Letter pubs.acs.org/JPCL
Circular Dichroism Measured on Single Chlorosomal LightHarvesting Complexes of Green Photosynthetic Bacteria Shu Furumaki,† Yu Yabiku,† Satoshi Habuchi,‡ Yusuke Tsukatani,§,+ Donald A. Bryant,§,¶ and Martin Vacha*,† †
Department of Organic and Polymeric Materials, Tokyo Institute of Technology, Ookayama 2-12-1-S8, Meguro-ku, Tokyo 152-8552, Japan ‡ Biological and Environmental Sciences and Engineering Division, King Abdullah University of Science and Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia § Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, United States ¶ Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717, United States ABSTRACT: We report results on circular dichroism (CD) measured on single immobilized chlorosomes of a triple mutant of green sulfur bacterium Chlorobaculum tepidum. The CD signal is measured by monitoring chlorosomal bacteriochlorphyll c fluorescence excited by alternate left and right circularly polarized laser light with a fixed wavelength of 733 nm. The excitation wavelength is close to a maximum of the negative CD signal of a bulk solution of the same chlorosomes. The average CD dissymmetry parameter obtained from an ensemble of individual chlorosomes was gs = −0.025, with an intrinsic standard deviation (due to variations between individual chlorosomes) of 0.006. The dissymmetry value is about 2.5 times larger than that obtained at the same wavelength in the bulk solution. The difference can be satisfactorily explained by taking into account the orientation factor in the single-chlorosome experiments. The observed distribution of the dissymmetry parameter reflects the well-ordered nature of the mutant chlorosomes. SECTION: Biophysical Chemistry and Biomolecules
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cylinders.11 The orientation of the sheets with respect to the cylinder axis is different for the wild-type and mutant forms.12 The molecular-level structure of chlorosomes results in the appearance of strong circular dichroism (CD) in the absorption of light. CD has been studied mainly on chlorosomes of Chloroflexus aurantiacus13−17 and partly also on C. tepidum.18,19 Unlike absorption and linear dichroism (LD), CD is a very sensitive tool to study slight structural changes, and different types of CD spectra have been observed even for chlorosomes isolated from the same species under different growing conditions.14,20 The changes in CD spectra have been suggested to originate either from changes in molecular-level BChl arrangement21 or from varying physical size of the aggregates.22−24 Here, we study for the first time the fluorescence-detected CD signal at a fixed absorption wavelength on single isolated chlorosomes of the well-characterized bchQ bchR bchU triple mutant of C. tepidum.25 The mutant was chosen over the wild type of the same species because its structure has been best characterized and because, unlike the wild type, it shows a strong CD value at the wavelength of the excitation laser (733
hlorosomes are light-harvesting complexes found in green bacteria1,2 and Acidobacteria.3,4 The cores of chlorosomes are comprised of up to 200 000 bacteriochlorophyll (BChl) c, d, or e molecules,1,5 which self-assemble into large aggregates,6 resulting in ovoid-shaped structures with dimensions of ∼150 × 60 × 30 nm. The large number of BChl molecules and the structure and size of the aggregates enable chlorosomecontaining organisms to capture and utilize incident light with very high efficiency. Because the absorption of chlorosomes extends well into the near-infrared region, they can serve as ideal models for artificial light-harvesting complexes in organic photovoltaic devices, where they would help to utilize the light energy in this part of the spectrum. There has been a continued interest and debate about the molecular-level structure of the BChl aggregates, and several models, such as one-dimensional rods7,8 or two-dimensional lamellae9 have been proposed for the structural arrangement. A cryoelectron microscopy study of chlorosomal cross sections10 showed that in a wild-type strain as well as in a well-ordered triple mutant of Chlorobaculum tepidum (C. tepidum), the BChl’s self-assemble into large aggregate sheets that form higher-order structures, such as concentric cylinders, scrolls (spirals), or curved lamellae. Solid-state nuclear magnetic resonance (NMR) suggested that the aggregates are comprosed of syn−anti dimer stacks that form mainly concentric © 2012 American Chemical Society
Received: October 16, 2012 Accepted: November 16, 2012 Published: November 16, 2012 3545
