Multi-Level, Multi Time-Scale Fluorescence Intermittency of

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Multi-Level, Multi Time-Scale Fluorescence Intermittency of Photosynthetic LH2 Complexes. a Precursor of Non-Photochemical Quenching? Mario Schörner, Sebastian Reinhardt Beyer, June Southall, Richard J. Cogdell, and Jürgen Köhler J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.5b06979 • Publication Date (Web): 29 Sep 2015 Downloaded from http://pubs.acs.org on October 3, 2015

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The Journal of Physical Chemistry

Multi-Level, Multi Time-Scale Fluorescence Intermittency of Photosynthetic LH2 Complexes. A Precursor of Non-Photochemical Quenching?

Mario Schörner1, Sebastian Reinhardt Beyer1, June Southall2, Richard J. Cogdell2, Jürgen Köhler*1

1

Experimental Physics IV and Bayreuth Institute for Macromolecular Research (BIMF), University of Bayreuth, Germany

2

Institute of Molecular, Cell & Systems Biology, College of Medical Veterinary and Life Sciences, University of Glasgow, United Kingdom *corresponding author: Prof. Dr. Jürgen Köhler, Experimental Physics IV University of Bayreuth Bayreuth Germany phone: +49 921 55 4000 email: [email protected]

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Abstract The light harvesting complex LH2 is a chromoprotein that is an ideal system for studying protein dynamics via the spectral fluctuations of the emission of its intrinsic chromophores. We have immobilized these complexes in a polymer film and studied the fluctuations of the fluorescence intensity from individual complexes over nine orders of magnitude in time. Combining time-tagged detection of single photons with a change-point analysis has allowed the unambigeous identification of the various intensity levels due to the huge statistical basis of the data set. We propose that the observed intensity level fluctuations reflect conformational changes of the protein backbone that might be a precursor of the mechanism from which non-photochemical quenching of higher plants has evolved.

Keywords: non-photochemical quenching, fluorescence intermittency, single-molecule spectroscopy, light-harvesting complex,


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Introduction A hallmark of single particle spectroscopy is fluorescence intermittency - blinking which refers to a random switching of the emission between emitting (on) and non-emitting (off) periods. It is an ubiquitous phenomenon that has been observed for a large variety of materials including semiconductor quantum dots1-3, organic dye molecules4,5, organic multichromophoric systems6-8, polymers9, or pigment-protein complexes10,11, where the bright and dark periods cover essentially all experimentally accessible time scales. Most of the studies on fluorescence intermittency have employed time-binned detection methods to record the fluorescence intentensity trajectories. The flow of emitted photons is integrated over a short time bin of 1-100 ms and then a signal threshold is defined that discriminates between the on- and the off-states. Despite the large variety of the investigated materials, the resulting probability distributions for finding on- and off-periods of distinct duration all follow an inverse power law P(τ) ~ t-m with an exponential cut-off for the on-states12. Typically, the exponents range from 1 < m < 2, and several models have been developed to explain blinking in general, or the power-law dependence in particular, see12,13. However, as has been shown in great detail previously, binning in combination with empirical thresholding can lead to severe artifacts14-16. Simulations revealed that the exponents obtained for the on- and off-time probability distributions depend significantly on the choice of the threshold preventing reliable statistical information beeing extracted from the experimental intensity traces17. This drawback constitutes a severe problem in particular for the analysis of data from weak emitters where only noisy data is available. On the one hand the binning time required to achieve a reasonable signal-to-noise ratio for proper thresholding has to be increased, while on the other hand longer binning times can give rise to a loss of information about the intrinsic dynamics of the systems. For example, variations of the intensity within an on-time cannot be distinguished from transitions to the off-state where these transitions are shorter than the binning time, or from transitions to "real" intermediate states with reduced fluorescence intensity. Experimental binning and empirical thresholding can be avoided using the change-



