Solid-state NMR for the characterization of retinal chromophore and Schiff base in TAT rhodopsin embedded in membranes under weakly acidic conditions

Abstract

TAT rhodopsin extracted from the marine bacterium SAR11 HIMB114 has a characteristic Thr-Ala-Thr motif and contains both protonated and deprotonated states of Schiff base at physiological pH conditions due to the low pK_a. Here, using solid-state NMR spectroscopy, we investigated the ¹³C and ¹⁵N NMR signals of retinal in only the protonated state of TAT in the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho (1'-rac-glycerol) (POPE/POPG) membrane at weakly acidic conditions. In the ¹³C NMR spectrum of ¹³C retinal-labeled TAT rhodopsin, the isolated 14-¹³C signals of 13-trans/15-anti and 13-cis/15-syn isomers were observed at a ratio of 7:3. ¹⁵N retinal protonated Schiff base (RPSB) had a significantly higher magnetic field resonance at 160 ppm. In ¹⁵N RPSB/λ_max analysis, the plot of TAT largely deviated from the trend based on the retinylidene-halide model compounds and microbial rhodopsins. Our findings indicate that the RPSB of TAT forms a very weak interaction with the counterion.

Significance

An observable ¹⁵N electronic environment of retinal protonated Schiff base (RPSB) using solid-state NMR spectroscopy is directly related to the hydrogen bond strength of RPSB in microbial rhodopsin. Here, we observed the solid-state NMR spectra of membrane-embedded TAT rhodopsin to analyze the structure of retinal Schiff base. The results of ¹³C NMR revealed that TAT rhodopsin has all-trans/15-anti and 13-cis/15-syn retinal at a ratio of 7:3 in the dark. ¹⁵N NMR signals of RPSB appeared in the higher magnetic field among microbial rhodopsins. Moreover, the results of ¹⁵N NMR suggested that interactions between RPSB and the counterion are weakened because of the TAT motif in helix C. Our study provided novel insights regarding the unusually low pK_a of the Schiff base in TAT rhodopsin.

Introduction

All microbial rhodopsins consist of seven-transmembrane helices with all-trans-retinal chromophores via retinal protonated Schiff bases (RPSBs) [1–3]. Numerous microbial rhodopsins have been identified using metagenomic technologies [4,5]. Functional conversions of microbial rhodopsins have been actively developed based on the knowledge of their molecular structure and photochemical properties [6,7]. Interaction of the RPSB with the negatively charged counterion(s) in microbial rhodopsins leads to electrostatic stabilization in the electronic ground state of the retinal, accompanied by an increase in the pK_a of RPSB [1]. The carboxyl group(s) of Asp and Glu constitute counterion(s), and bound water molecules inside the protein participate in the RPSB-counterion interactions. Bacteriorhodopsin (BR) has a characteristic sequence motif (DTD) composed of Asp85 (proton acceptor), Thr89, and Asp96 (proton donor) in helix C [2,8,9]. Asp85 and Asp212 with bound water molecules form a counterion complex, in which Asp85 acts as a primary counterion. In BR and proteorhodopsin (PR), the high pK_a of RPSB is reported to be 13.3 and 11.3, respectively [10,11].

TAT rhodopsin, discovered in the α-proteobacterial SAR11 HIMB114, has a unique TAT motif (Thr82, Ala86, and Thr93 in helix C), which contains no Glu or Asp in both the proton acceptor and donor positions (Figure 1) [12]. Asp227, corresponding to Asp212, is the only counterion because of the unknown crystal structure of TAT rhodopsin. TAT rhodopsin exhibits two absorptions at 561 nm and 400 nm, corresponding to the protonated and deprotonated states of the Schiff base at pH 8.0 [13]. The pK_a values of SB in a DDM detergent and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho (1'-rac-glycerol) (POPE/POPG) membrane were determined to be 7.3 and 8.4, respectively [13]. Both pK_a values were much lower than those of BR. Therefore, TAT rhodopsin is under a protonated/deprotonated SB equilibrium at physiological pH [14]. Moreover, TAT rhodopsin undergoes a unique pH-dependent photochemical reaction. Below pH 8 or less, no transient absorption of TAT rhodopsin was detected at microseconds and slower time scales. All-trans to 13-cis photoisomerization has been observed by photoexcitation of the protonated SB state using UV-visible and FT-IR spectroscopy at 77 K, and this photoreaction duration is in the order of 10^–5 s [13,14]. Therefore, the primary K intermediate rapidly returns to its initial state without the production of any of the following intermediates. Over pH 8.0, the deprotonated SB state of TAT is absorbed in the UV-blue region and undergoes a slow photocycle, which is advantageous for signal transduction [13,14]. In a study of the T82D TAT mutant, the pK_a of the RPSB increased (>10.5), resulting in Thr82 being the origin of the neutral pK_a of the SB in wild-type TAT rhodopsin [15]. In a recent report on the binding of Ca²⁺ to TAT, we found that the K_d value of Ca²⁺ binding to TAT rhodopsin is 0.17 mM at pH 8.0, and Ca²⁺ binding induces deprotonation of the SB and secondary structural changes [16]. Moreover, Glu54, which is distant from the retinal and Asp227, were identified as Ca²⁺-binding site residues [16].

