments directly and elucidate the physical mechanism underlying the opsin shift. Towards this end, we have obtained resonance Raman vibrational spectra of visual pigments absorbing in the blue, green and red by ex- ploiting preresonance and res- onance Raman microprobe techniques26,29–32. Comparison of these spectra shows that the interaction of dipolar residues with the chro- mophore ground- and excited- state charge distributions is the single most important physical interaction that de- termines the opsin shift. Figure 1 presents a compari- son of the Raman spectra of the 11-cis-retinal PSB chro- mophore in methanol (lmax = 440 nm), in a blue-rhodopsin analog (lmax = 438 nm) (Ref. 26)*, in the human green pigment (lmax = 530 nm) and in the human red pigment (lmax = 560 nm). The assignments of the retinal vibrational modes are summarized in Fig. 1, and Box 1 presents a brief tutorial on resonance Raman spec- troscopy and the concepts of vibrational structure determi- nation. A more detailed pres- entation of these assignments and of the properties of vari- ous pigment mutants is found elsewhere26,32,33. The first strik- ing observation is that the hy- drogen out-of-plane (HOOP) wagging, C–C stretch and C–H rocking, C=C stretching and C=NH Schiff-base modes of the 11-cis-retinal PSB in the blue Figure 1 pigment analog are nearly Resonance Raman vibrational spectra of the 11-cis-retinal protonated Schiff-base (PSB) chromophore in identical to those of the iso- methanol, in the blue-rhodopsin pigment analog*, in the human green cone pigment and in the human red lated PSB chromophore in cone pigment. The traces are broken at ~1600 cm–1 to facilitate presentation of expanded spectra of the † methanol . The similarity of Schiff-base region. These expanded insets also present portions of spectra recorded in D2O buffers. The the skeletal mode frequencies green 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 a indicates that there are no Lexel 479 Ti:sapphire laser pumped by the all-lines output from an Ar+ laser (Spectra-Physics 2020) into strong location-specific per- ~3 ␮l of pigment solution in a 300 ␮m I.D. circular capillary cooled to ~0°C (Ref. 32). The green pigment turbations of the chromo- samples were excited similarly with 30 mW of 795 nm light. The Raman microprobe apparatus used to phore imposed by the protein obtain 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 nine amino 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 from pigments that have regenerated to form a stable native and homogeneous structure. †The lower intensity of the C-14–C-15 stretching mode at 1187 cmϪ1 in the blue-rhodopsin pigment is due to a subtraction artifact. All reported pigment spectra are of 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 line at 1187 cmϪ1, this subtraction will unavoidably reduce the 1187 cmϪ1 band intensity. The shoulder at 960 cmϪ1 in the red pigment spectrum originates from a small population of the 9-cis isomer that is present in the photostationary state (see Ref. 32). 301 REVIEWS TIBS 24 – AUGUST 1999

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 the retinal 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, are delocalized and involve motion of many atoms throughout the molecule. Vibrational modes in the fingerprint region are mixtures of C–C stretching and 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 isotopic shifts that arise provide an additional probe of Schiff-base vibrational structure. (Online: see Fig. I)