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point analysis algorithm (CPA) developed by Watkins18, which relies on measuring the arrival time of each individual detected photon, i.e. so-called time tagging. This method is particularly useful to analyse changes in the fluorescence intensity at low signal levels. In order to retrieve those moments when changes in the emitted intensity occur, a generalized likelihood ratio test is recursively applied to the arrival times of the individual photons in the entire recorded fluorescence-intensity trace. The number of different intensity levels that are present in a photon time trace are then determined using a Bayesian information criterion together with expectation-maximization clustering. Recently, this algorithm has been applied to investigate the static and dynamic heterogeneity of single peripheral light-harvesting complexes (LH2) from the photosynthetic purple bacterium Rps. acidophila19. This pigmentprotein complex accommodates 27 BChl a molecules that are arranged in two concentric rings displaced with respect to each other along the common symmetry axis perpendicular to the plane of the rings. The two pigment pools are labeled B800 and B850, according to their room-temperature absorption maxima in the near infrared20. The B800 ring consists of nine well-separated BChl a molecules (B800) which have their bacteriochlorin plane aligned nearly perpendicularly to the symmetry axis. The other ring consists of eighteen BChl a molecules (B850) oriented with the plane of the molecules parallel to the symmetry axis21,22. Upon excitation of the B800 pigments the energy is transferred to the B850 molecules in less than 1 ps. The LH2 complexes were immobilised in solution by an anti-Brownian electrokinetic trap (ABEL)19, and the authors recorded simultaneously the fluorescence intensity, emission spectra and the fluorescence lifetime. This allowed them to identify different emissive states of LH2 that were associated with a photoactivated, reversible quenching pathway that most likely involves a conformational change of the protein. However, for the ABEL trap the feedback signal that compensates for the diffusion of the protein is generated from the emission of the sample. This limits the observation times to typically some ten seconds before the particle crosses over to a non-emitting state (off state) and gets lost from the trap. In previous work it has been shown that pigment-protein complexes from purple bacteria can be immobilized in a polymer film without significant


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perturbation of the protein23,24. This approach enables us to follow the dynamics of the LH2 fluorescence intermittency over unprecedented nine orders of magnitude in time, providing a huge statistical data base for statistical analysis. Together with the time-tagged time-resolved (t3r) detection of single photons and the subsequent change-point analysis this allows for an unambigeous identification of the various fluorescence intensity levels that can be observed for an individual LH2 complex. We suggest that these intensity level fluctuations reflect conformational changes of the protein that, given the previously observed conformational memory for these systems25, may represent a precursor of the mechanism from which nonphotochemical quenching (NPQ) seen in light harvesting complexes in higher plants could have evolved.



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Results and Discussion Examples of fluorescence-intensity traces from three different LH2 complexes are shown in fig.1. To allow comparison of the present data with that of previous studies, the red lines show time-binned traces that have been reconstructed from the unbinned data for a 100 ms dwell time.

Fig.1: Three representative fluorescence-intensity traces from different individual LH2 complexes. The traces are displayed as time-binned traces with 100 ms binning time that have been reconstructed from the data (red lines). For convenience the fluorescence intensity on the vertical axis is given as a time-averaged measure in counts per second. Left: Full data set recorded for one hour. Right: Expanded view of a short part of the trajectory, which is highlighted on the left hand side (yellow bar). From top to bottom the number of the different fluorescence-intensity levels that have been identified by the CPA algorithm are 2, 3 and 4. These levels (black lines and dots) are referred to as A, C, B, and OFF (vide infra). On the left hand side of the figure the data are shown for the full observation time of one hour, whereas on the right hand side an expanded view of a shorter part of the experiment, as indicated by the yellow bar on the left, is displayed. The black lines and dots correspond to the result from the analysis of the photon arrival times with the CPA algorithm.It is the strength of the CPA algorithm that it can uncover intensity levels that only appear for a short period, as indicated by the arrows in fig.1, and that obviously would be averaged out if the data was integrated over a finite binning time. For the examples shown in fig.1 this reveals


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changes of the fluorescence intensity between 2,3, and 4 distinct levels, respectively, only some of which would have been seen by the previous binning based analyses. Especially those transitions with short residence times would be missed and this would scew the statistics of these processes. Since the LH2 complex represents a strongly coupled multichromophoric system giving rise to delocalised electronic excitations26, it is not too surprising to find multiple discrete emission levels rather than the "classical" telegraph-like switching between just one on-state and one off-state. In total we recorded the fluorescence intensity from 40 individual LH2 complexes for 3600 s per complex, and the distribution of the number of the observed intensity levels is given in fig.2.

