Letter pubs.acs.org/JPCL
Two-Photon-Induced Selective Decarboxylation of Aspartic Acids D85 and D212 in Bacteriorhodopsin Martin Imhof,† Daniel Rhinow,‡ Uwe Linne,† and Norbert Hampp*,† †
Philipps-University of Marburg, Department of Chemistry, Hans-Meerwein-Str., D-35032 Marburg, Germany Max-Planck-Institute of Biophysics, Department of Structural Biology, Max-von-Laue-Str. 3, D-60438 Frankfurt, Germany
‡
ABSTRACT: The interest in microbial opsins stems from their photophysical properties, which are superior to most organic dyes. Microbial rhodopsins like bacteriorhodopsin (BR) from Halobacterium salinarum have an astonishingly high cross-section for two-photonabsorption (TPA), which is of great interest for technological applications such as data storage. Irradiation of BR with intense laser pulses at 532 nm leads to formation of a bathochromic photoproduct, which is further converted to a photochemical species absorbing in the UV range. As demonstrated earlier, the photochemical conversions are induced by resonant TPA. However, the molecular basis of these conversions remained unresolved. In this work we use mass spectroscopy to demonstrate that TPA of BR leads to selective decarboxylation of two aspartic acids in the vicinity of the retinal chromphore. These photochemical conversions are the basis of permanent two-photon data storage in BR and are of critical importance for application of microbial opsins in optogenetics. SECTION: Biophysical Chemistry and Biomolecules
R
of BR contains N-retinyl-bacterioopsin formed upon photochemical reduction of the Schiff base.20,21 However, the nature of the bathochromic photoproduct remained unresolved. We used purple membranes (PMs) from Halobacterium salinarum, containing BR (Figure 1a) and lipids only, to analyze
hodopsins are biological photoreceptors abundant among all kingdoms of life. A common feature of all 7transmembrane helix rhodopsins is that they contain a retinal chromophore as photoreceptive moiety. In their native hosts, microbial rhodopsins are key proteins of photosynthesis and signal transduction1−11 and harvest light with unprecedented quantum efficiency and long-term stability of the chromphore. Owing to their interesting photophysical properties, microbial rhodopsins hold great promise for applications in nanobiotechnology ranging from optical data storage12,13 to optogenetics.14,15 Most applications refer to conditions where a single photon is absorbed by a rhodopsin molecule. However, astonishingly high two-photon cross sections have been reported for bacteriorhodopsin (BR),16 and two-photon excitation of channelrhodopsin-2 (ChR2) has only recently been recognized as a valuable tool for optogenetics.17,18 The interest in studying two-photon absorption (TPA) of microbial rhodopsins is two-fold. First, because of the quadratic dependence on the field strength, two-photon-induced processes can be triggered with extremely high spatial selectivity. Second, TPA often causes photochemical transformations not accessible to single-photon photochemistry. In particular, obtaining chemical information about molecular changes in BR upon TPA is important to understand the mechanism of two-photon data storage in BR.19−21 Twophoton excitation of BR570 (initial state) with intense laser pulses leads to the formation of a red-shifted photoproduct (F620),20 which is subsequently photobleached to yield a photoproduct with an absorption maximum around 360 nm (P360). Previously, it has been demonstrated that the B570→F620 conversion is caused by resonant two-photon absorption of BR.20 The final product of two-photon-induced photobleaching © XXXX American Chemical Society
Figure 1. Color changes in BR suspensions upon irradiation with intense 532 nm laser pulses. With increasing irradiation time, the purple PM suspension (BR570 state) turns blue (F620 state) before it becomes completely colorless (P360 state).
