Identical Hydrogen-Bonding Strength of the Retinal Schiff Base

Mar 20, 2015 - Three aspects are generally considered in the color-tuning mechanism of vision: (I) chromophore distortion, (II) electrostatic interact...
0 downloads 0 Views 291KB Size
Download PDF
Letter pubs.acs.org/JPCL

Identical Hydrogen-Bonding Strength of the Retinal Schiff Base between Primate Green- and Red-Sensitive Pigments: New Insight into Color Tuning Mechanism Kota Katayama,† Takashi Okitsu,‡ Hiroo Imai,§ Akimori Wada,‡ and Hideki Kandori*,† †

Department of Frontier Materials and Sciences, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan Department of Organic Chemistry for Life Science, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658-8558, Japan § Primate Research Institute, Kyoto University, Inuyama 484-8506, Japan ‡

S Supporting Information *

ABSTRACT: Three aspects are generally considered in the color-tuning mechanism of vision: (I) chromophore distortion, (II) electrostatic interaction between the protonated Schiff base and counterion, and (III) polarity around the β-ionone ring and polyene chain. Primate green- and red-sensitive proteins are highly homologous but display maximum absorption at 530 and 560 nm, respectively. In the present study, the N−D stretching frequency of monkey greensensitive protein was identified by using C15-D retinal. The hydrogen-bonding strength between monkey green and red was identical. Together with a previous resonance Raman study, we conclude that the 30 nm difference originates exclusively from the polarity around the β-ionone ring and polyene chain. Three amino acids (Ala, Phe, and Ala in monkey green and Ser, Tyr, and Thr in monkey red, respectively) may be responsible for color tuning together with protein-bound water molecules around the β-ionone ring and polyene chain but not at the Schiff base region.

H

human green (HG) and red (HR) pigments, where O−H bearing residues are introduced in human red such as Ser, Tyr, and Thr (Figure 1b).10−12 This is also the case in monkey green (MG) and red (MR) pigments. As these groups are located near the β-ionone ring, this fact strongly suggests that polarity near the β-ionone ring is the dominant factor, owing to Mechanism III, which is supported by several theoretical calculations.14−18 It should be noted, however, that the mutation study does not necessarily exclude other possibilities such as (I) and (II). A previous resonance Raman study of HG and HR reported few differences in retinal vibrations such as C−C and hydrogen out-of-plane (HOOP) modes, thus excluding Mechanism I.19,20 On the other hand, Mechanism II remains uncertain, namely, an electrostatic interaction between the protonated Schiff base and the counterion. Mechanism II is more influential than Mechanism III for color tuning because the former involves a charge-pair interaction while the latter originates from neutral groups. Hydrogen-bonding strength of the protonated Schiff base is a good measure of the electrostatic interaction with the counterion. In the Raman study, the frequency difference between CNH and CND stretches of the Schiff base reflects the strength of the Schiff base hydrogen bond: a

umans have two types of vision: dim-light vision mediated by rhodopsin (Rh) and color vision achieved by three different cone pigments. We are able to distinguish various colors using blue-, green-, and red-sensitive proteins that maximally absorb at 425, 530, and 560 nm, respectively.1−3 Each cone pigment consists of a common chromophore molecule, 11-cis-retinal, and different chromophore−protein interactions allow preferential absorption at a selected range of wavelengths.4 The mechanism of color tuning has attracted many researchers as it can answer the question, “Why we are able to distinguish colors?”. In color tuning of visual pigments, three factors are mainly taken into account: (I) chromophore distortion, (II) electrostatic interaction between the protonated Schiff base and counterion, and (III) polarity around the βionone ring and polyene chain (Figure 1a).4 Structural information is a prerequisite for the computational reproduction of color vision. Even though X-ray crystallography was successfully employed to clarify the crystal structure of Rh,5−8 none of the cone pigments have yet been crystallized. During evolution, mammals lacked the normal color vision of vertebrate animals, but Catarrhini, including Old World monkeys and Hominoids, acquired green and red pigments by gene duplication.9 There is a ∼30 nm difference in the λmax with 98.1 and 98.6% amino acid identity (only seven and five amino acids differ) in monkeys and humans, respectively.3 A previous site-directed mutagenesis study revealed that three amino acids are responsible for the different λmax between © XXXX American Chemical Society