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nm). Absorption and fluorescence polarization spectroscopy on single chlorosomes has recently brought new insight into the excitonic structure and structural disorder in chlorosomes of C. tepidum.26−30 Given the high sensitivity of the CD technique, we may expect different values of CD parameters for different individual chlorosomes. In addition, the work was motivated by recent reports that indicate that fluorescence-monitored CD observed on the level of single chiral molecules may31 or may not32 largely exceed values of CD parameters observed on bulk samples. Cells of the C. tepidum bchQ bchR bchU triple mutant were grown, and chlorosomes were isolated and characterized as described elsewhere.25 The chlorosomes were stored in the dark in a freezer as a concentrated suspension in PBS buffer (pH 7.4, 10 mM Na-phosphate, 150 mM NaCl). Samples for single-chlorosome microscopic measurements were prepared by thawing a small aliquot (∼10 μL) of the suspension immediately before the experiment. The aliquot was diluted in 5 mL of the PBS buffer and mixed with 10 mM Na2S2O4. A 50 μL volume of the diluted solution was dropped onto a cleaned glass coverslip and left standing for 3−5 min to allow adsorption of the chlorosomes onto the substrate, and the remaining solution was washed away with a clean buffer. A thin (100 nm) layer of poly(vinyl alcohol) (PVA) was spin-coated on top of the chlorosomes from filtered 2 wt % PVA buffer solution to protect the chlorosomes from rapid oxidation. The samples were immediately used for experiments. Control experiments were also performed on dry samples without the PVA layer and on samples in a buffer solution. The samples without the PVA cover show faster photobleaching, lower emission intensity, and larger noise, but the CD results generally agree with the PVA-covered samples. Microscopic CD measurements on single chlorosomes were carried out using an inverted fluorescence microscope Olympus IX71. The setup is shown schematically in Figure 1a. Light from a diode laser (733 nm, PicoQuant) passes a fast-rotating diffuser to scramble its coherence and is linearly polarized by a Glan prism. It is then divided by a nonpolarizing beam splitter cube into two beams that undergo opposite phase shifts by π/2 after passing two quarter-wave plates. The beams are then realligned using a second nonpolarizing beam splitter cube and focused on the back focal plane of an oil-immersion lens (Olympus UPlanFLN100xO2, N.A. 1.3). The resulting left- (L) and right-handed (R) circular polarization is carefully checked at the sample plane above the objective lens using a linear polarizer, and the orientation of the quarter-wave plates is readjusted to precompensate for distortion of the circular polarization by the optical components of the microscope. The advantage of the present setup is that the L and R polarizations can be optimized independently by two separate optical components. The inset in Figure 1a shows the degree of circular polarization in the L and R beams. Using this degree and the LD parameters of the mutant chlorosomes,26 we estimate that the imperfection in circular polarization of the exciation beams would result in an artifactual CD dissymmetry value g of ∼0.005, which can be neglected in the context of the present experiments (see below). Fluorescence from the sample passes the same objective lens and is detected with an electronmultiplying CCD (EM CCD) camera (Andor iXon+). In a typical experiment, a series of CCD images is taken with alternating L and R circular polarization. The alternation is controlled by a pair of shutters synchronized with the CCD camera trigger.
Figure 1. (a) Scheme of the microscopic experimental setup for measurement of fluorescence-detected CD on single chlorosomes. The inset shows the degree of circular polarization. (b) Example of a correction image α used to compensate for different distributions of intensity in L- and R-polarized excitation light. (c) Distribution of the apparent dissymmetry factor g for an ensemble of achiral fluorescent beads emitting with an average intensity of 2300 cps. The inset shows the data (black dots) plotted against the fluorescence intensity, together with the simulated distribution expected due to photon shot noise only (blue dots). (d) Same as (c) for an average emission intensity of 330 000 cps.
One of the problems often overlooked in single-molecule CD experiments is the actual distribution of laser excitation intensity in the sample plane. Even though both L and R beams may have perfect overall circular polarization at the sample plane, there could be slight differences in their spatial intensity distributions, which would give rise to false CD signals. To minimize this problem, we used the rotating diffuser to decrease the laser coherence and the related interference patterns. Further, we used the dye 1,1′,3,3,3′,3′-hexamethylindotricarbocyanine iodide (HITIC) dispersed at bulk-level concentrations in a thin polymer film to check the intensity distribution in the L- and P-polarized beams immediately prior to and after the single-chlorosome experiments. After verifying that the distributions have not changed during the experiment, we calculated a correction factor αi for each pixel i in the image as a ratio of the average L and R polarization-excited bulk HITIC fluorescence intensities at the pixel. The dissymmetry CD value gi for pixel i is then calculated as I − αiIRi gi = 2 Li ILi + αiIRi (1) where ILi and IRi are the L and R polarization-excited intensities at pixel i in the actual single-chlorosome experiments. An example of the correction factor image is shown in Figure 1b. Apart from the intensity distribution variations, another factor that can cause false g values in CD experiments is the overall instrumental noise. To provide an idea of the magnitude of this problem, we measured the distribution of apparent g parameters for achiral fluorescent beads as control samples. The fluorescent beads were prepared using commercial non3546
