Photochemistry of Bacteriorhodopsin with Various Oligomeric Statuses

Article pubs.acs.org/JPCB

Cite This: J. Phys. Chem. B XXXX, XXX, XXX−XXX

Photochemistry of Bacteriorhodopsin with Various Oligomeric Statuses in Controlled Membrane Mimicking Environments: A Spectroscopic Study from Femtoseconds to Milliseconds Yu-Min Kao,† Chung-Hao Cheng,† Ming-Lun Syue,† Hsin-Yu Huang,† I-Chia Chen,*,† Tsyr-Yan Yu,*,‡,§ and Li-Kang Chu*,† †

Department of Chemistry, National Tsing Hua University, 101, Sec. 2, Kuang-Fu Road, Hsinchu 30013, Taiwan Institute of Atomic and Molecular Sciences, Academia Sinica, 1, Sec. 4, Roosevelt Road, Taipei 10617, Taiwan § International Graduate Program of Molecular Science and Technology, National Taiwan University, Taipei, Taiwan

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‡

S Supporting Information *

ABSTRACT: Preparing transmembrane protein in controllable lipid bilayers is essential for unravelling the coupling of the environments and its dynamic functions. Monomerized bacteriorhodopsin (mbR) embedded in covalently circularized nanodiscs was prepared with dimyristoylphosphatidylglycerol (DMPG) lipid and circular membrane scaffold proteins of two different sizes, cE3D1 and cΔH5, respectively. The retinal photoisomerization kinetics and thermodynamic photocycle were examined by femtosecond and nanosecond transient absorption, respectively, covering the time scale from femtoseconds to hundreds of milliseconds. The kinetics of the retinal isomerization and proton migration from the protonated Schiff base to Asp-85 were not significantly different for monomeric bR solubilized in Triton X-100 or embedded in circularized nanodiscs. This can be ascribed to the local tertiary structures at the retinal pocket vicinity being similar among monomeric bR in various membrane mimicking environments. However, the aforementioned processes are intrinsically different for trimeric bR in purple membrane (PM) and delipidated PM. The reprotonation of the deprotonated Schiff base from Asp-96 in association with the decay of intermediate M, which involved wide-ranged structural alteration, manifested a difference in terms of the oligomeric statuses, as well as a slight dependence on the size of the nanodisc. In summary, bR oligomeric statuses, rather than the environmental factors, such as membrane mimicking systems and nanodisc size, play a significant role in bR photocycle associated with short-range processes, such as the retinal isomerization and deprotonation of protonated Schiff base at the retinal pocket. On the other hand, the environmental factors, such as the types of membrane mimicking systems and the size of nanodiscs, affect those dynamic processes involving wider structural alterations during the photocycle.

1. INTRODUCTION The lipid nanodisc (ND) has been extensively employed for mimicking lipid bilayer environments in membrane protein researches1,2 due to its size adjustability3−5 and tunability in lipid compositions.1,6−8 Generally, lipid nanodiscs are selfassembled lipid bilayers wrapped by two copies of the membrane scaffold proteins (MSP) in linear form.3,4 Recently, the covalently circularized nanodisc (cND) was developed9,10 to enhance the stability. Bacteriorhodopsin (bR) is a light-driven proton pump protein composed of seven α-helices and one protonated retinal Schiff base covalently linked to Lys-216.11,12 The purple membrane (PM) is composed of three bRs in a unit hexagonal lattice,13 surrounded by ca. 10 native lipids per bR.14 The absorption of a yellow photon leads to Franck−Condon transition of the all-trans retinal from S0 to a 1Bu-like state, generating the short-lived intermediate H,15 followed by a relaxed intermediate I after 50−100 fs.15 The decay of intermediate I requires overcoming a barrier, which strongly © XXXX American Chemical Society

depends on the distribution of the charged residues at the retinal vicinity,16 to proceed with the cascading retinal isomerization at C13C14.15 The transition from intermediate I to intermediate J possesses a single lifetime of ca. 0.5 ps for PM.17,18 Consequently, a series of structural modulations trigger the proton migration19 and retinal reisomerization to the original all-trans configuration,20 in terms of a thermodynamically controlled photocycle characterized with spectrally distinguishable intermediates eq 1,21 ∼3 ps ∼ 2 μs hν ⎯ H, I, J states ⎯⎯⎯⎯⎯⎯⎯→ K 590 ⎯⎯⎯⎯⎯⎯⎯→ L550 bR 568 ⎯→ 50 μs 1 ms 2 ms 8 ms ⎯⎯⎯⎯⎯⎯→ M410 ⎯⎯⎯⎯⎯→ N560 ⎯⎯⎯⎯⎯→ O630 ⎯⎯⎯⎯⎯→ bR 568

(1)

Received: February 7, 2019 Revised: February 10, 2019 Published: February 11, 2019 A

DOI: 10.1021/acs.jpcb.9b01224 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B

absorption spectroscopy, respectively, to cover the temporal domains from hundreds of femtoseconds to milliseconds.