Fig.2: Distribution of complexes with a distinct number of intensity levels identified by the CPA algorithm For those complexes where the algorithm assigns 5,7, or 9 fluorescence intensity levels the differences between the signals in the various levels are very small, and can give rise to overinterpretation of the data. Moreover, for the six complexes that feature only a single fluorescence intensity level an unambigeous distinction between different intensity levels was impossible, either because of signal-to-noise limitations or simply because the particular complex did not change its emission status during the full observation period of 3600 s. Therefore we will focus in the following on the complexes that feature 2, 3, or 4 distinct fluorescence intensity levels and these represent 70% of the complexes studied. In order to allow the present data to be compared with the nomenclature used for the different fluorescence intensity levels seen in19, we will refer to the signal levels in the order of the signals strengths as A-OFF (2 level blinking), A-B-OFF (3 levels), and A-C-B-OFF (4 levels), 7 Environment ACS Paragon Plus


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respectively. For these complexes we find that the observed average intensities within each intensity level reproduce within experimental accuracy across the different types of complexes featuring 2,3, or 4-level blinking. The assignment of the intensity levels is corrobarated by the characteristic probability densities for the residence times that have been found for each level upon analysis of the blinking statistics (vide infra). Therefore, we obtained a histogram of the residence times for each intensity level. In order to consider the sparse statistics for long residence times, each entry in the residence times histograms was weighted with the mean temporal distance to its next-neighbour entries2. As a result we find the probability density distributions P(τ) of the residence times τ for each intensity level, as shown in fig.3 for 2, 3, and 4-level blinking from left to right, respectively. The bottom part of this figure shows an example for an individual complex, and the centre accumulates the entries from all complexes of the same blinking category. For comparison the top part displays the probability density of the residence times averaged over all histograms from the individual complexes, which is a common strategy in many other studies due to the lack of long time traces. We note that these data cover seven orders of magnitude for the probability densities and an unprecedented nine orders of magnitude for the residence times. This provides a rich amount of data that allows the deep analysis presented here.


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Fig. 3: Probability densities P(τ) of the residence times of the LH2 complexes in the states A (black), C (pink), B (blue) and OFF (red) for 2-level (a,b,c), 3-level (d,e,f), and 4-level (g,h,i) blinking systems, respectively. The bottom row displays typical examples from individual complexes, the centre row accumulates all entries from the respective individual complexes, and the top row displays the average of all distributions from the individual complexes of that category. For the 2-level blinking the residence times in A and OFF, fig.3a,b,c correspond to the commonly used "on" and "off" times in fluorescence blinking observed previously for single molecules or quantum dots. This figure reveals that the residence time in A is distributed over all experimentally accessible time scales from 10-6 to 103 s whereas the off-state distribution lacks the residence times shorter than about 100 ms. For the complexes that feature 3-level blinking the residence times in B are between 0.1 - 10 s, see fig.3d,e,f. Finally for 4-level blinking the residence times in C and B strongly overlap covering the range between 0.1 - 10 s, with a slight tendency of somewhat longer residence times in C, see fig. 3g,h,i. The cut-off for the off-time distributions towards time intervals shorter than about 100 ms is observed as well for complexes that feature 3- and 4-level blinking. Interestingly, within each category, all distributions from the individual complexes fall onto the same line. The data are not 9 Environment ACS Paragon Plus


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compatible with a single exponential yet can be described by an inverse power law P(τ) ~ τ-m with the exponents given in table 1.

Table 1: Exponents of the distributions of the mean residence times in the various intensity levels. mA

mC

2 levels

1,10 ± 0,07

3 levels

1,06 ± 0,08

4 levels

1,09 ± 0,08

1,08

mB

mOFF 1,20 ± 0,12

1,37 ± 0,09

1,22 ± 0,10

1,38 ± 0,14

1,34 ± 0,12

1,17 ± 0,13

1,38

1,35

1,20

The values of all the exponents determined here are clearly smaller than those found in previous studies on pigment-protein complexes using binned time traces and empirical thresholding10,25. In order to test whether this discrepancy might be caused by the previously used binning for data acquisition and thresholding for subsequent analysis, we have determined those exponents for the A-OFF systems also for binned time traces that were reconstructed from the raw data for dwell times of 50 ms, 100 ms, 500 ms, 1 s, and 5 s. This reveals a strong dependence of the resulting values for the exponents on the binning time. For increasing dwell times these vary between 2.6 and 1.2 for mA, and between 2.3 and 1.2 for mOFF (see also SI). Since the multilevel blinking systems can only be retrieved using the t3r data aquisition in combination with CPA analysis we refrained from testing this dependency as well for the 3- and 4-level blinking systems. It is clear for the CPA analysis of the present data that the values of the exponents that are associated with the same intensity level agree very closely with each other across the range of different blinking categories seen. The current results demonstrate the strength of the time-tagged data acquisition together with the CPA for analysing multichromophoric blinking of data with limited signal-to noise ratio. In particular the exponents mA and mOFF obtained from the 2-level blinking systems on the one hand and from the 3- and 4-level blinking systems on the other hand would differ strongly from each other if the discrimination of the intensity levels A/C and OFF/B was equivocal.