chemical changes in specific amino acids in BR upon TPA. PM suspensions were treated with intense nanosecond laser pulses (λ = 532 nm). Upon laser irradiation, the color of the suspension changes from purple to blue before it becomes colorless20,21 (Figure 1). Subsequently, we digested laser-irradiated BR using cyanogen bromide and analyzed the fragments by mass spectroscopy Received: August 29, 2012 Accepted: October 3, 2012
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Table 1. Calculated Masses of Peptides Obtained after Digestion with Cyanogen Bromide
a
fragment
calculated mass
amino acid number
helix
peptide sequence
1 2 3 4 5 6a 7 8 9 10a
2208.181 1250.714 2508.338 465.2344 819.4611 5397.845 2913.566 2081.099 5036.802 3843.029
1−20 21−32 33−56 57−60 61−68 69−118 119−145 146−163 164−209 210−248
A A B B turnC D, E E F G
EAQITGRPEWIWLALGTALM GLGTLYFLVKGM GVSDPDAKKFYAITTLVPAIAFTM YLSM LLGYGLTM VPFGGEQNPIYWARYADWLF TTPLLLLDLALLVDADEGTILALVGADGIM IGTGLVGALTKVYSYRFVWWAISTAAM LYILYVLFFGFTSKAESM RPEVASTFKVLRNVTVVLWS AYPVVWLIGSEGAGIVPLNIETLLFM VLDVSAKVGFGLILLRSRAI FGEAEAPEPSAGDGAAATS
Aspartic acids decarboxylated during TPA of BR are marked in bold.
Figure 2. Chemical changes in bacteriorhodopsin (BR) upon irradiation with intense laser pulses. (a) Model of BR (extracellular side up). Amino acids D85 and D212 in close proximity to the retinylidene moiety (orange) are shown. (b) Time-dependent mass spectra of BR fragments 10 and 6 obtained after digestion of laser-irradiated BR with cyanogen bromide. Two-photon-induced decarboxylation of fragments 10 (m/z = 3842.8 Da) and 6 (m/z = 5398.1 Da) is accompanied by gradual formation of two new species with lower mass (Δm = 44 Da). The cumulative laser energy is indicated. (c) Close-up view of D212 and D85 in the vicinity of retinal (RET). The nitrogen of the Schiff base is shown in blue. (d) Two-photon excitation of the retinal chromophore leads to selective decarboxylation of D85 and D212.
spectrum,22,23 two-photon-induced decarboxylation of D85, converting it to alanine, changes the purple color of BR to blue. MS gave no evidence of further photochemical modifications in BR. Only decarboxylation of amino acids D212 and D85 is observed. It is worth noting that single-photon excitation of BR with 532 nm causes fully reversible photochromic changes without any detectable chemical changes in the protein over many cycles,13 as distinguished from other antenna proteins like green fluorescent protein.24,25 Interestingly, MS gave evidence that TPA of BR-D85T, a blue mutant where D85 is replaced by threonine, leads to decarboxylation of D212, the only remaining aspartic acid in the vicinity of the retinal chromophore. This is further proof that in wildtype BR decarboxylation of D85 and D212 is decoupled. Furthermore, we did not observe photochemical modification of fragment 6 demonstrating that aspartic acids far away from the retinal chromophore are not decarboxylated upon two-photon-excitation.
(MS). Cyanogen bromide digestion of BR leads to the formation of 10 characteristic fragments, which are shown in Table 1. Figure 2b shows mass spectra of BR fragments 10 (m/ z = 3842.8 Da) and 6 (m/z = 5398.1 Da) containing D212 and D85, respectively, which reveal irradiation-dependent formation of new fragments with a mass difference of 44 Da to the native peptides. Both D85 and D212 are located in the vicinity of the retinal moiety (Figure 2a,c). Because the mass difference equals the molecular weight of CO2, we conclude that TPA of BR leads to selective decarboxylation of aspartic acids 85 and 212 in the vicinity of the retinal chromophore (Figure 2c,d). Decarboxylation of both D85 and D212 occurs to levels >50%, indicating that the antenna system of BR is preserved after decarboxylation of a first aspartic acid and thus still capable of promoting decarboxylation of the remaining second aspartic acid. We conclude that both decarboxylation events are decoupled. Because all BR mutants where D85 is replaced by a neutral amino acid show a bathochromic shifted UV−vis 2992
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Because two-photon-excitation is increasingly used in optogenetics,17,18 side reactions of BR and ChR2 upon TPA have to be considered. The question arises: What is the biological function of this intrinsic lightning arrester?