Figure 1 

The model structure of TAT rhodopsin with the 13-trans/15-anti retinal configuration. Amino-acid residues involved in the characteristic motif of TAT are indicated: Thr82/Ala86/Thr93 in helix C and Asp227 in helix G (TAT-D) correspond to Asp85/Thr89/Asp96 in helix C and Asp212 in helix G of BR (DTD-D), respectively. Glu54 of TAT rhodopsin is corresponding to Met56 in helix B of BR.

Solid-state nuclear magnetic resonance (SSNMR) studies of microbial rhodopsins have provided structural information regarding the local electronic environment of the RPSB and retinal configurations, as well as the conformation and dynamics of protein [17–19]. Since the electronic environment of RPSB is related to its hydrogen bond strength with the counterion relating to the regulation of the pK_a of RPSB and the color tuning, the ¹⁵N chemical shift values are particularly important for that detection and evaluation. Here, we have focused on the origin of unusually low pK_a of RPSB in TAT and performed solid-state NMR measurements of the sufficiently protonated SB state of TAT under weakly acidic conditions (pH 5.0, 6.0) in the dark to investigate the RPSB-counterion interaction. We show the ¹³C and ¹⁵N NMR signals of retinal and RPSB of TAT embedded in a membrane and highlight that NMR signals give us insights into TAT with a low pK_a value.

Materials and Methods

Materials

All chemicals were of analytical grade and were used without further purification. ¹⁵Nε lysine (Lys) was purchased from the Cambridge Isotope Laboratory (CIL, UK). 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho (1'-rac-glycerol) (POPG) were purchased from Avanti Polar Lipids. Isopropyl β-d-thiogalactopyranoside (IPTG) and n-dodecyl-β-D-maltoside (DDM) were purchased from Fujifilm Wako. The water was treated using a Milli-Q purification system.

Sample Preparation

[14,20-¹³C]-labeled retinal was synthesized as previously reported [20]. For expression of TAT rhodopsin, the plasmid containing the gene encoding TAT with a C-terminal 6×His tag was obtained as noted previously [13]. WT TAT rhodopsin with a C-terminal His-tag was overexpressed in E. coli BL21(DE3) cells cultured in M9 minimal medium containing 100 mg of [¹⁵Nε] Lys. At an OD₆₆₀ of 0.85, 1 mM IPTG and 5 μM [14,20-¹³C] all-trans retinal were added for induction, and then, we allowed the protein to be expressed at 37°C overnight. Then, the cells were lysed by ultrasonication, and TAT was solubilized using DDM. The solubilized protein was purified using Ni-NTA agarose via a batch procedure. TAT was reconstituted into (POPE)/(POPG) (3:1) membranes in a protein-to-lipid molar ratio of 1:25. The sample was suspended in 10 mM NaCl and 5 mM MES buffer at pH 5.0 or 6.0.