The generation of intermediate M, characterized at 410 nm absorption, is associated with the proton migration from the protonated Schiff base to Asp-85, while its decay correlates to the reprotonation of the deprotonated Schiff base with proton migration from Asp-96.22 The final intermediate O, which is characterized at 630 nm absorption,23 is correlated to the generation of the titled all-trans retinal.20 The photoisomerization kinetics of the all-trans retinal in bR can be altered by the mutated residues at the retinal pocket,24,25 monomerized configuration,26 delipidation of PM, 27 and reconstituted forms in various membrane mimicking environments.28 The cascading thermodynamic photocycle kinetics at a given temperature can be influenced by the environmental acidity,29 presence of surfactants,30−32 types of lipids in liposome to solubilize bR,33 and monomeric/ trimeric configuration.34 bR can be solubilized in surfactants26 and liposomes,33 as well as incorporated in lipid nanodiscs. Among the various methods to prepare different bR oligomers embedded nanodiscs,35−39 direct extraction from PM allows the preservation of the trimeric bR configuration.37 Previously, we reported that the composition of the lipids in nanodiscs influenced the photocycle kinetics38 and proton pump activity.39 The content of the natively charged lipids mostly ascertained the photocycle characteristics.38 Although the NMR spectral quality benefits from the usage of small-sized nanodiscs,4 the influences of nanodisc sizes in the dynamic biological function of membrane proteins have not been thoroughly discussed. In this work, monomeric bR (mbR), solubilized in surfactant Triton X-100 after removal of native lipids in solution, was incorporated into covalently circularized nanodiscs (cND)10 (Figure 1a) using two different circularized membrane scaffold

2. MATERIALS AND METHODS 2.1. Sample Preparations. Preparation of Monomeric bR in the Micelle of Triton X-100 (mbR_TX100). The PM from Halobacterium salinarum S9 was prepared and purified according to the established procedure.40 Monomeric bR solubilized in the micelle of Triton X-100 was prepared at a ratio of 1:10 (w/w). The solubilization process was performed in a dark environment for 72 h at 25 °C. The solubilized monomeric bR in the supernatants was collected after centrifugation at 21130g for 30 min and stored at 4 °C for further use. Preparation of Monomeric bR in the Micelle of CHAPSO. Monomeric bR solubilized in the micelle of 3-[(3cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) was prepared and purified according to a previous report.41 The solubilized bR in the micelle of Triton X-100 was applied to a size-exclusion column (Enrich 70 Analytical grade, GE Healthcare) to eliminate the remaining native lipid from the PM and replace the Triton X-100 with CHAPSO. The elution buffer consisted of 16.4 mM CHAPSO, 100 mM NaCl, 0.4 mM NaN3, and 20 mM CH3COONa at pH 5.5. The sample was eluted from the column upon detection of the absorption peaks at 580 and 280 nm. Then the sample was collected and concentrated to an appropriate volume in a concentration of bR of about 100 μM for further assembly in lipid nanodiscs. Preparation of Monomeric bR in lipid Nanodiscs. Circularized MSPs of different lengths, cΔH5 and cE3D1, were used to assemble the lipid nanodiscs. cΔH5 and cE3D1 were prepared and purified according to the established literature.10 MSP1D1ΔH5_srt and MSP1E3D1_srt, which contained an additional sortase A recognition motif and polyhistidine tag for the circularization reaction, were the homologues of linear MSPs of cΔH5 and cE3D1. The plasmids used to express MSP1D1ΔH5_srt and MSP1E3D1_srt were Escherichia coli BL21 (DE3) and Rosetta 2 (DE3), which were grown in a Winpact Bench-Top Fermenter (Major Science, New Taipei City) at 37 °C. The protein expression was induced with 1 mM isopropyl β-D-1thiogalactopyranoside (IPTG) when OD600 reached 1.5, and the protein was harvested after 5 h of protein expression. The cells were collected by centrifugation and purified as described previously.10 The polyhistidine tag at the N-terminal of MSP1D1ΔH5_srt and MSP1E3D1_srt was cleaved by Tobacco etch virus (TEV). The cleaved linear MSPs were then circularized by sortase A, forming the circularized MSPs, cΔH5 and cE3D1. The purification of cΔH5 and cE3D1 was carried out according to the literature using affinity chromatography,10 after which the cMSPs were concentrated to about 200 μM using protein concentrator spin columns (GE Healthcare, Vivaspin Turbo 15, 10000 MWCO PES) for further assembly of lipid nanodiscs. Dimyristoylphosphatidylglycerol (DMPG) was purchased from Avanti Polar Lipids (Alabaster, AL). DMPG lipids, monomeric bR solubilized in triton X-100 (mbR_TX100) and the two circular MSPs, cΔH5 and cE3D1, were used to prepare bR embedded nanodiscs according to the previous literature.10 The environment of monomeric bR in nanodisc samples was switched to phosphate buffer solution at pH 5.8, which consisted of 2 mM KH2PO4 and 10 mM Na2HPO4 in the

