Letter pubs.acs.org/JPCL
Cite This: J. Phys. Chem. Lett. 2018, 9, 672−676
Accessibility of Protein-Bound Chlorophylls Probed by Dynamic Electron Polarization Alessandro Agostini,†,‡ Daniel M. Palm,‡ Harald Paulsen,‡ and Donatella Carbonera*,† †
Department of Chemical Sciences, University of Padova, Via Marzolo 1, 35131 Padova, Italy Department of Molecular Physiology, Johannes-Gutenberg University Mainz, Johannes-von-Müller-Weg 6, 55128 Mainz, Germany
‡
ABSTRACT: The possibility to probe the accessibility of sites of proteins represents an important point to explore their interactions with specific substrates in solution. The dynamic electron polarization of nitroxide radicals induced by excited triplet states of organic molecules is a phenomenon that is known to occur in aqueous solutions. The interaction within the radical−triplet pair causes a net emissive dynamic electron polarization of the nitroxide radical, that can be detected by means of time-resolved electron paramagnetic resonance (TR-EPR) spectroscopy. We have exploited this effect to prove the accessibility of chlorophylls bound to a protein, namely, the water-soluble chlorophyll protein WSCP. The results have important implications for topological studies in macromolecules.
A
in virtue of its high ISC,13 intense polarization,14 and biological relevance.15,16 The investigated system is the water-soluble chlorophyllbinding protein (WSCP), a tetrameric complex17 characterized by the binding of four Chls and the lack of bound carotenoids that, in photosynthetic proteins, quench the Chl triplet (3Chl) avoiding singlet oxygen (1O2) formation.18−20 Despite the lack of carotenoids, WSCP displays an elevated resistance to photobleaching.21−23 The Chls are bound in a cavity in the interior of the complex, and in the crystallographic structure only a small portion of the pigment surface (less than 1%)17 is accessible to the solvent. Nevertheless, O2 has been shown to efficiently interact with the pigments in their triplet state,22 showing that the 3Chl is accessible and able to photosensitize 1 O2. The Chls are organized inside the complex in a “dimer of dimers”, with a Chl−Chl orientation leading to an unusual24 distribution of the oscillator strength, with the main excitonic band at higher energies.25 This distribution of the oscillator strength is the source of the observed lengthening of the lifetime of the excited singlet state,26 which causes an increase of the ISC probability.27 Actually, 3Chl in WSCP is formed with high yield;22 however, the photobleaching of the Chls is efficiently prevented by a steric shielding by the phytyl chains of the most reactive portions of the tetrapyrrole macrocycle.22 In this work we make use of TR-EPR to investigate the accessibility of the Chls bound in the WSCP complex through the observation of DEP arising from the quenching of 3Chl by nitroxide radicals free in solution.
spin state out of its thermal equilibrium is characterized by an electron paramagnetic resonance (EPR) spectrum in emission (Em) and/or enhanced absorption (Abs). A nonthermal distribution of population between the spin sublevels in the case of radicals can be induced by photochemical or photophysical processes and is commonly known as chemically induced dynamic electron polarization (CIDEP). The CIDEP signal of radicals is generated through the triplet mechanism,1 the radical pair mechanism (RPM),2 or the radical-triplet pair mechanism (RTPM).3 Among these CIDEP mechanisms, RTPM stands out in that it is able to polarize a stable radical and does not include any chemical reaction. In the following, we will refer to the polarization resulting from this process as dynamic electron polarization (DEP). Due to the selectivity of its formation and its applicability to water solutions at room temperature, DEP promises to be a powerful method to study the accessibility of excited triplet states in protein environments. EPR has assumed a prominent role in studies of the accessibility in macromolecules, thanks to its selectivity that allows assessment of the effect of various paramagnetic probes, like nitroxides introduced by site directed spin labeling,4−6 photoinduced radicals,7 flavin,8 and metal cofactors.9−11 Surprisingly, only recently Kawai et al. investigated the possibility to employ DEP to study the accessibility of photoexcited triplets in proteins, finding a good agreement between this new approach and previous results obtained by fluorescence spectroscopy when studying the accessibility of tryptophan residues in folded and unfolded α-lactoalbumin.12 Here we explore the possibility to expand this methodology to proteins binding cofactors, which can be exited in their triplet state under photoexcitation, selecting chlorophyll (Chl) © XXXX American Chemical Society
Received: December 28, 2017 Accepted: January 23, 2018 Published: January 23, 2018 672
DOI: 10.1021/acs.jpclett.7b03428 J. Phys. Chem. Lett. 2018, 9, 672−676
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The Journal of Physical Chemistry Letters
Figure 1. Molecular structure of 4-oxo-TEMPO (A). TR-EPR spectrum taken after 1.7 μs from the laser photoexcitation of 25 μM WSCP/1 mM 4oxo-TEMPO mixture in presence (black) and absence (red) of oxygen (B). Transient EPR traces measured at 3328 G in the presence (black) and absence (red) of oxygen (C). T = 298 K.