Received: February 10, 2015 Accepted: March 11, 2015

1130

DOI: 10.1021/acs.jpclett.5b00291 J. Phys. Chem. Lett. 2015, 6, 1130−1133

Letter

The Journal of Physical Chemistry Letters

those of water because of the isotope effect of water, and the Schiff base N−D stretch should be insensitive to D218O water hydration (black-tagged frequencies). Among various blacktagged bands in Figure S1 in the SI, the most plausible candidates are those at 2233 (−)/2216 (+) cm−1 for MR, and at 2227 (−)/2210 (+) cm−1 for MG (Figure 2), as retinal

Figure 2. Light-minus-dark difference FTIR spectra of MR (red) and MG (green) in the 2260−2050 cm−1 region measured at 77 K. Positive and negative bands originate from the all-trans (bathointermediate) and 11-cis forms, respectively. The spectra are reproduced from ref 24. One division of the y axis corresponds to 0.0001 absorbance units.

Figure 1. (a) Chromophore structure in visual pigments and color tuning mechanism. Three regions are highlighted in the mechanism: (I) chromophore distortion in the polyene chain, (II) electrostatic interaction between the protonated Schiff base and the counterion, and (III) polarity around the β-ionone ring and polyene chain. (b) Left: X-ray crystal structure of the chromophore binding site of bovine rhodopsin (Protein Data Bank entry: 1U1913), which is viewed from the helix VII side. The upper and lower regions correspond to the extracellular and cytoplasmic sides, respectively. Two helices (helices IV and VI) and a loop connecting from helix IV are shown in gray. Retinal chromophore and side chains of three key residues for color tuning are illustrated by orange stick and magenta space-filling models, respectively. Right: 90° rotated crystal structure, which is viewed from the extracellular side.

vibrations possibly appear as prominent bands. If this is the case, a lower frequency for MG indicates that the Schiff base hydrogen bond is stronger in MG than in MR. This is consistent with the general feature of color tuning because a stronger hydrogen bond causes greater localization of the positive charge into the Schiff base,4 leading to a spectral blue shift in MG compared with MR. It is intriguing that different residues are located near the ring between MG and MR (Figure 1b) and that the molecular interaction of the Schiff base side differs as well. It is noted that the previous discussion is only possible after vibrational assignment. We have identified the Schiff base N−D stretching frequencies of light-driven proton-pump bacteriorhodopsin (2173 and 2123 cm−1),27 chloride-pump halorhodopsin (2488 cm−1),28 pharaonis sensory rhodopsin II (2140 and 2091 cm−1),29 and Anabaena sensory rhodopsin (2163 and 2125 cm−1)30 by use of low-temperature FTIR spectroscopy. In these studies of microbial rhodopsins, we labeled the Schiff base nitrogen by adding [ζ-15N] labeled lysine into the culture media of bacteria such as H. salinarum and E. coli. In contrast, isotope labeling of proteins in HEK293 cells is not well established, which was a serious problem in the FTIR study of animal rhodopsins. Therefore, in the present study, we attempted a different approach to assign the Schiff base mode of color pigments. Because the Schiff base N−D stretching mode is possibly coupled to other retinal vibrations, we used a C15-Dsubstituted retinal derivative (Figure 3a and Figure S2 in the SI), expecting an isotope effect on the Schiff base N−D stretch. In this study, we expressed MG in HEK293 cells, as described previously,25,31 while C15-D retinal was added to the culture. Then, light-induced difference FTIR spectra of MG were compared between C15-D-labeled and unlabeled retinal. Figure 3b compares the difference FTIR spectra of MG between C15-D (dotted line) and unlabeled (solid line) retinal. (See Figure S3 in the SI for other frequency regions.) Unexpectedly, there are no isotope effects on the 2227

stronger hydrogen bond results in a larger difference frequency.21−24 The previous resonance Raman reported a similar Schiff base hydrogen bond between HG and HR,19 although there was no strict comparison in that study. The hydrogen-bonding strength of the Schiff base (Mechanism II) thus remains uncertain for primate green- and red-sensitive pigments. In addition, our recent FTIR analysis of MG and MR revealed a different hydrogen-bonding network of proteinbound water molecules between them (Figure S1 in the SI).25 Different hydrogen-bonding conditions of water possibly originate from the three O−H-bearing residues (Mechanism III), although it is also possible that these water molecules affect the hydrogen-bonding strength of the Schiff base. Thus, it is important to experimentally determine the hydrogen-bonding strength. The Schiff base N−H stretch is a direct and highly sensitive measure to monitor hydrogen-bonding strength of the protonated Schiff base in which a stronger hydrogen bond results in a lower frequency. As the Schiff base N−H group is deuterated in D2O, the N−D stretch appears at 2600−2000 cm−1, being clearly isolated from O−H, N−H, and C−H stretches.26 Figure S1 in the SI compares X−D stretching vibrations in the light-minus-dark difference FTIR spectra of MR and MG, where green-tagged frequencies correspond to 1131