Figure 1. Schemes of (a) the assembly of bR and DMPG in circularized nanodiscs (cND) and (b) the constitutions of two cMSPs, cΔH5 and cE3D1.

proteins (cMSP) (Figure 1b), cΔH5 and cE3D1,10 to prepare monomeric bR in nanodiscs of different sizes. The negatively charged lipid, 1,2-dimyristoyl-sn-glycero-3-phosphorylglycerol (DMPG), was chosen to construct the lipid bilayer because the negatively charge lipids assisted in preserving the photocycle characteristics.38 The kinetics of retinal photoisomerization and thermodynamic photocycle were characterized at pH of 5.8 using the femtosecond and nanosecond transient B


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pellet, and 2 mL of 20 mM CHAPS containing 5 mM sodium acetate buffer (pH 5.5) was added to dissolve the pellet. The solution was left to equilibrate overnight. Then, it was spun down at 21,130 × g for 30 min (Centrifuge 5424 R, rotor model FA-45-24-11, Eppendorf) and the supernatant was discarded. The aforementioned process was repeated three times. Finally, the dPM was dissolved in solution of 12 mM CHAPS containing phosphate buffer (pH 5.8) and characterized with the absorption maximum at 560 nm.34 2.2. Femtosecond Transient Absorption Spectroscopy. The femtosecond transient absorption spectra were measured using a femtosecond transient absorption spectrometer (ExciPro, CDP), shown in Figure S2. A Ti:sapphire-based amplifier system (Hurricane, Spectra Physics) provided 800 nm laser pulses of a full-width at half-maximum (fwhm) of 300 fs at a repetition rate of 1 kHz. About 70% of the power of the amplified laser beam was introduced into the optical parametric amplifier (TOPAS, Light Conversion) to generate the signal and idler beams. The excitation wavelengths at 550 and 575 nm were generated by the sum frequency generation of the fundamental beam with the idler wavelengths at 1760 and 2044 nm, respectively. Laser pulses provided by Hurricane will be slightly broadened after passing through TOPAS and other optics. The cross correlation of the instrumental response function in this system has a full-width at halfmaximum (fwhm) of ∼400 fs, which was used to deconvolute the temporal profiles for determining the time constant of the decay of intermediate I. The laser power was attenuated to 60 nJ pulse−1 to avoid sample photobleaching. The residual 800 nm beam was focused onto a flowing water cell of 2 mm optical length to generate continuous white light in a wavelength range of 440−670 nm, which passed through a wedge reflector and was split into two beams for reference and probe. The pump beam traversed the delay line and then was focused and overlapped with the probe beam at the sample cell. The pump beam was further decoupled at a repetition rate of 500 Hz with an external chopper. The sample cell (silica windows, optical length of 1 mm) was mounted on a motor to prevent thermal damage and overshooting of the samples. The probe and reference beams propagated through the focusing lenses and were collected with two optical fibers. A monochromator was used to disperse the wavelengths, which were recorded with a photodiode array detector. The intensity of the probe beam was divided by the intensity of the reference beam to obtain the difference spectrum. The cross correlation of the instrumental response function in this system has a fullwidth at half-maximum (fwhm) of ∼400 fs, which was used to deconvolute the temporal profiles for determining the time constant of the decay of intermediate I. The concentrations of the PM, dPM, and mbR_TX100 were ca. 30 μM, and those of the nanodisc samples were ca. 10 μM. All measurements were performed at 22 °C. 2.3. Nanosecond Transient Absorption Spectroscopy. The nanosecond transient absorption spectroscopy followed the previous experimental arrangement with slight modifications,38,39 as shown in Figure S3. A frequency-doubled Nd:YAG laser (LS-2134UTF, LOTIS TII) provided 532 nm photons, and its repetition rate was adjusted to avoid overshooting. The laser flux was controlled at 0.5 mJ cm−2. A deuterium/tungsten halogen lamp (DH-2000, Ocean Optics) served as the stationary probing light and was introduced via an optical fiber (QP400−025-SR, Ocean Optics) to a cell compartment with a thermostat (qpod,