Figure 2. Molecular structure of 4-amino-TEMPO (A) and 3-carboxy-proxyl (D), with the protonation state expected at pH 7.8. TR-EPR spectra taken after 1.7 μs from the laser photoexcitation of 1 mM 4-amino-TEMPO (B) or 1 mM 3-carboxy-proxyl (E) in the presence of 25 μM WSCP. TR-EPR spectrum of 1 mM 4-oxo-TEMPO/25 μM WSCP (Figure1B) is reported for comparison (gray). Transient EPR traces measured at 3328 G of 1 mM 4-amino-TEMPO (C) or 1 mM 3-carboxy-proxyl (F) in the presence of 25 μM WSCP. T = 298 K. The same vertical scale as in Figure1 is adopted for better comparison.
Illumination of Lepidium virginicum WSCP, reconstituted with four Chl a molecules per tetramer, leads to the formation of a 3Chl a population that can be detected by TR-EPR at low temperature, as previously reported.22 The spectrum, taken at short times after excitation, is polarized due to the anisotropy of the ISC population mechanism.28 The values of the ZFS parameters |D| and |E| (|D| = 30.7 mT and |E| = 3.8 mT) and the triplet sublevel populations (0.12:0.36:0.52),22 are typical for monomeric 3Chl a.29−31 The TR-EPR spectrum of a WSCP (25 μM)/4-oxo-TEMPO (1 mM) mixture obtained immediately after a 532 nm pulse laser excitation is reported in Figure 1. Since 3Chl is not detectable at room temperature, due to fast spin relaxation and orientation averaging in solution, only a TR-EPR spectrum characterized by the typical three-line splitting of nitroxides was observed, which can be assigned to the 4-oxo-TEMPO. The three lines were all emissive, which can only be explained on the basis of a DEP mechanism created by the interaction with 3 Chl. The negative sign of the signal is due to RTPM,3,32 since polarization transfer through dipolar mechanisms is expected to lead to signals opposite in sign.33 The observed spectral effect shows that the 4-oxo-TEMPO effectively reaches the chlorin ring (in the order of the van der Waals radii) and mix its
electronic spin with the triplet state, proving a remarkable accessibility of the Chls inside the complex. Since in the presence of oxygen a lifetime of less than 10 μs has been determined for the 3Chl in WSCP,22 the polarization transfer is expected to be in the microsecond time scale, as also pointed out from the analysis of the transient EPR traces of the polarized nitroxides, for which the maximum of the signal is achieved 1.7 μs after the laser pulse. When oxygen was removed from the solution, no increase of the nitroxide signal intensity was observed, pointing toward a negligible competition of the oxygen in quenching the 3Chl, in the experimental conditions adopted. Moreover, the absence of line broadening in the oxygen containing samples indicates that the line width of the EPR lines is determined mainly by relaxation processes due to spin− spin interactions among nitroxides, which are present in solution at higher concentration compared to oxygen. In order to extract more information regarding the accessibility of the bound Chls, water-soluble spin probes characterized by similar molecular size but different charge, have been tested (Figure 2). In the case of a positively charged spin probe, as 4amino-TEMPO, an emissive signal, similar to the one observed in the case of 4-oxo-TEMPO, has been observed. The lower 673
DOI: 10.1021/acs.jpclett.7b03428 J. Phys. Chem. Lett. 2018, 9, 672−676
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The Journal of Physical Chemistry Letters
Figure 3. Distribution of the electrostatic potential over the L. virginicum WSCP17 surface (red: negative, blue: positive). The electrostatic calculations was performed with the Adaptive Poisson−Boltzmann Solver (APBS)37 tool in PyMOL, which employs a nonlinear Poisson− Boltzmann solver and the AMBER atomic charges and radii (with side chains protonation defined for pH 7.8). The inset is a close-up view of the pore facing Chl-1.