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fluorescent polystyrene beads (PS02N 5378, Bangslabs, diameter 300 nm) by immersing them in an ethanol/aqueous solution of the dye HITIC for 3 weeks and purifying by repeated centrifugation. The measurements were done with two largely different excitation powers to induce different levels of noise. The two powers resulted in the beads emitting in the range between 1200 and 3150 counts per second (cps) with an average fluorescence intensity of 2300 cps (low-intensity), and in the range between 235 000 cps and 525 000 cps with average intensity of 330 000 cps (high-intensity), respectively. Distributions of the corresponding g values are shown in Figure 1c and d. While the means of the distributions are similar (0.0055 and 0.0057, respectively) and close to zero, as expected for nonchiral species, the width of the distribution (in terms of the standard deviation) for the low-intensity measurement is 2.6 times larger than that for the high-intensity measurement (0.063 and 0.024, respectively). This result shows that one must be careful to evaluate samples of comparable fluorescence intensity when discussing widths of g value distributions. The insets in Figure 1c and d show the data plotted against the fluorescence intensity, together with simulated distributions expected due to photon shot noise at the corresponding emission intensity levels. It is obvious that the measured distributions are much broader than those expected from the shot noise only and that other factors, such as background noise, signal noise other than the shot noise, effects from the data analysis (2D fitting of the images, residual imperfection in the intensity distribution correction), imperfectly adjusted L and R circular polarization of the excitation beams, and so forth, contribute to the measured distributions and make singlemolecule and single-particle CD experiments, and polarization experiments in general,33 challenging. Absorption, fluorescence, and CD spectra of the mutant chlorosomes at the ensemble level in buffer solution are shown in Figure 2. The CD spectra are of the type I in the terminology
Figure 3. Single-chlorosome CD characterization. (a) Typical fluorescence microscopic image showing three individual chlorosomes. (b) Fluorescence intensity of chlorosomes labeled 1 and 2 in the image excited with alternating L and R circularly polarized light. The inset shows dissymmetry g values calculated from the two fluorescence traces using the correction factor α. (c) Distributions of the g parameter obtained for ensembles of chlorosomes (orange, excitation power 9.6 W/cm2) and fluorescence beads (black, excitation power 0.69 W/cm2) emitting with comparable average intensity. Blue lines are Gaussian fits to the distributions. In the inset, the same data are plotted as the intensity versus the g parameter.
by alternate excitation with L and R circularly polarized light for chlorosomes labeled 1 and 2 in the image are presented in Figure 3b. Each point in the trace represents a backgroundcorrected intensity average from a 3 × 3 pixel area centered on a particular chlorosome. The points correspond to individual frames taken continuously by the CCD camera with an accumulation time of 200 ms. The raw intensity data presented in the traces appear to be completely different for the two chlorosomes 1 and 2 and even point to opposite signs of the CD values. However, after taking into account the intensity distribution correction factor αi for each pixel and calculating the g value from each pair of consecutive points using eq 1, the difference between the chlorosomes is much smaller. The inset of Figure 3b shows time traces of the calculated g values for the two chlorosomes. The time-averaged mean g values obtained from the traces are −0.037 ± 0.035 for chlorosome 1 and −0.025 ± 0.039 for chlorosome 2, respectively. The analysis method described above was used to evaluate the g parameter for a large ensemble of 460 individual chlorosomes. The experiments were done over an extended period of time on different sample preparations and using different experimental conditions (mainly different fluorescence excitation power). After combining the CD values of all measured chlorosomes into one statistical sample, we obtained the mean value of the parameter gs of −0.0246. This value was then compared with a subensemble of 84 chlorosomes characterized by the highest emission rate (60 000 cps on average) for which we can expect minimized effect of the experimental noise. The mean gs value for this subensemble was −0.0245, which is identical within an error with the overall
Figure 2. Spectroscopic characterization of chlorosomes in bulk solution. Absorption (black), fluorescence (gray), and CD (red) spectra; the blue, vertical line indicates the wavelength of 733 nm.