presence of 27 mM KCl using protein concentrator spin columns (GE Healthcare, Vivaspin Turbo 15, 10000 MWCO PES). Then the bR embedded nanodisc samples were applied to a Resource Q anion exchange column (GE Healthcare, Buckinghamshire, U.K.) using the elution buffer, which consisted of linearly increased concentrations of NaCl from 50 mM to 1 M, 25 mM Tris-HCl and 0.5 mM EDTA at pH 5.8. The aforementioned samples were eluted from the column upon detection of the absorption peaks at 560 and 280 nm. The resultant chromatogram is shown in Figure S1a. Then the three fractions containing mbR_cE3D1_ND and mbR_cΔH5_ND samples were collected and concentrated to about 500 μL using protein concentrator spin columns (GE Healthcare, Vivaspin Turbo 15, 10000 MWCO PES). Sequentially, these samples were applied to a size-exclusion column (Superdex 200 Prep grade, GE Healthcare) to separate unwanted aggregates. The elution buffer consisted of 25 mM Tris-HCl, 0.5 mM EDTA and 100 mM NaCl at pH 7.5. These bR embedded nanodisc samples were eluted from the column upon monitoring the absorption peaks at 580 and 280 nm. The three fractions of SEC, centered at the 560 nm absorption peak, were collected for each sample. The resultant chromatogram is shown in Figure S1b. The molecular weights of mbR_cΔH5_ND and mbR_cE3D1_ND were estimated with size exclusion chromatography, as shown in Figure S1b, to be 164 kDa and 195 kDa, respectively. Similarly, the molecular weight of mbR_TX100 was estimated to be 129 kDa. Since the molecular masses of cΔH5, cE3D1, and bR are reported to be 19.826 kDa, 30.314 kDa and 26.5 kDa,10,38 the masses of DMPG lipids in mbR_cΔH5_ND and mbR_cE3D1_ND were estimated to be around 98 kDa and 108 kDa, respectively. Accordingly, the numbers of DMPG lipids in mbR_cΔH5_ND and mbR_cE3D1_ND were calculated to be 142 and 156, respectively. Then these two nanodisc samples were collected and the environment of these samples was switched to phosphate buffer solution at pH 5.8, which consisted of 2 mM KH2PO4 and 10 mM Na2HPO4 in the presence of 27 mM KCl using protein concentrator spin columns (GE Healthcare, Vivaspin Turbo 15, 10000 MWCO PES). After the exchange of buffer solution, these nanodisc samples were concentrated to about 10 μM for further spectral and time-resolved experiments. The absorption maxima and the corresponding retinal configurations are listed in Table 1. Preparation of Delipiated PM (dPM). Delipidated PM was prepared as previously described42 using 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) treatment. Approximately 10 mg PM was centrifuged to a Table 1. Retinal Configuration and Absorption Maximum in Light-Adapted States of PM, dPM, mbR_TX100, mbR_cE3D1_CD, and mbR_cΔH5_CD at pH 5.8 all-trans, 15-anti/13-cis, 15-syn PM dPM mbR_TX100 mbR_cE3D1_ND mbR_cΔH5_ND

95/5−98.5/1.526,52 62/3844 84/16−50/50a,56

absorption maximum (nm) 56826 560b,42 55326 557 558

a This value depends on the concentration of Triton X-100. bThis work.