Solid-State NMR Measurements

The hydrated membrane sample with TAT was concentrated by centrifugation and packed into the central part of a 4.0-mm outer diameter zirconia rotor under dim light. The rotor was maintained in the dark for two days. ¹³C and ¹⁵N solid-state NMR experiments (n=4) were performed using a 600 MHz Bruker Avance III spectrometer equipped with a ¹H-¹³C-¹⁵N triple-resonance probe. Furthermore, ¹³C and ¹⁵N cross-polarization and magic-angle spinning (CP-MAS) experiments were performed at setting temperatures of 264.5, 278, and 300 K under a MAS frequency of 12.0 kHz, while ¹³C-¹³C dipolar-assisted rotational resonance (DARR) experiments were performed for ¹³C homonuclear recoupling using a mixing time of 500 ms with 64 and 2048 acquisition points in the f1 and f2 dimensions [21]. A spinal-64 proton high-power decoupling field of 76 kHz was used during each acquisition. ¹³C chemical shifts were externally referenced to the methylene resonance of adamantane at 40.48 ppm (sodium 3-(trimethylsilyl) propane-1-sulfonate (DSS) at 0.0 ppm), while ¹⁵N chemical shifts were externally referenced to the ¹⁵NH₄Cl crystal at 38.34 ppm. The acquired NMR data were phased and baseline-corrected using TOPSPIN software. A deconvolution procedure was applied to the ¹³C and ¹⁵N CP-MAS spectra to obtain the values of the chemical shift, line width, and integrals in the spectral region of 14-¹³C and ¹⁵N RPSB.

Results and Discussion

¹³C NMR of Retinal in TAT

¹³C NMR signals of 14- and 20-¹³C retinal are very sensitive to 15-syn/anti and 13-trans/cis configurations, respectively [17]. First, the ¹³C NMR signals of the retinal of TAT rhodopsin in the POPE/POPG membrane were obtained to investigate the retinal configuration. Figure 2(A) shows the ¹³C CP-MAS NMR spectrum of [14,20-¹³C] retinal-labeled TAT at 264.5 K and pH 5.0 in the dark. Intense NMR signals of 14- and 20-¹³C of retinal were observed together with those of proteins and lipids. Two isolated 14-¹³C signals appeared at 121.6 and 113.0 ppm, indicating the presence of two retinal isomers with different isomer ratios. The ratio of the two isomers was estimated to be 7:3 using the integrals of individual peaks. At the higher temperatures of 278 K and 300 K, the ratio of isomers was mostly unchanged in the temperature range, although the observed components at 119 ppm may correspond to unsaturated carbons of POPG (Figure S1). Figure 2 (B) shows the ¹³C-¹³C DARR spectrum of [14,20-¹³C] retinal-labeled TAT at 278 K and pH 6.0. Two cross-peaks between 20-¹³C and 14-¹³C were observed at 15.7/121.6 and 25.3/113.0 ppm, indicating 13-trans/15-anti and 13-cis/15-syn isomers, respectively. Thus, our NMR results showed that TAT in the membrane primarily takes the 13-trans/15-anti (all-trans) form in the dark. This is consistent with the HPLC analysis of the all-trans retinal isomer of dark-adapted TAT rhodopsin [13]. Although a MES buffer shows medium pH changes with temperature [22], almost no buffer pH in this temperature range (264.5–300 K) affects the charged state of the titratable Asp and Glu residues, such as the carboxylate (COO^–) of Asp227 in the vicinity of retinal.

Figure 2 

(A) Chemical structure of 13-trans/15-anti and 13-cis/15-syn retinal isomers, and the ¹³C CP-MAS NMR spectrum of [14,20-¹³C] retinal-labeled TAT rhodopsin at pH 5.0 and 264.5 K. (B) ¹³C-¹³C DARR NMR spectrum at pH 6.0 and 278 K using a mixing time of 500 ms.