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Formation of triplet states as a possible origin of the observed blinking processes can be excluded because the respective time scales of these provesses are beyond the temporal resolution of our experiment27. In photosynthesis photons are harvested by pigment-protein complexes that serve as antennae for absorption of excitation energy which is then transported with high efficieny to a reaction centre where it is used to generate a stable charge-separated state. However, under oxygenic conditions excess light energy is very dangerous28,29. In order to overcome this problem nature has developed protection mechanisms that allow the light-harvesting complexes of higher plants to dissipate excess energy under conditions of light saturation30,31. (Note: In baterial photosynthesis light harvesting complexes are referred to as LH and numbered by Arabic numerals 1,2, whereas in photosynthesis of higher plants the abbreviation LHC together with Roman numerals I, II is common practice.) This protective mechanism is generally referred to as non-photochemical quenching (NPQ). In other words, depending on the light conditions the LHCs must be able to switch reversibly between an efficient light-harvesting state, and an almost perfectly quenched state. Yet, the mechanisms that regulate the conflicting demands between efficient energy transfer on the one hand, and dissipation of unused photoenergy on the other hand, are only poorly understood to date. It has been suggested that conformational changes of the protein matrix play an important role in this task. In recent studies on single LHC II complexes32-34 it has been found that both the switching rate between the light-harvesting and the quenched state, as well as the dwell times can be controlled by the environmental conditions, like pH and irradiation intensity. Accordingly, this suggests that nature has learned how to exploit specific protein motions for efficient switching between these two modes. In (19) it was found that transitions from A → B in single LH2 complexes were light induced, whereas the back transition was most probably thermally activated. These authors associated the level B with a photoactivated conformational change of the pigmen-protein complex, and level C with a photobleached BChl a chromophore in the protein. The rare transitions from C → B were interpreted as a conformational change of a LH2 with a photobleached BChl a molecule. Unfortunately the ABEL trap does not allow the recording of 11 Environment ACS Paragon Plus


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long time trajectories, because quenched fluorescent states of LH2 are no longer confined within the trap. This then prevented the monitoring of the OFF state.

Fig.4: Sketch of a precursor mechanism from which NPQ might have evolved. The minimum A is associated with the native (light-harvesting) state of the protein. The population of the second shallow minimum occurs as a light induced process, whereas depopulation can be thermally activated. Under light saturation conditions this state gets stabilized and forms the quenched state B. This stabilization could be stimulated for example by a change of pH35, or mixed charge transfer character of the excited states, that is removed when the light is no longer saturating.

Imagine the situation where light can drive the conformation of a light-harvesting complex from one where it is strongly fluorescent (acting as an efficient light harvester) to one where it is highly quenched (acting as an inefficient light harvester). Then allow the transition from the quenched state to the fluorescent state to be thermally activated, see fig.4. If evolution could act to stabilize the quenched state under conditions of light saturation, and to remove that stabilization when the light is no longer saturating then you have the basis for nonphotochemical quenching (NPQ). Even though of course LH2 is not LHC II the phenomenon we show here gives a good indication how this process could have evolved in LHC II. The effects of pH and Carotenoid type that now seem to control NPQ could then have been used to stabilise the different conformations. Moreover, if the system retains conformational memory such that the quenched state has a high probability of reverting when it does revert to the same strongly fluorescent state then switching between efficient light harvesting and efficcient photoprotection will be established. The conformational switching behaviour seen with the LH2 complexes in the present study suggests that the properties described here and in the previous paper36 could provide a pathway by which NPQ evolved.