C


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The Journal of Physical Chemistry B Quantum Northwest) at 25 °C. Then the probe beam was further dispersed by a monochromator (Model 218, McPherson) to define the detection of the recovery of parent depletion at 568 nm (for PM and dPM) /550 nm (for other monomeric bR samples), intermediates M at 410 nm and O at 650 nm, respectively. The excitation laser beam propagated perpendicularly to the probe beam and overlapped in the center of the cell compartment. A notch filter (532/355 nm notch filter, CVI Laser Optics) was mounted in front of the photomultiplier (R928, Hamamatsu) to avoid scattering of the 532 nm excitation laser. The voltage signal was recorded with a 200 MHz digital oscilloscope (24MXs-B, LeCroy). The repetition rates and numbers of laser shots to increase the signal-to-noise ratios are listed in Table S1 for different samples. The evolution of the absorbance difference, ΔAt, was derived as follows, ΔA t = −log(St /S0)

where St and S0 denoted the dc-coupled voltages in the presence and absence of the excitation laser, respectively.

3. RESULTS AND DISCUSSION 3.1. Steady-State Spectroscopic Characterization. The bR-embedded nanodisc samples were purified using anion exchange chromatography followed by size exclusion chromatography. Anion exchange chromatogram of mbR_cE3D1_ND and mbR_cΔH5_ND, denoting the monomeric bR embedded in nanodiscs composed of cE3D1 and cΔH5, respectively, at pH 5.8 using a linearly increasing salt gradient manifested a single dominant component for both samples (Figure S1a). Chromatograms monitored at two different wavelengths, 560 and 280 nm, were attributed to the retinal of bR, and the aromatic residues of bR and cMSP, respectively. The coincidence of these two peaks suggested the successful assembly of bR embedded nanodiscs using cMSP. Further purification using size exclusion chromatography (Figure S1b) was performed to differentiate the samples on the basis of their corresponding sizes. The steady-state absorption contour of the protonated retinal Schiff base after the light adaptation was influenced by the chemical environments in the vicinity of the retinal chromophore43 and the population of the all-trans, 15-anti and 13-cis, 15-syn retinals.44 Circular dichroism spectroscopy (CD) in the visible region was employed to differentiate the monomeric and trimeric bR in the samples in terms of the biphasic ellipticity.45 The steady-state absorption and CD spectra of the samples at pH 5.8 after 1 h of light adaptation are shown in parts a and b of Figure 2, respectively. The monophasic lob of the CD spectra of mbR_cΔH5_ND and mbR_cE3D1_ND indicated that bR embedded nanodiscs were indeed the monomeric form. Since the CD signal from the trimer, with the biphasic feature, is much stronger than monomeric bR, the CD spectra of mbR in Figure 2b would mainly be attributed to the monomeric bR. The light-adapted protonated retinal Schiff base of PM, dPM, and mbR peaked at 568, 560, and 553 nm, respectively, consistently with previous reports.26,34,46 In contrast, the light-adapted mbR_cE3D1_ND and mbR_cΔH5_ND both peaked at 557 ± 1 nm. The retinal configurations and absorption maxima for different bR oligomers after light adaptation are different, as summarized in Table 1. The spectral shift could probably be attributed to the combinational effects of the alteration in the interaction of the amino acids and the retinal Schiff base at the binding

Figure 2. (a) Normalized UV−vis absorption and (b) CD spectra of the light-adapted PM (black), dPM (cyan), mbR_TX100 (blue), mbR_cΔH5_ND (green), and mbR_cE3D1_ND (red) at pH 5.8. The buffer contained 2 mM KH2PO4, 10 mM Na2HPO4, and 27 mM KCl.

pocket,47 loose helical packing in monomerized bR,48 and different populations of the all-trans, 15-anti and 13-cis, 15-syn retinals in the light adapted form.44 Thus, we were not able to exclusively ascribe the spectral shift to the aforementioned reasons at the present stage. The decay kinetics of intermediate I is influenced by the interaction of the retinal and its adjacent amino acids;16 hence, it is tightly associated with the potential structural change of monomeric bR in nanodiscs. Moreover, the decay kinetics of intermediate I could be altered upon changing the excitation wavelengths, which are correlated with different absorption wavelength maxima of bR embedded nanodiscs with different retinal isomer populations in the light adapted state. 3.2. Kinetics of Intermediate I at 460 nm. The intermediate I upon photoexcitation of the all-trans, 15-anti retinal of light-adapted PM was characterized at 460 nm absorption, and the corresponding decay kinetics was mainly characterized with a single time constant of ca. 500 fs,25,49,50 whereas the 13-cis, 15-syn retinal had an accelerated decay kinetics.51 Logunov et al. observed 60%−100% slower lifetimes of the intermediate I in various bR mutants, such as W182F and V94A, and ascribed the change in the retinal environment to the altered kinetics.25 Wang et al. observed a ca. 37% slower decay of intermediate I at 490 nm in mbR_TX100, as compared to trimeric bR in PM, upon 555 nm excitation.26 Cembran et al. demonstrated that an energy barrier along the reaction coordinate of the decay of intermediate I will increase as the charged residue approaches either the end of the retinal or the protonated Schiff base.16 The excitation energy was confirmed to avoid the photobleaching and multiphoton excitation of bR in PM in the femtosecond transient absorption difference experiments in the D