pocket that alter the frequencies or in- because no such vibrational alterations (1559 to 1531 cmϪ1) in the green pig- tensities of specific vibrational modes. are observed. Instead, the protein envi- ment. This is expected because the al- In particular, the similarity of the Schiff- ronment solvates the positively charged tered chromophore–protein interac- base vibrational properties shows that Schiff-base group in a similar manner to tions produce a more delocalized the Schiff-base group in the blue pig- methanol. These dielectric interactions electronic structure that has less bond ment analog experiences a dielectric stabilize the Schiff-base complex ion, alternation. In addition, there is a signifi- and hydrogen-bonding environment prevent delocalization of the charge on cant shift in the Schiff-base mode (1660 that is equivalent to that of the chro- the retinal chromophore and blue shift to 1641 cmϪ1). The shift induced by mophore in methanolic solution. The the absorption maximum. deuteration (see Box 1) is also reduced identity of the HC-11=C-12H HOOP fre- Comparison of the spectrum of the from ~28 cmϪ1 in the blue pigment ana- quencies and intensities at 970 cm–1 in- blue pigment analog with that of log to Ϫ21 cmϪ1 in the green pigment. The dicates that the skeletal twists in the the green pigment reveals several im- frequency of the Schiff-base mode and C-10–C-13 region of the chromophore portant differences associated with the magnitude of the shift induced by are also very similar33. These obser- amino acid residue side-chain alter- deuteration correlate with the strength vations reveal that the absorption maxi- ations. Although the ‘fingerprint’ modes of the hydrogen bonding and electro- mum of the chromophore in the blue (1200–1300 cmϪ1) and the HOOP modes static interaction between the Schiff- pigment analog is not determined by (970 cmϪ1) of the two pigments are base imine cation and its counterion11. strong local perturbations of the chro- nearly identical, there is a dramatic fre- Thus, formation of the green pigment mophore structure by the protein, quency reduction of the C=C mode involves a significant reduction of the 302 TIBS 24 – AUGUST 1999 REVIEWS dielectric interaction between the Schiff-base group and its protein environment. Further- more, the lack of other pertur- bations in the vibrational structure tells us that this change in Schiff-base environ- ment is the primary structural difference between the chro- mophore in the blue pigment and the chromophore in the green pigment. Comparison of the green- pigment spectrum with the red-pigment spectrum reveals yet another pattern. The red- pigment ‘fingerprint’ modes are found at frequencies that are nearly the same as those of the green and blue pig- ments. The alteration in inten- sity of the C–C modes in the red pigment is a consequence of the altered resonance- enhancement conditions. The Figure 2 Structural model of the human green-cone pigment viewed from the cytoplasmic side of the membrane. HOOP mode in all these pig- The 11-cis-retinal chromophore and its Glu113 counterion are shown in green. The green-pigment ments is found between 970 Ϫ residues whose alteration is important for the color shift from the green-to-blue pigment are shown in and 973 cm 1, which indicates blue; green-pigment residues whose alteration is important for the color shift from the green-to-red pig- that the C-10–C-13 region ex- ment are shown in red. The model was built in Insight, and the protein structure was minimized in periences a similar skeletal Discover. Transmembrane helices I–VII are indicated. twist (Ref. 33; see † on p. 301) The ethylenic mode has shifted down to 1526 cmϪ1, which is consistent Origin of the opsin shift are thought to be present in the vicinity with the red-shifted absorption and By considering the sequences of these of the Schiff base34. Finally, the fact that delocalized electronic structure. Band pigments and molecular models of their additional helix 3 mutations (Ala117Gly deconvolution demonstrates that the structures, we have produced a new and Glu122Leu) in rhodopsin are necess- C=NH mode in the red pigment is at model for the origin of the opsin shift ary for the fully blue-shifted pigment ab- 1644 cmϪ1. The Schiff-base frequency of that explains these structural obser- sorption suggests that a slight movement this pigment in D2O buffers was not de- vations. Figure 2 presents a structural of the counterion towards the Schiff base termined. The slightly elevated fre- model of the green visual pigment. The also helps to stabilize the ground-state quency of the C=NH mode compared 11-cis-retinal chromophore in the green charge distribution26. This synergistic di- with that of the green pigment is prob- pigment is surrounded mainly by non- electric stabilization leads to reduced ably due to the fact that the spectrum of polar residues‡. Replacement of these electron delocalization and the blue- the red pigment was taken at 77 K. The non-polar residues (Gly90, Ala292 and shifted absorption. reduced temperature induces a 3-cmϪ1 Ala295) by more polar groups (Ser90, Conversion of the human green visual frequency increase in the Schiff-base Ser292 and Ser295) on transmembrane pigment into a red-absorbing pigment mode of the green pigment32. Assuming helices 2 and 7 in the blue pigment ana- requires seven amino acid replace- a similar shift for the red pigment, we log and in the human blue pigment ex- ments13. In rhodopsin, the replacement conclude that the Schiff-base vibrational poses the chromophore’s Schiff-base of three conserved non-polar residues structures of the green and red pig- group to a much more polar, methanol- (Ala164, Phe261 and Ala269) by hy- ments are identical. Thus, although like environment. This is illustrated more droxyl amino acids produces shifts of there are clear indications from the ab- clearly in Fig. 3, where the nine residue 75, 400 and 550 cmϪ1, respectively; this sorption spectrum and from the alterations that convert rhodopsin to the suggests that these residues play an im- ethylenic mode frequency that the elec- blue pigment analog are indicated26. portant role in tuning the color from tronic structure is more delocalized, These polar residues might also re- green to red27. The fact that no specific there is no evidence of any specific arrange or stabilize water molecules that local perturbations of the vibrational alterations in the skeletal or Schiff-base structure are observed in the red pig- vibrational structure that are caused by ment is consistent with the distance of ‡An exception to this trend is the presence of Glu86 strong local interactions. The interactions 5 Å from the protonated Schiff base. However, the these residues from the chromophore that generate the difference between the absence of strong perturbations of the C=N–D (Fig. 2). This suggests that these dipolar absorption maxima of the green and stretching mode in the green and red pigments residues interact electrostatically with red pigments must involve some de- provides structural evidence that the electrostatic the chromophore charge distribution in interaction between Glu86 and the chromophore localized perturbation of the molecules’ is strongly shielded, possibly by the presence of the ground and excited states and electronic structure (see below). several water molecules in the binding site. thereby shift the absorption maximum. 303 REVIEWS TIBS 24 – AUGUST 1999