¹⁵N NMR of Protonated Retinal Schiff Base

Although ¹⁵N NMR measurements of the ¹⁵N Lys-enriched TAT for signal enhancement were also performed at 278 K and 300 K, RPSB intensities were substantially low (data not shown). CP-MAS experiments at lower temperatures can improve signal sensitivity. Therefore, the ¹⁵N CP-MAS NMR experiment of [¹⁵Nε] Lys-labeled TAT was performed in the dark at 264.5 K. TAT has 13 Lys residues, and Lys231 forms a Schiff base that is covalently bound to retinal. The peaks at 32.3 and 118.3 ppm correspond to free Lys residues and backbone amide nitrogen, respectively. Moreover, the unresolved peaks of the protonated Schiff base in TAT rhodopsin appeared at approximately 160 ppm, and no peaks were observed in the range over 230 ppm, corresponding to the deprotonated state. Thus, TAT rhodopsin forms a significantly protonated SB state under weakly acidic conditions (data not shown at pH 6.0), as reported in the pH-titrated absorption data [13,14]. Peak deconvolution analysis revealed that the RPSB peaks were separated into two peak components with an intensity ratio of 7:3 (Figure S2). Consequently, the intensity ratio of the ¹⁵N RPSB peaks is consistent with that of the 14-¹³C retinal peaks. Thus, one ¹⁵N peak identified at 160.6 ppm could be assigned to the 13-trans/15-anti isomer as a major form, while another peak at 159.0 ppm could be attributed to the 13-cis/15-syn isomer (Figure S2). The deviation of ¹⁵N RPSB chemical shift values (|Δ¹⁵N([13-trans]-[13-cis])|) between the 13-trans/15-anti and 13-cis/15-syn isomers of TAT was very low (1.6 ppm) as compared to those of BR (7.0 ppm) and MR (3.0 ppm) (Table 1). The electronic environments of the RPSB nitrogen of both isomers are similar, implying that the two isomers have similar hydrogen bonding strength. In addition, at higher temperatures (278 and 300 K), the ¹⁵N peaks also appeared at approximately 160 ppm but they were difficult to separate because of the more heavily overlapping signals (data not shown).

Table 1  ¹³C and ¹⁵N chemical shift values (ppm) of 20-¹³C and 14-¹³C of retinal and ¹⁵N RPSB of TAT, BR, and MR. Difference of ¹⁵N RPSB between 13-trans/15-anti and 13-cis/15-syn isomers*

	Configuration	20-13C	14-13C	15N	|Δ15N([13-trans]-[13-cis])|
TAT	13-trans/15-anti	15.7	121.6	160.6	1.6
	13-cis/15-syn	25.3	113.0	159.0
BR1)	13-trans/15-anti	15.1	125.1	168.8	7.0
	13-cis/15-syn	24.2	112.0	175.8
MR2)	13-trans/15-anti	14.6	124.5	184.7	3.0
	13-cis/15-syn	23.0	116.1	181.7

¹⁾ From references of [17], [18].

²⁾ From reference of [19].

* The ¹³C and ¹⁵N chemical shifts in this table were calibrated to the reference chemical shifts of DSS and ¹⁵NH₄Cl.

Figure 3 (B) shows the plot of ¹⁵N chemical shift of RPSB and the maximum absorption (λ_max) for the all-trans isomer. A linear relationship was observed between the all-trans-retinylidene model compounds, which have different halide ion species as counterions [23,24]. The λ_max at 561 nm was used with an ¹⁵N shift at 160.6 ppm (all-trans form) for the plot of TAT [13]. Plotting the observed data of TAT and the microbial rhodopsins, several plots showed a similar trend with a linear relationship. The plot of each rhodopsin can be characterized using the corresponding residues, such as the DTD of BR in Figure 3 (B). All rhodopsins, except for the TAT rhodopsin plotted in Figure 3 (B), have at least one Asp residue in helix C as a counterion. Interestingly, the plot of TAT significantly deviated from the linear relationship, resulting in the highest magnetic field resonance compared with those of other microbial rhodopsins, such as BR, MR, sensory rhodopsin II from Natronomonas pharaonis (NpSRII), Thermophilic rhodopsin (TR), Anabaena sensory rhodopsin (ASR), and Krokinobacter rhodopsin 2 (KR2) [19,23–28]. In particular, the unique ¹⁵N isotropic chemical shift value of TAT suggests that the electronic environment of the RPSB nitrogen in TAT is considerably different from that of other microbial rhodopsins and that the hydrogen bonding strength of the RPSB with counterions is largely weakened. Interaction of the RPSB with the counterion leads to electronic stabilization in the electronic ground state of the retinal, accompanied by an increase in the pK_a of RPSB [1]. Although Asp227 is the presumed negatively charged counterion on TAT rhodopsin, Thr82 in helix C may influence RPSB destabilization. In the T82D mutant, the pK_a (RPSB, T82D) is increased to up to ~10, implying that Asp82 acts as a primary counterion and stabilizes RPSB [15]. Halorhodopsin (HR), which is a light-driven inward Cl^– pumping rhodopsin, contains a Thr residue corresponding to Asp85 in BR and Thr82 in TAT. In the case of NpHR from Natronomonas pharaonis with Thr126, the pK_a of SB was determined to be 11.7 for 1 M NaCl concentration, while it was 9.2 in the absence of Cl^– [29]. For RmHR from Rubricoccus marinus, the pK_a was also determined to be 10.0 for 4 M NaCl and 7.3 for free Cl^– [30]. Thus, there is a phenomenon that the RPSB in HR is stabilized by the bound Cl^– as a counterion, resulting in the high pK_a values [31]. In a previous study, the pK_a for model RPSB compound in a methanol/water (1:1) solution was estimated to be 7.2 [32]. Those imply that the pK_a of RPSB tends to be low when it is exposed to the solvent itself or has a weak interaction with the counterion.