In addition, we compared photochemical modification of BR upon two-photon excitation with possible modifications caused by single-photon excitation upon irradiation with UV light. PM suspensions were irradiated with 266 nm UV light using the excitation light path of a fluorescence spectrometer. Subsequently, we analyzed the changes in the UV/vis spectra of BR and recorded mass spectra after digestion of UV-irradiated BR with cyanogen bromide. Figure 3 shows dose-dependent UV−
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EXPERIMENTAL METHODS HPLC-grade solvents were purchased from Sigma-Aldrich. PMs were supplied by Dieter Oesterhelt’s group at the Max-PlanckInstitute of Biochemistry (Martinsried, Germany) as a crude material and purified according to standard procedures.26 PM suspensions were irradiated with a frequency doubled Nd:YAG laser (Infinity 40−100, Coherent) operating at 532 nm (Pulse length 3 ns, repetition rate 20 Hz). PM samples were delipidated, and BR was digested with cyanogen bromide and analyzed by ESI-MS and MALDI-MS. For ESI-MS, fragments were separated by reverse phase HPLC with a Phenomenex C18 Kinetek chromatography column (3 mm × 150 mm) using an isopropanol/acetonitrile/aqueous solvent system and analyzed with an Orbitrap Velos Pro (Thermo Fisher Scientific). For MALDI-MS, BR-fragments were mixed with an equal amount of 2,5-dihydroxybenzoic acid as matrix substance and were analyzed by MALDI-TOF/TOF-MS (Ultraflex, Bruker).
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AUTHOR INFORMATION
Corresponding Author
Figure 3. Dose-dependent UV−vis spectra of PM suspensions treated with (a) UV light (single-photon excitation) and (b) intense laser pulses at 532 nm (two-photon excitation). Both samples were successively irradiated until the absorbance of the main peak at 570 nm decreased to ∼75% of the initial absorbance. (c,d) Close-ups of the UV−vis spectra revealing differences between the samples in the nearUV and UV range.
*E-mail: hampp@staff.uni-marburg.de. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Nina Schneider and Natalia Fritzler for technical assistance.
vis spectra of PM suspensions irradiated with UV light (Figure 3a,c) and 532 nm laser pulses (Figure 3b,d), respectively. Both spectra show the formation of a bathochromic state absorbing at 640 nm and photochemical species absorbing around 360 nm. This indicates that irradiation with UV light leads to decarboxylation of D85 and D212 as well as photochemical reduction of the Schiff base as observed upon two-photonexcitation with 532 nm laser light. However, the close-up view (Figure 3c) reveals differences in the fine structure of the photochemical species formed upon SPA with UV light compared with the species formed upon two-photon excitation with 532 nm laser light (Figure 3d). With increasing irradiation time the UV−vis spectra of UV-irradiated PM suspensions reveal the formation of photochemical species absorbing at wavelengths lower than 340 nm as well as consumption of a species absorbing at 400 nm, not observed upon TPA of BR. Mass spectra of UV-irradiated PM suspensions, recorded after digestion with cyanogen bromide, revealed the formation of numerous photoproducts, which precluded a unique assignment of the spectra. Obviously, single-photon excitation with UV light causes many unspecific molecular changes in the protein backbone, whereas decarboxylation of D85 and D212 are minor side reactions only. In conclusion, we have shown that two-photon excitation of BR with 532 nm laser light causes selective decarboxylation of two aspartic acids. Two-photon-induced decarboxylation is a kind of post-translational modification (D85 → A85, D212 → A212) likely to occur in all rhodopsins containing aspartic acids or glutamic acids in close proximity to the chromophore.
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