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The Journal of Physical Chemistry B energy range of 60−200 nJ pulse−1 (cross section = 0.22 mm2). The corresponding normalized temporal profiles of intermediate I integrated at 450−470 nm upon 575 nm excitation are shown in Figure 3, and the normalized steady-state

Figure 3. Normalized traces of the integrated absorbance difference at 450−470 nm of PM at pH 5.8 upon 575 nm femtosecond excitation with different pulse energy (60−200 nJ pulse−1). The buffer contained 2 mM KH2PO4, 10 mM Na2HPO4, and 27 mM KCl. The open circles and solid lines denote the observed and fitted data, respectively. Steady-state absorption spectra of the samples before and after the laser exposure are shown in Figure S4a.

Figure 4. Normalized temporal profiles of the integrated absorbance difference at 450−470 nm of PM, dPM, mbR_TX100, mbR_cE3D1_ND, and mbR_cΔH5_ND (from Figure S5) at pH 5.8 upon excitation at (a) 575 and (b) 550 nm. The laser energy was controlled as ca. 60 nJ pulse−1. The open circles and solid lines denote the observed and fitted data, respectively. Steady-state absorption spectra of the samples before and after the laser exposure are shown in Figure S4, parts b and c.

absorption spectra before and after laser excitation are shown in Figure S4a. At 60 nJ pulse−1, the decay of intermediate I was fitted as 0.66 ps using a single exponential component, which is consistent with previous reports,25,49,50 and the steady-state absorption contours remained unchanged before and after the laser irradiation, with slight aggregation that caused a minute drift toward the short wavelengths (Figure S4a). However, the higher excitation energy caused the decelerated decay of intermediate I (Figure 3) and the change in the absorption contours (Figure S4a). As a result, pulse energy of 60 nJ pulse−1 was employed for the retinal photoisomerization experiments to avoid the photobleaching and unexpected photochemical pathways. The femtosecond pump−probe difference spectra of PM, dPM, mbR_TX100, mbR_cE3D1_ND, and mbR_cΔH5_ND at pH 5.8 upon excitation at 575 nm are shown in Figure S5a. The normalized temporal profiles of the integrated absorbance difference at 450−470 nm of the aforementioned spectra were fitted with a single exponential component upon deconvoluting the temporal profiles with the instrumental response function, which has a full-width at half-maximum (fwhm) of ∼400 fs, as shown in Figure 4a, and the results are summarized in Table 2. We found that the decay kinetics of intermediate I in PM and dPM were similar and that in mbR_TX100, mbR_cE3D1_ND and mbR_cΔH5_ND were retarded with respect to PM and dPM. The mbR_TX100 possesses a prolonged decay lifetime, ca. 50% slower than that of PM, close to the previous report (37%) by Wang et al.26 The decays of intermediate I of mbR_cE3D1_ND and mbR_cΔH5_ND were similar and were much slower at 460 nm than those of PM, dPM, and mbR_TX100. The mbR_TX10026 and recombinant bR in a DMPC−CHAPS micelle system28 show increased lifetimes of intermediate I of ca. 37% and 51% with respect to PM, respectively, suggesting the diverse retinal photoisomerization kinetics in different solubilized environments of the monomeric bR. Moreover, compared to PM and dPM which are composed of trimeric bR, these monomeric bR configurations manifested the prolonged lifetimes of intermediate I, indicating

Table 2. Observed Lifetime τ (ps) of Intermediate I in PM, dPM, mbR_TX100, mbR_cE3D1_ND, and mbR_cΔH5_ND at pH 5.8 upon 550 and 575 nm λEx Excitation PM dPM mbR_TX100 mbR_cE3D1_ND mbR_cΔH5_ND

τ550 nm

τ575 nm

τ550 nm/τ575 nm (%)