Figure 3 

(A) ¹⁵N CP-MAS NMR spectrum of [¹⁵Nε] Lys-labeled TAT rhodopsin at pH 5.0 and 264.5 K. Expanded spectral region in the 90 and 210 ppm region in the inset. ¹⁵N RPSB signals appeared at around 160 ppm as a signal/noise (S/N) ratio of 15:1 in 60,000 scans. (B) Correlation between ¹⁵N chemical shift of RPSB and maximal absorption wavelength (λ_max) for all-trans isomer. The black dotted line is the linear relationship of the all-trans retinylidene halide (Cl^–, Br^–, I^–) model compounds [23,24]. The motif in helix C of each rhodopsin is indicated like BR (DTD).

The photolyzed wild-type BR experiences a transient drop in RPSB pK_a with the event of proton transfer. In the D85N mutant of BR, the pK_a value of RPSB is lowered from above 13 to 9 and the replacement prevents the formation of M-intermediate with the deprotonated state of SB in the photolyzed D85N [33]. ¹⁵N RPSB signal of all-trans isomer in D85N exhibits a higher field magnetic resonance of approximately a maximum of 15 ppm than that of BR [34]. Therefore, the higher field resonance trends of RPSB are exhibited for systems with weaker counterion interactions. No transient absorption was detected at microseconds and slower time scales for the protonated SB state of TAT rhodopsin, as the primary K intermediate rapidly returns to its initial state without the production of any of the following intermediates [14]. This indicates that the activation barrier for thermal 13-cis to all-trans isomerization is much lower than the relaxation of the 13-cis state (to the next intermediate), which is achieved by the specific chromophore−protein interaction in TAT rhodopsin. Very weak interaction of the protonated Schiff base with the counterion in TAT rhodopsin, found in the present NMR study, is possibly correlated with the unique reaction dynamics in the early photointermediates. The dynamics of the retinal and surrounding residues may also contribute to the structure of the retinal-binding pocket. As mentioned above, by plotting the λ_max/¹⁵N RPSB chemical shift, the variation tendency over the individually plotted data was visualized, as shown in Figure 3(B). The plot of TAT in Figure 3(B) deviated from the linear relationship of the model compounds. Additional factors may contribute to the deviation, such as the twist of the retinal polyene chain or polar/aromatic amino acids close to the chromophore. In summary, the weak interaction of RPSB suggested by the NMR results can be considered as one of the reasons for the low pK_a of TAT rhodopsin.

In this study, the ¹³C and ¹⁵N NMR measurements of TAT rhodopsin in the membrane under weakly acidic conditions showed that the hydrogen bonding strength of RPSB is largely weakened, suggesting a lower pK_a of the RPSB. However, TAT rhodopsin contains unique features, such as a UV-dependent pH sensor and a Ca²⁺ receptor based on the TAT motif [13–16]. For instance, the deprotonated state of TAT rhodopsin generated over pK_a exhibits a characteristic photocycle [13,14]. To understand the specificity of TAT rhodopsin, ¹³C and ¹⁵N solid-state NMR measurements of its deprotonated SB and its surrounding residues under alkaline conditions or in the presence of Ca²⁺ must be performed.

