Ultrafast fluorescence of photosynthetic crystals and light-harvesting complexes

B.F. van Oort

Research output: Thesisinternal PhD, WU


This thesis focuses on the study of photosynthetic pigment protein complexes using time resolved fluorescence techniques. Fluorescence spectroscopy often requires attaching fluorescent labels to the proteins under investigation. With photosynthetic proteins this is not necessary, because these proteins contain fluorescent pigments. Each pigment’s fluorescence is influenced by its environment, and thereby may provide information on structure and dynamics of pigment protein complexes in vitro and in vivo. Another way to probe protein structure is X ray diffraction of crystals of the pigment protein complexes. In this work fluorescence was measured of crystals of Light Harvesting Complex II (LHCII), of which the structure is known from X ray diffraction on similar crystals. Analysis of spectral properties and structure of the crystals yielded important insights in the process of nonphotochemical quenching (NPQ). The insights were supplemented by studies of aggregated LHCII, and LHCII under high hydrostatic pressure. The largest photosynthetic pigment protein complex to be crystallized to date (PSI LHCI) was also studied. A minor light harvesting complex (CP29), which may be important for NPQ, and which has eluded crystallization, was studied by site directed fluorescent labeling combined with FRET to obtain structural information. This summarizing discussion first treats CP29 (section 7.2), then LHCII and NPQ (section 7.3), and finally PSI (section 7.4). The general conclusions are in section 7.5, followed by the recommendations for future study.

7.1 Streak camera setup: Structure of CP29
A valuable detection method of time resolved fluorescence is the synchroscan streak camera system. Such a setup was built, using a set of lasers and amplifiers of Coherent (U.S.A.), a spectrograph of Chromex (U.S.A.) and a streak camera of Hamamatsu (Japan). Chapter 2 describes the details of the setup, and analysis of the data obtained with it. In Chapter 3 the value of the setup is illustrated for CP29. CP29 is one of the minor light harvesting complexes of PSII. Its exceptionally long N terminal domain1 is phosphorylated2 under cold stress induced photoinhibition3. Phosphorylation leads to spectral changes, that were assigned to conformational changes of the transmembranal part of CP294. Later CP29 was suggested to be a site for quenching during NPQ5, and in Arabidopsis thaliana plants that lack CP29 (and CP24) NPQ is decreased compared to wild type plants6. The structure of CP29 has not been resolved, however the high sequence homology with LHCII, especially in the transmembrane domain, suggests an organization similar to that of LHCII, the structure of which has been obtained at 2.5-2.72 Å7,8. However, no information is available about the organization of the N-terminal domain, which differs completely from that of LHCII. In Chapter 3 specific amino acids in the tail were replaced by a cysteine, which was then labeled with a rhodamine type dye (TAMRA), which can be selectively excited around 530 550 nm. Förster resonance energy transfer was measured from TAMRA to the Chl molecules, providing information about distances between specific sites of the N-terminal domain and the chlorophyll molecules. The N terminal domain seems to fold back to the transmembranal part of CP29. Although these results require further substantiation, the experiments demonstrate the feasibility of this approach to study protein structure.

7.2 Nonphotochemical quenching and LHCII
Nonphotochemical quenching is an important mechanism that plants and algae use to prevent photodamage under conditions of high light intensity9. It has been proposed to take place in LHCII10-13 (or CP295, see above). An unresolved issue is the mechanism of quenching. Currently there are two views: (i) quenching occurs via energy transfer from Chl a to Lut and subsequent rapid relaxation to the ground state13; (ii) quenching occurs via cation radical formation by charge separation in a Chl Zea dimer, and subsequent rapid relaxation to the ground state14. Both candidates have in common that a light harvesting complex (LHC) can switch between a state with a long excited state lifetime, and a state in which this lifetime is reduced. Chapters 1, 4 and 5 describe experiments on this switching in LHCII.