Figure 3 Structural model of the region around the chromophore in the blue visual pigment indicating the chromophore–protein inter- actions that are responsible for the absorption difference be- tween green and blue visual pigments. The polyene color coding represents the calcu- lated ground-state-charge distri- bution difference between the green and blue pigments from semiempirical INDO (intermedi- ate neglect of differential over- lap) electronic structure calcu- lations32. The introduction of the polar amino acid residues in the vicinity of the Schiff-base group, in synergy with a slight movement of the Glu113 coun- terion, creates an electrostatic potential that stabilizes the positive charge near the proto- nated Schiff-base group in the blue pigment, thereby lowering the ground-state energy and shifting the absorption from green to blue.

Retinals and retinal PSBs experience a there is a shift of net positive charge to- calculations predict that there is an ab- dramatic change in charge distribution wards the ionone ring upon excitation35,36. sorption red shift of 1150 cmϪ1 if the pro- upon electronic excitation. Stark shift In the structural model in Fig. 4, the di- tein dipoles are placed in the orientation (electric field perturbation) measure- polar residues Ser164, Tyr261 and Thr269 shown in Fig. 4, establishing our model as ments have shown that this change in are ideally positioned to stabilize this a fully competent mechanism for explain- dipole moment is as large as 10–15 debye excited-state charge distribution and ing the ~1000 cmϪ1 absorption shift (1 debye = 3.336 ϫ 10Ϫ30 C m) and that produce a red shift. Semiempirical between green and red visual pigments32.

Conclusions The vibrational data pre- sented here, together with se- quence alignment and molecu- lar modeling, allow us to advance the understanding of color-pigment tuning beyond previous explanations that are based on differential point- charge perturbations9. Given that the chromophore–pro- tein-binding-site complex is electrically neutral35, charged amino acid side-chains other than the Schiff-base counter- ion cannot play an important role in the opsin-shift mecha- nism. Instead, direct dipolar electrostatic interactions with the ground-state chromo- phore charge distribution Figure 4 dominate the green-to-blue Structural model of the region around the chromophore in the red visual pigment, indicating the chro- shift in the pigment absorp- mophore–protein interactions that are responsible for the absorption difference between green and tion maximum11,12,28 in synergy red visual pigments. The orientation of the hydroxyl groups was constrained by INDO calculations mod- 32 with movement of the counter- eling the green–red opsin shift . The color coding of the polyene chain represents the INDO-calculated 26 charge distribution difference between the ground and first-excited states of the chromophore in the ion . Longer-range, dipolar red pigment. The three hydroxyl-bearing amino acid residues in the red pigment interact preferentially interactions between polar with the excited-state charge distribution to lower the excited-state energy and shift the absorption protein hydroxy dipoles, and from green to red. the change in electric dipole 304 TIBS 24 – AUGUST 1999 REVIEWS moment upon electronic excitation are 26 Lin, S. W. et al. responsible for the shift of the absorp- (1998) J. Biol. Chem. 273, tion maximum from green to red pig- 24583–24591 ments. Nature thus exploits the dielec- 27 Chan, T., Lee, M. tric interaction of polar protein residues and Sakmar, T. P. with the asymmetric and highly polariz- (1992) J. Biol. Chem. 267, able charge distribution of the retinal 9478–9480 36,37 prosthetic group to give us the vivid 28 Sun, H., Macke, sensation of color. J. P. and Nathans, J. (1997) Proc. Natl. Acad. Sci. Acknowledgements U. S. A. 94, We thank Daniel Oprian and Lubert 8860–8865 Stryer for constructive comments on 29 Barry, B. and this manuscript, and the NIH (EY 02051) Mathies, R. A. and the Allene Reuss Memorial Trust for (1987) Biochemistry 26, support. 59–64 30 Loppnow, G. R., References Barry, B. A. and 1 Nathans, J. (1989) Sci. Am. 260, 42–49 Mathies, R. A. 2 Oprian, D. D., Asenjo, A. B., Lee, N. and (1989) Proc. Natl. Pelletier, S. L. (1991) Biochemistry 30, Acad. Sci. U. S. A. 11367–11372 86, 1515–1518 3 Merbs, S. L. and Nathans, J. (1992) Nature 31 Lin, S. W. et al. 356, 433–435 (1992) 4 Mathies, R., Oseroff, A. R. and Stryer, L. Biochemistry 31, (1976) Proc. Natl. Acad. Sci. U. S. 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