Conclusions

In this study, we investigated the NMR signals of retinal and Schiff bases of TAT rhodopsin in a membrane under weakly acidic conditions using solid-state NMR. The ¹³C NMR results revealed that TAT rhodopsin in the POPE/POPG membrane contains 13-trans/15-anti and 13-cis/15-syn isomers at a ratio of 7:3 in the dark. We found that the ¹⁵N NMR signal of RPSB of the all-trans isomer appeared at 160.6 ppm, indicating that the hydrogen bonding strength of the RPSB with the counterion was largely weakened. It is possible that the weak interaction between RPSB and TAT rhodopsin is one of the reasons for the low pK_a of TAT rhodopsin.

Conflict of Interest

The authors declare that no competing interests exist.

Author Contributions

I.K., K.K., and H.K. directed the project. S.A. and I.K. prepared the manuscript. T.S. and K.K. prepared the expression plasmid. S.A. expressed the isotope-labeled protein and prepared solid-state NMR samples. S.A. and I.K. performed solid-state NMR measurements and analyzed the ¹³C and ¹⁵N NMR data. T.O. and A.W. chemically synthesized 14, 20-¹³C stable isotope-labeled retinal. All authors discussed and commented on the manuscript.

Data Availability

The evidence data generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Acknowledgements

This work was supported in part by JSPS KAKENHI Grant Numbers (JP21H05229 to I.K., JP21H04969 to H. K., JP21H01883 to K.K., JP21K06466 to T. O.), JST CREST (JPMJCR21B2 to I.K.), and Public Interest Yokohama Kogyokai (to I.K.). The authors thank Nobuko Yamaguchi for the financial support.