Aggregation of LHCII in vitro leads to fluorescence quenching that is very similar to that observed in vivo under conditions of NPQ15,16. Therefore aggregated LHCII has been used extensively as a model system for studying the role of LHCs in NPQ10,17-20. However, it is still unknown whether LHCII aggregation leads to the formation of quenchers (excitation traps), as proposed for instance by Horton et al.11,19, or that increased connectivity between trimers upon aggregation leads to efficient quenching by a small population of permanently quenched LHCII21,22. If quenchers are formed, the question remains whether strong quenchers form in a small fraction of LHCII18, or whether weaker quenchers form in a large fraction of LHCII11. In Chapter 4 excited state lifetimes of monomers, trimers and aggregates of LHCII are compared. The quenching in aggregates was so strong, that it could not be explained quantitatively by enhanced trapping by quenchers that were present before aggregation. So quenchers are created upon aggregation. These quenching traps do not only trap excitations in the trimer in which they are located, but also excitations originating in complexes that do not contain traps themselves. The fluorescence quenching in monomers was found to be even stronger than that in trimers, suggesting an intramonomeric origin of this process.
In Chapter 5 hydrostatic pressure was used to study the switching between quenched/unquenched states of LHCII. At atmospheric pressure a small fraction of isolated LHCII is quenched, and this fraction is in thermodynamic equilibrium with the bulk unquenched fraction23. Applying high hydrostatic pressure shifts the equilibrium more to the quenched conformation and this allows determination of the energy difference between both states and the change in volume. The volume difference between the two states is very small: 5 ml/mol; less than 0.006% of the volume of one trimeric LHCII complex, which indicates a local conformational switch between the two states. The switch is accompanied by a small change in energy: 7.0 kJ/mol; high enough to keep the quenched state population low under normal conditions, but low enough to switch in a controlled way by environmental changes (such as pH, membrane structure, aggregation) induced by high light intensities.
In addition, at high pressure a state forms that is approximately 100 fold more quenched than the other two states. This state has a fluorescence lifetime of ~25 ps, reflecting the average time to reach an extremely efficient quencher somewhere within the trimer (the excitation equilibration time)24. At 400 MPa (4 kbar) less than 1% of all Chls are highly quenched, whereas in LHCII in which the pigments were uncoupled by detergent treatment this was 47%. These pressure experiments demonstrate that at least two types of quenchers can be formed in LHCII in vitro, very strong and relatively weak ones. In vivo a small number of strong quenchers could quench fluorescence of many connected LHCs. Alternatively a large number of weak quenchers could lead to the same amount of quenching.

A third approach to gain insight in the switching of LHCII between quenched and unquenched states involves the study of crystals of LHCII, as described in Chapter 1 and by Pascal et al.12. The crystal structure of LHCII from spinach at 0.272 nm7 aided in the understanding of its spectroscopic features; extensive modeling based on this structure explained many steady stated and time resolved spectral properties of LHCII in solution25. It is not known a priori, however, whether the structure of LHCII is the same when LHCII is dissolved in buffer, and when it is crystallized. This is particularly relevant, because LHCII has the ability to switch between conformations with different fluorescence lifetimes10,17,18. Fluorescence lifetime imaging microscopy showed that the fluorescence lifetime of LHCII crystals was ~850 ps. When the crystals were dissolved the fluorescence lifetime switched to ~4 ns. Subsequent aggregation switched LHCII back to a quenched state, with a lifetime of ~650 ps (Chapter 1 and ref. 12). Thus, it is clear that the crystal structure does not correspond to the unquenched state of LHCII, but more resembles the quenched state of aggregated LHCII, and/or that of LHCII under hydrostatic pressure. Also the Raman and low temperature fluorescence emission spectra of the crystals differed from those of LHCII in solution12. Those differences indicated a higher degree of homogeneity and stronger twisting of the Neo in the crystals as compared to LHCII in solution. Also the interactions of Chls b and their environment are different. The crystal packing was not dense enough to induce these changes directly by trimer trimer interactions, nor can the quenching be caused by intertrimeric pigment interactions. Therefore the quencher must be sought for inside the trimeric units. Based on the crystal structure7, two sites were proposed: a Chl a dimer in close proximity of a Lut, and a Chl b close to Neo. It was later confirmed that in aggregated LHCII excited state energy is transferred from Chl to Lut, supporting the role of Lut in fluorescence quenching in the crystals13.