carotenoids, and flavins, but also to any other protein, in virtue of well established protein labeling techniques. Indeed, labels consisting of chromophores with high triplet yield, such as eosin or erythrosine, can in principle be introduced to any position of interest to be checked in terms of accessibility. This opens the way to an interesting new approach to topological characterization, surface studies, and conformational changes of macromolecules.
intensity of the signal, obtained with the same protein concentration and excitation power as reported above, can be attributed to the width difference of the EPR spectra of the two spin probes,34 as the integral of the two spectra are similar. Therefore, the efficiency of the polarization transfer appears to be comparable for the two spin probes. In marked contrast, no DEP has been observed with the negatively charged nitroxide 3-carboxy-proxyl (Figure 2), showing that it is unable to access the bound Chls of WSCP. With the aim of rationalizing these findings, the electrostatic potential of the surface of WSCP has been analyzed (Figure 3). The potential is predominantly negative at pH 7.8, congruently with the low theoretical isoelectric point (pI = 4.5) (uniprot: O04797).35 Remarkably, the electrostatic potential is negative in the region surrounding the pores from which, in the crystallographic structure, the Chls bound in the interior of the complex appear to face the solvent. Moreover, in close proximity of the pores, hydrophobic and weakly polar side chains of several residues are present. This analysis nicely fits with the observed accessibility of the triplet state of the bound Chls to positively charged and neutral nitroxides and with the scarce accessibility to the negatively charged 3-carboxy-proxyl. It has been shown that the lifetime of the encounter complex in electron−electron spin communication may play a role when the direct orbital overlap between the two molecules is poor.36 Thus, the comparable DEP effect observed for neutral and positively charged nitroxides in WSCP is likely due to the higher penetration of the first in the hydrophobic/weakly polar pores and to the longer lifetime of the encounter complex of the second, which is attracted by the negative potential of the protein surface. The results also explain the capability of Chls bound to WSCP to photosensitize 1O2, which is a neutral substrate able to easily reach the negatively charged pore. Our investigation of the WSCP protein complex shows the potentiality of the experimental approach based on DEP to probe the accessibility of cofactors taking advantage of the extensive library of nitroxides with different size, shape, and charge. Moreover, it is important to note that the method can be applied not only to those proteins which contain cofactors able to populate their triplet states, as hemes, chlorins,
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EXPERIMENTAL METHODS Protein overexpression in Escherichia coli and protein purification have been performed as previously reported.22 Chl a was isolated and purified from pea plants (Pisum sativum) according to Booth and Paulsen.38 The purified WSCP apoprotein was reconstituted with Chl a as previously reported.22 The samples were prepared in 20 mM phosphate buffer (pH 7.8) with a 1 mM concentration of the soluble nitroxide (4-oxoTEMPO, 4-amino-TEMPO, or 3-carboxy-proxyl) and a 25 μM concentration of the WSCP complexes (100 μM Chl a). Oxygen was removed from the samples by flushing nitrogen in the EPR capillary before sealing. X-band TR-EPR experiments were performed on a modified Bruker ER200D spectrometer with an extended detection bandwidth (6 MHz) and response time of 150 ns. Laser excitation at 532 nm (5 mJ per pulse and repetition rate of 50 Hz) was provided by the second harmonic of a Nd:YAG laser (Quantel Brilliant) in a high-Q cylindrical TE011 resonant cavity. The experiments were carried out with a microwave power in the cavity of 10 mW. The temperature was controlled with a nitrogen flow cryostat. The signal was recorded with a LeCroy LT344 digital oscilloscope, triggered by the laser pulse. The spectra were obtained averaging 3000 transient signals at each field position. To correct for the background signal, transients accumulated under off-resonance field conditions were subtracted from those on resonance.
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AUTHOR INFORMATION
Corresponding Author
*Telephone: +39 0498275144. Fax: +39 0498275161. E-mail:
[email protected]. 674
DOI: 10.1021/acs.jpclett.7b03428 J. Phys. Chem. Lett. 2018, 9, 672−676
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Alessandro Agostini: 0000-0002-8877-315X Harald Paulsen: 0000-0003-0532-3004 Donatella Carbonera: 0000-0002-5499-1140 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank Lorenzo Franco for insightful discussion. This work has been funded by a grant from the Deutsche Forschungsgemeinschaft to H.P. (Pa 324/10-1). A.A. gratefully acknowledges the Ing. Aldo Gini foundation for supporting his stay in Germany with a travel grant.
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