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Materials and Methods The LH2 complexes of Rps. acidophila (strain 10050) were isolated and purified as described previously37. After purification the LH2 complexes were aliquoted and stored at 80°C in a buffer (20 mM TRIS/HCL, pH 8, 0.2% LDAO) in the dark. The stock solution was diluted with pure buffer in three steps to a concentration of 10-11 M, where in the last dilution step 2% polyvinyl alcohol (PVA) was added. Subsequently the material was spincoated on a fused quarz substrate forming a thin film with a thickness of about 100 nm38 in which the LH2 complexes were embedded. For the optical experiments the sample was mounted in a recipient that was flushed with helium gas which reduced the concentration of oxygen to less than 1 ppb and minimizes irreversible photobleaching of the complexes. The LH2 complexes were excited at 800 nm with the light from a continuous Titanium:Sapphire laser (Model 3900s, Spectra Physics) that was reflected into a home-built scanning confocal microscope (objective: Euromex Holland, S.PlanM 60x, NA = 0.85) via a fused quarz wedge that served as beam splitter. The excitation intensity was 85 W/cm² (corresponding to a fluence of 2.2×1020 photons/s) to avoid saturation of the LH2 complexes39. The red-shifted emission from the LH2 complexes was collected with the same objective and transmitted through a long-pass filter (LP808, AHF Analysentechnik AG) towards an avalanche photo diode (Micro Photon Devices, PD5CTC). In order to select the individual complexes a confocal image of a sample region of about 30 × 30 µm2 was acquired with the microscope. Subsequently, the confocal spot of the microscope was adjusted to coincide with the position of one of the complexes found in the image and the fluorescence intensity of this complex was registered as a function of time. For the time-tagged time-resolved single-photon detection a TCSPC module (Pico Quant Timeharp 200) in TTTR (t3r) mode has been used, providing a temporal resolution of 100 ns. The flow of the emitted photons from an individual LH2 complex was analysed according to the change-point analysis (CPA) algorithm developed by Watkins et al.18 and that was adapted for handling the timeharp-t3r-files. All experiments were carried out at room temperature. 13 Environment ACS Paragon Plus


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Acknowlegement M.S., S.R.B., and J.K. thankfully acknowledge financial support by the Deutsche Forschungsgemeinschaft (DFG) within the project GRK1640 and the State of Bavaria within the initiative “Solar Technologies go Hybrid”. J.S. and R.J.C. thank the BBSRC for support.

Supporting Information Available - Exponents of the inverse Power Law as a function of the Binning Time This information is available free of charge via the Internet at http://pubs.acs.org


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(32) Krüger, T. P. J.; Ilioaia, C.; Valkunas, L.; van Grondelle, R. Fluorescence Intermittency from the Main Plant Light-Harvesting Complex: Sensitivity to the Local Environment. J. Phys. Chem. B 2011, 115, 5083–5095. (33) Valkunas, L.; Chmeliov, J.; Krüger, T. P. J.; Ilioaia, C.; van Grondelle, R. How Photosynthetic Proteins Switch. J. Phys. Chem. Lett. 2012, 3, 2779–2784. (34) Schlau-Cohen, G. S.; Yang, H.-Y.; Krüger, T. P. J.; Xu, P.; Gwizdala, M.; van Grondelle, R.; Croce, R.; Moerner, W. E. Single-Molecule Identification of Quenched and Unquenched States of LHCII. J. Phys. Chem. Lett. 2015, 6, 860–867. (35) Gall, A.; Ilioaia, C.; Krüger, T.P.J.; Novoderezhkin, V.I.; Robert, B.; van Grondelle, R. Conformational Switching in a Light-Harvesting Protein Followed by Single-Molecule Spectroscopy. Biophys. J. 2015, 108, 2713-2720. (36) Schörner, M.; Beyer, S. R.; Southall, J.; Cogdell, R. J.; Köhler, J. Conformational Memory of a Protein Revealed by Single-Molecule Spectroscopy. J. Phys. Chem. B 2015 this is the accompanying paper (37) Cogdell, R. J.; Hawthornthwaite, A. M. Preparation, Purification, and Crystallization of Purple Bacteria Antenna Complexes. In The Photosynthetic Reaction Center; Deisenhofer, J.; Norris, J. R., Eds.; Academic Press, 1993. (38) Beyer, S. R.; Ullrich, S.; Kudera, S.; Gardiner, A. T.; Cogdell, R. J.; Köhler, J. Hybrid Nanostructures for Enhanced Light-Harvesting: Plasmon Induced Increase in Fluorescence from Individual Photosynthetic Pigment-Protein Complexes. Nano Letters 2011, 11, 4897–4901. (39) Trinkunas, G.; Herek, J. L.; Polívka, T.; Sundström, V.; Pullerits, T. Exciton Delocalization Probed by Excitation Annihilation in the Light-Harvesting Antenna LH2. Phys. Rev. Lett. 2001, 86, 4167-4170.



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