7.3 Photosystem I
The structural and spectral differences between LHCII in crystal form and in solution triggered the study of PSI LHCI crystals in Chapter 6. PSI LHCI is a pigment protein complex that is more complicated than LHCII: PSI LHCI binds more pigments (~160 vs. 42), it performs photochemical quenching, and it contains “red Chls”, with an excited state energy lower than that of the reaction centre26. The fluorescence decay is rather complex due to the presence of energetically coupled Chls with very different excited state energies. The excited state energy of the Chls and their coupling may depend on plant species, preparation method and measuring conditions27-33. Consequently, a wide range of fluorescence decay kinetics have been reported over the years27-33. In Chapter 6 it is shown that the picosecond fluorescence of intact crystals is identical to that of dissolved crystals, but differs considerably from most kinetics presented in literature27-33. Caution should therefore be taken in using the crystal structure to model those kinetics.
The data of dissolved crystals were described quantitatively by a simple model that required only two pigment clusters: PSI core and LHCI. This model yielded rates of photosynthetic trapping from the core, and wavelength dependent excitation energy transfer from LCHI to PSI core and vice versa. The model differs from previous models with respect to the reduced number of pigment clusters, and the introduction of the wavelength dependence of the transfer rates. The modeling yields spectra and rate constants that originate specifically from excitation of pigments in PSI core or LHCI, and can therefore serve as a starting point for detailed modeling at the molecular level, using the PSI LHCI crystal structure.
7.4 General conclusions
This thesis presents important information on the mechanisms by which LHCII (or LHCs of PSII in general) can contribute to NPQ. Better understanding of this photoprotective process may in time lead to strategies to increase crop yields and/or plant fitness. It may further aid systems that mimic photosynthesis in vitro, aiming at energy production. The experiments with LCHII crystals showed that its crystal structure was that of a quenched conformation, as compared to LHCII in buffer. Next it was shown that upon aggregation of LHCII quenchers are formed, which consequently also quench fluorescence of LHCII trimers without quenchers. Under high hydrostatic pressure, two quenching mechanisms were observed: (i) Strong quenching, limited by the excitation equilibration time of a trimeric unit; and (ii) weaker quenching, caused by a conformational change that is associated with very small energy and volume changes. These results mark the broad dynamic range of quenching that LHCII can undergo. With CP29, a start has been made to study the structure of its N terminal domain, which is phosphorylated under stress.
This thesis further deals with the aptness of using X ray crystal structures to model spectral properties of pigment protein complexes. Often it is tacitly assumed that the structure of these complexes is the same in solution and in crystal form. For LHCII this assumption is not completely valid, because the fluorescence of LHCII crystals is quenched as compared to that of LHCII in solution. For PSI LHCI the fluorescence decay was identical for crystals and dissolved crystals. The decay was however different from most of those reported before, and the crystal structure may not be suited to model those data. A new type of model provides a simple description of the fluorescence kinetics, based on only two compartments and wavelength dependent excited state energy transfer among them. The experiments on these crystals illustrate the need of caution when using crystal structures to model specific spectral parameters of pigment protein complexes.

7.5 Recommendations
7.5.1 CP29 structure
With regard to the study of the structure of the N terminal tail of CP29 I recommend the following:
1. More detailed structural information can be obtained by using more labeling positions.
2. The effect of the fluorescent label on the structure should be checked, by comparing, for example, (low temperature) linear and circular dichroism, with and without label attached.
3. Another label can be used, for example a smaller fluorescent label, or a paramagnetic label for electron paramagnetic resonance (EPR) experiments.
4. The N terminal domain of CP29 is phosphorylated under stress3. Therefore it would be interesting to study the effect of phosphorylation on the structure of this domain. If in vitro phosphorylation is impossible, its effect may be mimicked by introduction of a negatively charged amino acid.