DOI: 10.1021/acs.jpclett.5b00291 J. Phys. Chem. Lett. 2015, 6, 1130−1133

Letter

The Journal of Physical Chemistry Letters

contribute to the spectral tuning between MG and MR. Previous resonance Raman19,20 and the present FTIR studies reveal no contribution of Mechanism I and II for color tuning between green- and red-sensitive pigments, respectively, indicating that the 30 nm difference originates exclusively from Mechanism III. We previously reported different frequencies (O−D stretch in D2O) of protein-bound water molecules between MG and MR.25 In fact, among the seven and six water vibrations observed for MG and MR, respectively, only two bands showed almost the same frequencies (2583 cm−1 and 2671/2670 cm−1; Figure S4 in the SI). At that time, we had no information about their locations, but the present study provided possible locations of such water molecules. Because the hydrogenbonding network near the Schiff base region is identical between MG and MR, water molecules in this region must possess very similar frequencies as well as the Schiff base N−D stretch. Therefore, water molecules with O−D stretches at 2583 cm−1 and 2671/2670 cm−1 can only be involved in the hydrogen-bonding network in the Schiff base region. Other water molecules whose O−D stretches at 2650−2600 and 2550−2450 cm−1 are located in other regions and different frequencies for highly homologous proteins (MG and MR) suggest that such water molecules are located near the three residues that differ between MG and MR. The color-tuning mechanism for MG and MR is exclusively explained by polarity around the β-ionone ring and polyene chain (Mechanism III), but the origin is not ascribed to only the O−H-bearing residues. Protein-bound water molecules probably assist color discrimination (Figure 4). Our previous analysis found a

Figure 3. (a) Chemical structure of C15-D retinal. (b) Light-minusdark difference FTIR spectra of MG in the 2245−2050 cm−1 region measured at 77 K. Solid and dotted lines represent the spectra with unlabeled and C15-D retinal, respectively. One division of the y axis corresponds to 0.00003 absorbance unit. (c) Light-minus-dark difference FTIR spectra of MR (red) and MG (green) in the 2135− 2050 cm−1 region measured at 77 K. One division of the y axis corresponds to 0.00003 absorbance unit.

Figure 4. Mechanism of color discrimination between green- and redsensitive pigments in primates, which is concluded from the present study.

correlation between the averaged frequencies of water and the color of visual pigments, where the red-shifted pigments possess protein-bound water molecules at a low frequency.24 This observation is consistent with the present interpretation. Before this study, we believed that the 2227 (−)/2210 (+) cm−1 bands originated from the Schiff base N−D stretch, but this was clearly incorrect. The origin of the bands thus remains intriguing because the vibrational bands come from protein while being sensitive to colors. This vibration, either an O−D or N−D stretch of protein, is positively correlated with its absorption, and the identification of the band is our future focus. In summary, the present FTIR analysis identified the Schiff base N−D stretching frequency of MG by use of C15-D retinal. We found that the Schiff base hydrogen-bonding strength is identical between MG and MR. Together with a

(−)/2210 (+) cm−1 bands. Instead, a clear isotope shift was observed for the bands at 2099 (−)/2083 (+) cm−1, being downshifted to 2093 (−)/2078 (+) cm−1 for C15-D retinal. We thus assigned the Schiff base N−D stretches of MG at 2099 cm−1, whose frequency is lowered upon retinal photoisomerization. More importantly, MR has an identical negative band at the same frequency (2099 cm−1; Figure 3c). Although we did not assign the band for MR, very similar spectral features at these frequency regions strongly suggest the same origin. Thus, from the identical Schiff base N−D stretching frequency at 2099 cm−1 for both MG and MR, it is concluded that the hydrogen-bonding strength of the Schiff base is identical between MG and MR. In other words, an electrostatic interaction in the Schiff base region (Mechanism II) does not 1132

DOI: 10.1021/acs.jpclett.5b00291 J. Phys. Chem. Lett. 2015, 6, 1130−1133

Letter

The Journal of Physical Chemistry Letters previous resonance Raman study,19,20 it is safely concluded that the 30 nm difference between green- and red-sensitive pigments originates exclusively from the polarity around the β-ionone ring and polyene chain. Three O−H-bearing amino acid residues in red-sensitive pigments but not in greensensitive pigments and protein-bound water molecules in this region must be directly responsible for color tuning.