0.56 0.69 0.84 1.10 1.00

0.66 0.64 0.99 1.23 1.19

85 108 85 89 84

that the inter-bR interactions in trimeric configuration in PM probably ensured a specific protein structure and led to more efficient photoisomerization kinetics than the monomerized forms. In comparison, the steady-state absorption spectra (Figure 2a) of mbR_cE3D1_ND and mbR_cΔH5_ND were different from that of mbR_TX100. If the spectral shift is attributed to great changes in the populations of the all-trans, 15-anti and 13-cis, 15-syn retinals, the decay kinetics of intermediate I upon excitation at different wavelengths for exciting all-trans, 15-anti and 13-cis, 15-syn retinals, respectively, should be characterized with two kinetic components, since the lifetime of intermediate I upon excitation of 13-cis,15-syn retinal was three times faster than that of all-trans,15-anti.51 The time-resolved difference spectra of PM, dPM, mbR_TX100, mbR_cE3D1_ND, and mbR_cΔH5_ND at pH 5.8 upon excitation at 550 nm are shown in Figure S5b. The normalized temporal profiles of the integrated absorbance difference at 450−470 nm were also fitted with a single exponential component in Figure 4b, and the results are summarized in Table 2. Because the population of the all-trans retinal in PM is higher than 90%,26,52 the larger decay lifetime of intermediate I in PM upon excitation at 575 nm (0.66 ps) than at 550 nm (0.56 ps) can be ascribed to a wavelength-dependent photoisomerization quantum yield of the all-trans protonated retinal Schiff base. This observation is E


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The Journal of Physical Chemistry B consistent with the reported wavelength-dependent yield of the intermediate K, the cascading product of intermediate I, which decreased from 45.3 ± 0.8% to 42.1 ± 1.0% as the excitation wavelengths elongated from 562 to 580 nm.53 If the populations of all-trans, 15-anti and 13-cis, 15-syn in the light-adapted mbR_TX100 and monomeric bR in nanodiscs were greatly changed with respect to PM, the lifetime of intermediate I upon excitation at shorter wavelengths will be shortened, and the τ550 nm/τ575 nm, denoting the ratio of the lifetimes of intermediate I upon excitation at 550 and 575 nm, will decrease noticeably. Comparing the τ550 nm/τ575 nm in Table 2, the values of all monomeric bR samples were about 85% and did not show significant differences. This result probably indicates that populations of all-trans, 15-anti and 13cis, 15-syn retinals were not greatly different among these samples. Concurrently, the minute shifts of the absorption maxima of the retinal Schiff base and the kinetics of intermediate I of mbR_cE3D1_ND and mbR_cΔH5_ND with respect to mbR_TX100 indicated the slight change in the potential energy surfaces of the ground state and electronically excited state of the retinal due to the slight helical alteration of monomeric bR in nanodiscs. The decay of intermediate I of dPM is 0.69−0.64 fs that is similar to PM but different from monomeric bR in different solubilized conditions. The minute structural change of the constituent bRs might result in the slight hypsochromic shift of the light-adapted dPM, probably referring to the slight lift-up of the excited state potential energy curve and the concomitant reduction in the energy different between the well and the energy barrier along the isomerization coordinate. Accordingly, the wavelength-dependent kinetics is slightly weakened and the ratio of τ550 nm/τ575 nm is close to one within the uncertainty of 50 fs. It is noteworthy that the decay of intermediate I of dPM is still distinguishable from monomeric bR, further suggesting that the oligomeric status of bR is the dominant factor in the retinal isomerization in a short-ranged interaction domain. 3.3. Kinetics of Intermediates M at 410 nm and O at 650 nm. In addition to the retinal pocket, which denotes a localized regime of bR, the rise of intermediate M is associated with the deprotonation of the protonated Schiff base to Asp8522,54 and slight structural change at helix C.54 Tests of repetition rates of the nanosecond pulsed laser excitation by monitoring the recovery of the parent state were performed, as shown in Figure S6. The repetition rates at which the time required for each excitation can be shortened without overshooting are listed in Table S1. The recovery kinetics of parent state for different samples were shown in Figure 5a. The rise of intermediate M (Figure 5b) of monomeric bR within 100 μs was greatly accelerated with respect to the trimeric bR in PM and dPM, consistent with the reports by Wang et al.26 and Heyes and El-Sayed.34 Meanwhile, three monomeric bRs, mbR_TX100, mbR_cE3D1_ND, and mbR_cΔH5_ND, possessed the similar M intermediate generation kinetics, suggesting a specific protein structure in the monomeric state. Like the decay kinetics of intermediate I, the photocycle kinetics were categorized in terms of the monomeric and trimeric bR; that is, the structural differences of bR in different oligomeric configurations altered the potential energy surfaces of the ground and excited states of the retinal and the interaction of the protonated Schiff base with its adjacent residues, leading to the difference in the steady-state absorption, photoisomerization kinetics of the retinal, and deprotonation of the protonated Schiff base. Moreover, the