of ref 14 (i.e., positive/negative) and are similar to those measured on chlorosomes from wild-type C. tepidum.19 At the wavelength of 733 nm of the excitation laser, the CD signal is ∼−380 mdeg, corresponding to gb = −0.010 in terms of the dissymmetry parameter. This value is considerably larger than the sensitivity of the microscopic CD setup obtained as the average microscopic g values of 0.005−0.006 from the achiral fluorescent beads (previous paragraph), making it meaningful to proceed with the microscopic CD measurement on single chlorosomes. A typical fluorescence image of single chlorosomes is shown in Figure 3a. Examples of fluorescence intensity traces obtained 3547
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sample. Distribution of the g parameter values for the highestintensity chlorosomes is shown in the histogram in Figure 3c. To evaluate the width of this ditribution, we first analyzed a sample of 76 fluorescent beads emitting with comparable intensity (70 000 cps on average). The histogram of the g parameters for the beads is also shown in Figure 3c. The mean g value for this ensemble was 0.0062, and the standard deviation was 0.037. These values can be taken as the instrument-limited g distribution mean and width for this particular emission intensity level. The width (standard deviation) of the chlorosome ensemble ditsribution in Figure 3c was 0.043. To estimate the instrinsic distribution of the g values of the chlorosomes that would be free of the instrumental noise effects, we used the distribution width (0.037) of the fluorescent beads and subtracted it from the distribution width of the chlorosomes. We may thus conclude that the g parameter analyzed from an ensemble of individual chlorosomes is gs = −0.025 ± 0.006. To interpret this value, we first compared it to the bulk solution value of gb = −0.010 at 733 nm obtained from the CD spectrum in Figure 2. The main reason behind the obvious discrepancy is an orientation factor. The bulk solution of randomly oriented chlorosomes is an isotropic sample. In the microscopic experiments, individual chlorosomes are oriented mainly with their long axis parallel to the sample plane.26 Each individual chlorosome thus represents an anisotropic system in the CD measurement. It is known34 that CD signal CDiso of an isotropic system, which is composed of anisotropic components, is given by weighted sum of three contributions (CDxan, y z , CDan ), each representing the CD signal of the CDan anisotropic component corresponding to the propagation of light along the axes x, y, and z, that is, CDiso = 1/3(CDxan + CDyan + CDzan). In the case of chlorosomes adsorbed on a substrate, the usual notation corresponds to the x and z axes being in the sample plane, the y axis perpendicular to the plane, and the z axis parallel with the chlorosome long axis.26 Theoretical study of the effect of the molecular structure and the overall geometry of BChl molecular aggregates on CD spectra24 has shown that for sufficiently long tubular or spiral macrostructures (the ones that form the core of the mutant chlorosomes), the lowest exciton state has the highest oscillation strength, is delocalized along the tube long axis, and provides the strongest contribution to the CD signal. For chlorosomes adsorbed on the substrate, the long axis (z-axis) of the tubular aggergates is parallel with the sample plane, and light propagating along the z-axis is always polarized mainly perpendicular to the strong lowest exciton component. In contrast, light propagating along the x or y axes can have the polarization component along the z axis of the tubes and thus excite the lowest exciton state. We may thus use the approximation CDxan ≅ CDyan ≫ CDzan, which would give us a factor of approximately 2/3 between the values of the bulk solution and single-chlorosome g parameters, that is, gb = 0.66gs. Experimentally, this factor is 0.4. The difference may partly originate from the fact that the chlorosomes are not symmetric in the x−y plane, and the approximation CDxan ≅ CDyan may not be completely valid. A systematic error resulting from the slight ellipticity of the exciation R and L beams can also be a contributing factor to the difference. Overall, however, the agreement between the bulk and single-chlorosome data is satisfactory. The intrinsic distribution of the g parameter of ±0.006 for single chlorosomes is surprisingly narrow and indicates that
there is no contribution of, for example, type II (negative/ positive) CD spectra. This fact reflects the well-ordered nature of the mutant chlorosomes found previously.26,27 The existing distribution could be easily explained using the concept of variations in the physical size of the aggregates22,23 due to residual disorder,26 even though it should be noted that none of the theoretical models presented in refs 22 and 23 managed to reproduce the pure type I CD spectra. Another possibility is that the g distribution is a result of a distribution of the absorption peaks of individual chlorosomes. The width of ±0.006 would correspond to the absortion peak distribution of approximately ±6 nm. Distributions of florescence peaks on much larger scales have been reported before,26,28 and a distribution of absorption spectral peaks of single chlorosomes at low temperature has recently also been observed (Jendrny, M.; Köhler, J., private communication), making this intepretation plausible. In summary, we have studied CD on single-chlorosomal light-harvesting complexes isolated from a triple mutant of green photosynthetic bacterium Chlorobaculum tepidum. The results are reproducible over extended periods of time and for different sample preparations. The average value of the dissymmetry parameter g obtained on a large statistical sample of individual chlorosomes is consistent with bulk solution measurement of the same chlorosomes when the orientation factor is taken into account. The g parameter measured for chlorosomes with the highest fluorescence emission rate shows relatively narrow intrinsic distribution. This is on one hand surprising in light of the many reports of the high sensitivity of CD spectra to slight changes in chlorosome aggregate structure or sample preparation (bacteria growing) conditions. On the other hand, it reflects the well-ordered structure of the mutant chlorosomes and provides yet another independent confirmation of the reduced structural disorder in these light-harvesting complexes.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Present Address +
Graduate School of Life Sciences, Ritsumeikan University, Shiga 525-8577, Japan; PRESTO, Japan Science and Technology Agency, Saitama 332-0012, Japan. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The work was supported by a Grant-in-Aid for Scientific Research No. 23651107 of the Japan Society for the Promotion of Science (M.V.).
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REFERENCES
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