References

• [1]   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. 114, 126–163 (2014). https://doi.org/10.1021/cr4003769
• [2]   Kandori,  H. Ion-pumping microbial rhodopsins, Front. Mol. Biosci. 2, 1–11 (2015). https://doi.org/10.3389/fmolb.2015.00052
• [3]   Kurihara,  M.,  Sudo,  Y. Microbial rhodopsins: Wide distribution, rich diversity and great potential. Biophys. Physicobiol. 12, 121–129 (2015). https://doi.org/10.2142/biophysico.12.0_121
• [4]   Rozenberg,  A.,  Inoue,  K.,  Kandori,  H.,  Béjà,  O. Microbial rhodopsins: The last two decades. Annu. Rev. Microbiol. 75, 427–447 (2021). https://doi.org/10.1146/annurev-micro-031721-020452
• [5]   Inoue,  K.,  Tsukamoto,  T.,  Sudo,  Y. Molecular and evolutionary aspects of microbial sensory rhodopsins. Biochim. Biophys. Acta 1837, 562–577 (2014). https://doi.org/10.1016/j.bbabio.2013.05.005
• [6]   Kaneko,  A.,  Inoue,  K.,  Kojima,  K.,  Kandori,  H.,  Sudo,  Y. Conversion of microbial rhodopsins: Insights into functionally essential elements and rational protein engineering. Biophys. Rev. 9, 861–876 (2017). https://doi.org/10.1007/s12551-017-0335-x
• [7]   Kojima,  K.,  Shibukawa,  A.,  Sudo,  Y. The unlimited potential of microbial rhodopsins as optical tools. Biochemistry 59, 218–229 (2020). https://doi.org/10.1021/acs.biochem.9b00768
• [8]   Luecke,  H.,  Schobert,  B.,  Richter,  H. T.,  Cartailler,  J. P.,  Lanyi,  J. K. Structure of bacteriorhodopsin as 1.55Å resolution. J. Mol. Biol. 291, 899–911 (1999). https://doi.org/10.1006/jmbi.1999.3027
• [9]   Shibata,  M.,  Tanimoto,  T.,  Kandori,  H. Water molecules in the Schiff base region of bacteriorhodopsin. J. Am. Chem. Soc. 125, 13312–13313 (2003). https://doi.org/10.1021/ja037343s
• [10]   Sheves,  M.,  Albeck,  A.,  Friedman,  N.,  Ottolenghi,  M. Controlling the pK_a of the bacteriorhodopsin Schiff base by use of artificial retinal analogues. Proc. Nati. Acad. Sci. U.S.A. 83, 3262–3266 (1986). https://doi.org/10.1073/pnas.83.10.3262
• [11]   Imasheva,  E.,  Balashov,  S.,  Wang,  J.,  Dioumeav,  A.,  Lanyi,  J. Selectivity of retinal photoisomerization in proteorhodopsin is controlled by aspartic acid 227. Biochemistry 43, 1648–1655 (2004). https://pubs.acs.org/doi/10.1021/bi0355894
• [12]  Philosof, A., Béjà, O. Bacterial, archaeal and viral-like rhodopsins from the red sea. Environ. Microbiol. Rep. 5, 475−482 (2013). https://doi.org/10.1111/1758-2229.12037
• [13]   Kataoka,  C.,  Inoue,  K.,  Katayama,  K., Béjà, O. Kandori, H. Unique photochemistry observed in a new microbial rhodopsin. J. Phys. Chem. Lett. 10, 5117–5121 (2019). https://doi.org/10.1021/acs.jpclett.9b01957
• [14]   Kataoka,  C.,  Sugimoto,  T.,  Shigemura,  S.,  Katayama,  K.,  Tsunoda,  S. P.,  Inoue,  K., et al. TAT rhodopsin is an ultraviolet-dependent environmental pH sensor. Biochemistry 60, 899–907 (2021). https://doi.org/10.1021/acs.biochem.0c00951
• [15]   Sugimoto,  T.,  Katayama,  K.,  Kandori,  H. Role of Thr82 for the unique photochemistry of TAT rhodopsin. Biophys. Physicobiol. 18, 108–115 (2021). https://doi.org/10.2142/biophysico.bppb-v18.012
• [16]   Sugimoto,  T.,  Katayama,  K.,  Kandori,  H. Calcium binding to TAT Rhodopsin. J. Phys. Chem. B 126, 2203–2207 (2022). https://doi.org/10.1021/acs.jpcb.2c00233
• [17]   Naito,  A.,  Makino,  Y.,  Shigeta,  A.,  Kawamura,  I. Photoreaction pathways and photo intermediates of retinal-binding photo-receptor proteins as revealed by in-situ photoirradiation solid-state NMR spectroscopy. Biophys. Rev. 11, 167–181 (2019). https://doi.org/10.1007/s12551-019-00501-w
• [18]   Bajaj,  V. S.,  Mak-Jurkauskas,  M. L.,  Belenky,  M.,  Herzfeld,  J.,  Griffin,  R. G. Functional and shunt states of bacteriorhodopsin resolved by 250 GHz dynamic nuclear polarization-enhanced solid-state NMR. Proc. Nati. Acad. Sci. U.S.A. 106, 9244–9249 (2009). https://doi.org/10.1073/pnas.0900908106
• [19]   Kawamura,  I.,  Seki,  H.,  Tajima,  S.,  Makino,  Y.,  Shigeta,  A.,  Okitsu,  T., et al. Structure of a retinal chromophore of dark-adapted middle rhodopsin as studied by solid-state nuclear magnetic resonance spectroscopy. Biophys. Physicobiol. 18, 177–185 (2021). https://doi.org/10.2142/biophysico.bppb-v18.019
