Peridinin - chlorophyll a - protein (PCP) complex in its native form is a trimer. The monomer contains two chlorophyll a molecules and eight peridinins, which are highly substituted carotenoids. Such a high ratio between the chlorophylls and carotenoids distinguishes this system from most of other light-harvesting complexes, where the dominant absorption occurs through the chlorophylls. In single molecule experiments described below we use the broad absorption band associated with the peridinins in order to excite the fluorescence of the chlorophylls. In addition to native PCP complex, which contains six identical chlorophylls, we have studied PCP complexes reconstituted with various chlorophyll mixtures. We show that they are equivalent to the monomers of native PCP. The reconstituted PCP complexes have been produced in collaboration with the group of Professor H. Scheer at the Department of Biology, LMU Munich. This important degree of freedom, which enables us to exchange - and to some extent control - the chlorophylls in PCP significantly enhances our abilities to extract information about the protein energy landscape, which is the central focus of the ongoing reserach project.

1. Ensemble spectroscopy of native and reconstituted PCP.
2. Single molecule spectroscopy of native PCP.
3. Single molecule spectroscopy of PCP reconstituted with Chl a.
4. Single molecule spectroscopy of PCP reconstituted with Chl a nd Chl b.


1. Ensemble spectroscopy of native and reconstituted PCP.

In Figure 1 we show absorption and fluorescence measured for three variuos reconstituted PCP complexes: red - PCP reconstituted with chlorophyll a (identical to the one present in the native form), blue - PCP reconstituted with chlorophyll b, and red-blue - PCP reconstituted with the mixture of chlorophyll a and chlorophyll b. The absorption of the PCP reconstituted with chlorophyll a is almost identical to the one measured for the native PCP. The spectrum features a broad and intense band (from 350 nm to 550 nm) associated predominantly with the absorption of peridinins. The main contribution from chlorophylls, besides the QY band - seen around 670 nm - is through Soret band at 437 nm. On the other hand, in the case of PCP reconstituted with chlorophyll b, the absorption spectrum is dominated by the Soret band, while the peridinin contribution is somewhat reduced.

The fluorescence spectra measured for PCP reconstituted with either Chl a or Chl b feature strong and narrow emission lines at 673 nm and 651 nm, respectively. Interestingly, in the case of PCP complexes where both types of chlorophyll molecules were added during the reconstitution, we observe two well - separated lines (~20 nm) at the two above energies, which correspond to Chl a and Chl b emissions. However, on the basis of the ensemble experiments it is impossible to conclusively determine whether the complexes contain identical chlorophyll molecules (Chl a and Chl a, or Chl b and Chl b) or if there are two different chlorophylls present in some of them. We address this point using single molecule techniques, which in addtion allows us to gain unique information about the energy transfer in this system.


2. Single molecule spectroscopy of native PCP.

Figure 2 shows a summary of single molecule spectroscopy results obtained for single native PCP complexes. The image displays 30x30 micron area with a large number of fluorescing PCP complexes (bright spots). The fluorescence is relatively stable and features limited blinking during the scan. A typical spectrum of the native PCP fluorescence is given in Figure 2b. The linewidth, estimated using a Gaussian fit, is equal to 14nm, which is considerably smaller than for the most of the biomolecules, including other light harvesting complexes. Further, in Figure 2c we compare the distribution of emission energies with the fluorescence spectrum of the PCP ensemble. Although the central wavelength of the fluorescence exhibits some variation from complex to complex, the overall distribution reasonably well mirrors the spectrum measured at ambient temperature for the PCP ensemble, both in terms of the central energy and the linewidth. This correlation indicates that the photo-physical properties of single PCP complexes are not affected by the surface and/or preparation procedure in any significant way.
 


Surprisingly, the fluorescence of PCP complexes is very stable. The signal could be observed for tens of seconds. Figure 2d shows the hystogram of survival times measured for single native PCP complexes. As one can see, some of them could be inspected for more than a minute. This is important as it allows us to study in detail both the spectral dynamics and subsequent photobleaching of all the chlorophylls within the complex.

An example of the intensity trace measured for a single PCP complex is shown in Figure 3. In this case we observe several steps of the total fluorescence intensity of the complex (color-coded), which we attribute to subsequent bleaching of the chlorophylls within the complex. Indeed, as shown on the lower panel, we could identify six steps in the intensity, which correspond to the number of chlorophyll a molecules in the native PCP. Most of the complexes studied features between 4 and 6 steps. We also observe some complexes where only up to two steps could be identified. We attribute them to monomeric PCP, which is suggested to coexist with the trimeric form.
 


The most important conclusion drawn from this data is that the chlorophylls within PCP are very weakly coupled to each other. This property further distinguishes this complex from all the other light-harvesting systems, where the chlorophylls are strongly coupled and form an exciton. This conclusion is reinforced by the results of single molecule spectroscopy obtained for reconstituted complexes.


3. Single molecule spectroscopy of PCP reconstituted with Chl a.

Qualitatively, the fluorescence spectra measured for PCP reconstituted with chlorophyll a are identical to the ones of native complex, the emission lines are very narrow and the fluorescence is quite intense and stable in time. However, as can be seen in Figure 4, the fluorescence of these complexes features less number of steps in the intensity. Namely, we observe either one single step or, as in most cases, two steps in the fluorescence intensity. This indicates, that the reconstituted PCP is monomeric, which makes it very attractive for various single molecule studies. Moreover, such a behavior provides a clear proof for weak coupling between the chlorophylls. The resulting ability to independently trace each and every chlorophyll in the complex allows us to gain important information about energy scheme of PCP complex.

An example is given in Figure 5, which shows spectra of PCP reconstituted with Chl a before and after subsequent bleaching of the chlorophylls. For the complex displayed in the upper row, the energy difference between the two chlorophylls within the same monomer is very large, around 9 nm. In contrast, for the complex shown in the lower row, the splitting is negligible. This indicates that the two chlorophylls within a single monomer, which - from the structural point of view - should feel the same environment, are energetically distinguishable.


4. Single molecule spectroscopy of PCP reconstituted with Chl a and Chl b.

Particularly interesting are the complexes reconstituted with chlorophyll mixtures. In Figure 5 we show some of the spectra taken for PCP complexes reconstituted with both chlorophyll a and chlorophyll b. Clearly, we observe various situations. Sometimes, the fluorescence features single line, corresponding to one of the chlorophylls, but most of the times we observe two emission lines, whose energy splitting reaches up to 20 nm. We attribute them to the complexes containing two different chlorophylls.

MORE STILL TO COME :-)

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