■

(12) Asenjo, A. B.; Rim, J.; Oprian, D. D. Molecular Determinants of Human Red/Green Color Discrimination. Neuron 1994, 12, 1131− 1138. (13) Okada, T.; Sugihara, M.; Bondar, A. N.; Elstner, M.; Entel, P.; Buss, V. The Retinal Conformation and its Environment in Rhodopsin in Light of a New 2.2 Å Crystal Structure. J. Mol. Biol. 2004, 342, 571− 583. (14) Trabanino, R. J.; Vaidehi, N.; Goddard, W. A., III. Exploring the Molecular Mechanism for Color Distinction in Humans. J. Phys. Chem. B 2006, 110, 17230−17239. (15) Coto, P. B.; Strambi, A.; Ferre, N.; Olivucci, M. The Color of Rhodopsins at the ab Initio Multiconfigurational Perturbation Theory Resolution. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 17154−17159. (16) Fujimoto, K.; Hasegawa, J.; Nakatsuji, N. Origin of Color Tuning in Human Red, Green, and Blue Cone Visual Pigments: SACCI and QM/MM Study. Chem. Phys. Lett. 2008, 462, 318−320. (17) Rajamani, R.; Lin, Y. L.; Gao, J. The Opsin Shift and Mechanism of Spectral Tuning in Rhodopsin. J. Comput. Chem. 2011, 312, 855− 865. (18) Sekharan, S.; Katayama, K.; Kandori, H.; Morokuma, K. Color Vision: The “OH-Site” Rule for Seeing Red and Green. J. Am. Chem. Soc. 2012, 134, 10706−10712. (19) Kochendoerfer, G. G.; Wand, Z.; Oprian, D. D.; Mathies, R. A. Resonance Raman Examination of the Wavelength Regulation Mechanism in Human Visual Pigments. Biochemistry 1997, 36, 6577−6587. (20) Kochendoerfer, G. G.; Lin, S. W.; Sakmar, T. P.; Mathies, R. A. How Color Visual Pigments Are Tuned. Trends Biochem. Sci. 1999, 24, 300−305. (21) Baasov, T.; Friedman, N.; Sheves, M. Factors Affecting the C N Stretching in Protonated Retinal Schiff Base: A Model Study for Bacteriorhodopsin and Visual Pigments. Biochemistry 1987, 26, 3210− 3217. (22) Lin, S. W.; Imamoto, Y.; Fukada, Y.; Shichida, Y.; Yoshizawa, T.; Mathies, R. A. What Makes Red Visual Pigments Red? A Resonance Raman Microprobe Study of Retinal Chromophore Structure in Iodopsin. Biochemistry 1994, 33, 2151−2160. (23) Smith, S. O.; Pardon, J. A.; Mulder, P. P. J.; Curry, B.; Lugtenburg, J.; Mathies, R. A. Chromophore Structure in Bacteriorhodopsin’s O640 Photointermediate. Biochemistry 1983, 22, 6141− 6148. (24) Smith, S. O.; Marvin, M. J.; Bogomolni, R. A.; Mathies, R. A. Structure of the Retinal Chromophore in the hR578 Form of Halorhodopsin. J. Biol. Chem. 1984, 259, 12326−12329. (25) Katayama, K.; Furutani, Y.; Imai, H.; Kandori, H. Protein-Bound Water Molecules in Primate Red- and Green-Sensitive Visual Pigments. Biochemistry 2012, 51, 1126−1133. (26) Kandori, H. Hydration Switch Model for the Proton Transfer in the Schiff Base Region of Bacteriorhodopsin. Biochim. Biophys. Acta 2004, 1658, 72−79. (27) Kandori, H.; Belenky, M.; Herzfeld, J. Vibrational Frequency and Dipolar Orientation of the Protonated Schiff Base in Bacteriorhodopsin Before and After Photoisomerization. Biochemistry 2002, 41, 6026−6031. (28) Shibata, M.; Muneda, N.; Sasaki, T.; Shimono, K.; Kamo, N.; Demura, M.; Kandori, H. Hydrogen-bonding Alterations of the Protonated Schiff Base and Water Molecule in the Chloride Pump of Natronobacterium pharaonis. Biochemistry 2005, 44, 12279−12286. (29) Shimono, K.; Furutani, Y.; Kamo, N.; Kandori, H. Vibrational Modes of the Protonated Schiff Base in pharaonis Phoborhodopsin. Biochemistry 2003, 42, 7801−7806. (30) Kawanabe, A.; Furutani, Y.; Jung, K. H.; Kandori, H. FTIR Study of the Photoisomerization Processes in the 13-cis and All-trans Forms of Anabaena Sensory Rhodopsin at 77 K. Biochemistry 2006, 45, 4362−4370. (31) Katayama, K.; Furutani, Y.; Imai, H.; Kandori, H. An FTIR Study of Monkey Green- and Red-Sensitive Visual Pigments. Angew. Chem., Int. Ed. 2010, 49, 891−894.