• [20]   Lugtehburg,  J. The synthesis of ¹³C-labelled retinals. Pure Appl. Chem. 57, 753–762 (1985). https://doi.org/10.1351/pac198557050753
• [21]   Takegoshi,  K.,  Nakamura,  S.,  Terao,  T. ¹³C-¹H dipolar-assisted rotational resonance in magic-angle spinning NMR. Chem. Phys. Lett. 344, 631–637 (2001). https://doi.org/10.1016/S0009-2614(01)00791-6
• [22]   Fukuda,  H.,  Takahashi,  K. Enthalpy and heat capacity changes for the proton dissociation of various buffer components in 0.1 M potassium chloride. Proteins 33, 159–166 (1998). https://onlinelibrary.wiley.com/doi/10.1002/(SICI)1097-0134(19981101)33:2%3C159::AID-PROT2%3E3.0.CO;2-E
• [23]   Harbison,  G. S.,  Herzfeld,  J.,  Griffin,  R. G. Solid-state nitrogen-15 nuclear magnetic resonance study of the Schiff base in bacteriorhodopsin. Biochemistry 22, 1–5 (1983). https://doi.org/10.1021/bi00270a600
• [24]   De Groot,  H. J. M.,  Harbison,  G. S.,  Herzfeld,  J.,  Griffin,  R. G. Nuclear magnetic resonance study of the Schiff base in bacteriorhodopsin: Counterion effects on the ¹⁵N shift anisotropy. Biochemistry 28, 3346–3353 (1989). https://doi.org/10.1021/bi00434a033
• [25]   Shi,  L.,  Kawamura,  I.,  Jung,  K. H.,  Brown,  L. S.,  Ladizhansky,  V. Conformation of a seven-helical transmembrane photosensor in the lipid environment. Angew. Chem. Int. Ed. 50, 1302–1305 (2011). https://doi.org/10.1002/anie.201004422
• [26]   Shionoya,  T.,  Mizuno,  M.,  Tsukamoto,  T.,  Ikeda,  K.,  Seki,  H.,  Kojima,  K., et al. High thermal stability of oligomeric assemblies of thermophilic rhodopsin in a lipid environment. J. Phys. Chem. B 122, 6945–6953 (2018). https://doi.org/10.1021/acs.jpcb.8b04894
• [27]   Tomonaga,  Y.,  Hidaka,  T.,  Kawamura,  I.,  Nishio,  T.,  Osawa,  K.,  Okitsu,  T., et al. An active photoreceptor intermediate revealed by in situ photoirradiated solid-state NMR spectroscopy. Biophys. J. 101, L50–L52 (2011). https://doi.org/10.1016/j.bpj.2011.10.022
• [28]   Shigeta,  A.,  Ito,  S.,  Kaneko,  R.,  Tomida,  S.,  Inoue,  K.,  Kandori,  H., et al. Long-distance perturbation on Schiff base-counterion interaction by His30 and the extracellular Na⁺-binding site in Krokinobacter rhodopsin 2. Phys. Chem. Chem. Phys. 20, 8450–8455 (2018). https://doi.org/10.1039/C8CP00626A
• [29]   Hayashi,  S.,  Tamogami,  J.,  Kikukawa,  T.,  Okamoto,  H.,  Shimono,  K.,  Miyauchi,  S., et al. Thermodynamic parameters of anion binding to halorhodopsin from Natronomonas pharaonis by isothermal titration calorimetry. Biophys. Chem. 172, 61–67 (2013). https://doi.org/10.1016/j.bpc.2013.01.001
• [30]   Nakajima,  Y.,  Tsukamoto,  T.,  Kumagai,  Y.,  Ogura,  Y.,  Hayashi,  T.,  Song,  J., et al. Presence of a haloarchaeal halorhodopsin-like Cl^– pump in marine bacteria. Microbes Environ. 33, 89–97 (2018). https://doi.org/10.1264/jsme2.ME17197
• [31]   Varo,  G.,  Zimanyi,  L.,  Fan,  X.,  Sun,  L.,  Needleman,  R.,  Lanyi,  J. K. Photocycle of halorhodopsin from Halobacterium salinarium. Biophys. J. 68, 2062–2072 (1995). https://doi.org/10.1016/S0006-3495(95)80385-1
• [32]   Rousso,  I.,  Friedman,  N.,  Sheves,  M.,  Ottolenghi,  M. pK_a of the protonated Schiff base and aspartic 85 in the bacteriorhodopsin binding site is controlled by a specific geometry between the two residues. Biochemistry 34, 12059–12065 (1995). https://doi.org/10.1021/bi00037a049
• [33]   Kataoka,  M.,  Kamikubo,  H.,  Tokunaga,  F., Brown L. S.,  Yamazaki,  Y.,  Maeda,  A., et al. Energy coupling in an ion pump: The reprotonation switch of bacteriorhodopsin. J. Mol. Biol. 243, 621–638 (1994). https://doi.org/10.1016/0022-2836(94)90037-X
• [34]   Hatcher,  M. E.,  Hu,  J. G.,  Belenky,  M.,  Verdegem,  P.,  Lugtenburg,  J.,  Griffin,  R. G. et al. Control of the pump cycle in bacteriorhodopsin: Mechanisms elucidated by solid-state NMR of the D85N mutant. Biophys. J. 82, 1017–1029 (2002). https://doi.org/10.1016/S0006-3495(02)75461-1