Figure 5. Normalized temporal profiles of (a) recovery of parent state at 550 nm, (b) rise of M intermediate at 410 nm, (c) decay of intermediate M at 410 nm, and (d) the evolution of intermediate O at 650 nm of PM (black), dPM (cyan), mbR_TX100 (blue), mbR_cΔH5_ND (green), and mbR_cE3D1_ND (red) at pH 5.8 in buffer of 2 mM KH2PO4, 10 mM Na2HPO4, and 27 mM KCl upon excitation at 532 nm. The laser energy was controlled as ca. 0.5 mJ cm−2. Steady-state absorption spectra of the samples before and after the laser exposure are shown in Figure S7.

size of the nanodiscs did not play a role in the aforementioned processes. The decay kinetics of the intermediate M is associated with the reprotonation of the deprotonated Schiff base from Asp9622 and the movement of helix F.55 The intermediate M decay in monomeric bR in nanodiscs is retarded with respect to that in mbR_TX100, as shown in Figure 5c. Moreover, the M intermediate decay of dPM is greatly decelerated, consistent with the results by Heyes and El-Sayed.34 It probably referred to the different couplings between the structures of monomeric bR and the chemical environment in different immobilized conditions. Moreover, the decay of intermediate M in mbR_cΔH5_ND is slightly faster than that in mbR_cE3D1_ND, indicating a weak size effect of circularized nanodiscs. Since the charge density of mbR_cΔH5_ND is slightly higher than that of mbR_cE3D1_ND (Figure S1a), the larger polarized environment probably facilitated the longrange proton migration from Asp-96 to deprotonated Schiff base, i.e., a quick decay of intermediate M. The last photocycle intermediate O, which involved the retinal reisomerization from 13-cis to all-trans, was characterized at 650 nm (Figure 5d). Since the intermediate O was generated after successive processes, its kinetics does not purely reflect the dynamic process of the retinal reisomerization. Its transient population in two mbR in nanodiscs confirmed the completion of the photocycle.

4. CONCLUSION To conclude our work, the monomeric bR in different solubilization conditions and trimeric bR in PM and dPM have distinguishable retinal isomerization kinetics and proton migration from protonated Schiff base to Asp-85, attributed to F


Article


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the different oligomeric statuses of bR. These two events in association with retinal isomerization and the localized structural change at the vicinity of the retinal pocket do not deviate in the Triton X-100 solubilized condition and covalently circularized nanodiscs. However, the reprotonation of the Schiff base during the decay kinetics of intermediate M, which involved a wide-range of structural alteration, manifested a slight dependence on nanodisc size. These results might imply that nanodisc size could affect the dynamic functions of a given membrane protein involving the long-range processes.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.9b01224. Chromatograms of the samples (Figure S1), experimental setup of femtosecond pump−probe spectroscopy (Figure S2), experimental setup of nanosecond transient absorption (Figure S3), steady-state absorption spectra before and after femtosecond and laser exposure (Figure S4), femtosecond transient absorption contours (Figure S5), repetition rate test for nanosecond transient absorption (Figure S6 and Table S1), and steady-state absorption spectra before and after nanosecond laser exposure (Figure S7) (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*(I-C.C.) Telephone: +886-3-5715131 ext. 33339. E-mail: [email protected]. *(T.-Y.Y.) Telephone: +886-2-23668210. E-mail: [email protected]. Present Address: P.O. Box 23-166 Taipei, 10617, Taiwan. *(L.-K.C.) Telephone: +886-3-5715131 ext. 33396. E-mail: [email protected]. ORCID

I-Chia Chen: 0000-0002-5821-6416 Li-Kang Chu: 0000-0001-6080-9598 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the grants from the Ministry of Science and Technology of Taiwan (I-C.C., MOST 106-2119018; T.-Y.Y., MOST 105-2113-M-001-021-MY2; L.-K.C., MOST 105-2113-M-007-014 and MOST 106-2113-M-007007). T.-Y.Y. was also supported by an iMate grant (ASiMATE-107-31) from Academia Sinica.

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Photochemistry of Bacteriorhodopsin with Various Oligomeric Statuses

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