ASSOCIATED CONTENT

* Supporting Information S

Materials and methods, difference FTIR spectra of MR and MG in the 2750−1800 cm−1 region (S1), HPLC chromatograph of 11-cis C15-D labeled retinal (S2), spectral comparison between unlabeled and C15-D retinal in the low frequency region (S3), and possible location of protein-bound water in MR and MG (S4). This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This study was partially supported by grants from the Japanese Ministry of Education, Culture, Sports, Science, and Technology to H.K. (25104009).

■

REFERENCES

(1) Wald, G. Molecular Basis of Visual Excitation. Science 1968, 162, 230−239. (2) Nathans, J.; Thomas, D.; Hogness, D. S. Molecular Genetics of Human Color Vision: The Genes Encoding Blue, Green, and Red Pigments. Science 1986, 232, 193−201. (3) Oprian, D. D.; Asenjo, A. B.; Lee, N.; Pelletier, S. L. Design, Chemical Synthesis, and Expression of Genes for the Three Human Color Vision Pigments. Biochemistry 1991, 30, 11367−11372. (4) Ernst, O. P.; Lodowski, D. T.; Elstner, M.; Hegemann, P.; Brown, L. S.; Kandori, H. Microbial and Animal Rhodopsins: Structures, Functions and Molecular Mechanisms. Chem. Rev. 2014, 114, 126− 163. (5) Palczewski, K.; Kumasaka, T.; Hori, T.; Behnke, C. A.; Motoshima, H.; Fox, B. A.; Trong, I. L.; Teller, D. C.; Okada, T.; Stenkamp, R. E.; Yamamoto, M.; Miyano, M. Crystal Structure of Rhodopsin: A G Protein-Coupled Receptor. Science 2000, 289, 739− 745. (6) Scheerer, P.; Park, J. H.; Hildebrand, P. W.; Kim, Y. J.; Krauss, N.; Choe, H. W.; Hofmann, K. P.; Ernst, O. P. Crystal Structure of Opsin in its G-Protein-Interacting Conformation. Nature 2008, 455, 497− 502. (7) Choe, H. W.; Kim, Y. J.; Morizumi, T.; Pai, E. F.; Krauss, N.; Hofmann, K. P.; Scheerer, P.; Ernst, O. P. Crystal Structure of Metarhodopsin II. Nature 2011, 471, 651−655. (8) Standfuss, J.; Edwards, P. C.; D’Antona, A.; Fransen, M.; Xie, G.; Oprian, D. D.; Schertler, G. F. X. The Structural Basis of AgonistInduced Activation in Constitutively Active Rhodopsin. Nature 2011, 471, 656−660. (9) Shichida, Y.; Imai, H. Visual pigment: G-protein-Coupled Receptor for Light Signals. Cell. Mol. Life Sci. 1998, 54, 1299−1315. (10) Yokoyama, R.; Yokoyama, S. Convergent Evolution of the Redand Green-Like Visual Pigment Genes in Fish, Astyanax Fasciatus, and Human. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 9315−9318. (11) Neitz, M.; Neitz, J.; Jacobs, G. H. Spectral Tuning of Pigments Underlying Red-Green Color Vision. Science 1991, 252, 971−974. 1133

DOI: 10.1021/acs.jpclett.5b00291 J. Phys. Chem. Lett. 2015, 6, 1130−1133