7.5.2 Nonphotochemical quenching and LHCII
With regard to the study of LHCII as a model system of nonphotochemical quenching in vivo, I recommend the following:
1. Chapter 5 shows that hydrostatic pressure can be used to controllably and reversibly switch LHCII between quenched and unquenched states. This switching can now be studied by many other spectroscopic methods to gain insight in the quenching mechanism.
2. Most experiments on isolated LHCII have been done with micellar systems, where individual monomers or trimers are solubilized by detergent molecules. In vivo LHCII is in a crowded membrane, which is a quite different environment. The in vivo state can be mimicked by reconstitution of LHCII in lipid vesicles (as done by Moya et al.23). The effect of crowding can be studied by changing the protein/lipid ratio of the vesicles. Optical and structural properties of LHCII should be studied in such vesicles. Also the experiments on CP29 could be repeated in lipid vesicles. The structure of the N terminal domain may be affected by the membrane via steric hindrance or electrostatic interactions.
3. In vivo the pH gradient across the photosynthetic membrane is required for full NPQ. Indeed, lowering pH induces fluorescence quenching of isolated LHCII19. However, in vivo the formation and relaxation of qE and ΔpH occur with different kinetics34. With LHCII in lipid vesicles a pH gradient can be applied across the membrane (ideally all LHCII units in a vesicle should be oriented with the same side to the interior). Then the quenching effect of the gradient can be studied in detail in a system that is more in vivo like than LHCII in micelles.
4. Study how connectivity of photosynthetic units in the native membrane influences the effect of quenchers. Native photosynthetic membranes are densely packed35, enabling energy transfer among photosynthetic complexes, which results in migration of excitation energy through multiple complexes. A quencher is more effective in a highly connected network of pigments than in a weakly connected network21. The connectivity is related to the speed of excited state energy migration, but this speed is still under debate36-38, and may change in response to a plant’s environment39,40. Knowledge about the connectivity will therefore help to understand to which extent fluorescence quenching of individual LHCs can contribute to the overall quenching in vivo. Connectivity should be measured (using rapid fluorescence induction41) in unquenched samples and during NPQ formation.

7.5.3 Photosynthetic pigment protein crystals
With regard to the study of crystals of photosynthetic pigment protein complexes, I recommend the following:
1. The use of total internal reflection microscopy instead of confocal microscopy, to avoid reabsorption effects.
2. Study other (optical and electronic) properties than fluorescence lifetimes. A start has been made with measuring Raman and fluorescence spectra of LHCII12, variable fluorescence and electron transfer rates in cyanobacterial PSII cores42, EPR spectroscopy of cyanobacterial PSI43 and PSII44 cores. Such experiments can give more insight in which properties of pigment protein complexes are conserved in crystals and which differ from those of the same complexes in solution. Further experiments are then required to compare those properties with those of the complexes in vivo.
3. “Manipulate” crystals, and measure the effect on optical and electronic properties. For example, it is possible to infiltrate photosynthetic crystals with chemicals such as DCMU42 (which inhibits QA to QB electron transfer). Crystals can be “manipulated” in many other ways, such as by magnetic or electric fields or by light. Also temperature can be varied (note that X ray diffraction experiments are generally performed at cryogenic temperatures). Measurements of pigment protein complex properties under these different conditions can aid the comparison with the complexes in solution and in vivo.

7.5.4 Crystals of biomolecules
The question whether a structure obtained by X ray crystallography is the same as that in solution, is appropriate for many biomolecules, not just photosynthetic proteins. Often it is tacitly assumed that the structure is the same in crystal and solution. This is not always true for pigment protein complexes (see LHCII). Also the fluorescence of several fluorescent proteins of the GFP family is quenched compared to that of the same proteins in solution45. Also the fluorescence of ethidium bromide bound to DNA is quenched more in crystals than in solution45. Therefore a systematic comparison of spectroscopic and structural properties of many types of biomolecules in crystal and in solution can provide valuable missing information.

7.5.5 Photosynthesis research in broader perspective
The production of enough food to feed and energy to power the world’s growing population are key issues of our society. At this moment all food, and most energy, originates from biomass (either directly from “fresh” biomass (~14%) or indirectly from fossil biomass, e.g. oil, coal, gas (~74%)46). The growth of the world’s population and its increasing prosperity, boost the demand for food and energy. Meanwhile the amount of easily accessible natural reserves of fossil fuels is decreasing, and CO2 released by burning fossil fuels is proposed to contribute to climate changes47. Therefore alternative energy sources are required.
A wide range of energy sources is available, for example energy from biomass, wind energy, tidal energy and energy from nuclear fission. Each of these has its own advantages and disadvantages. The main advantages of energy from biomass is that biomass production uses an abundant energy source (the sun) and existing production facilities (i.e. photosynthetic organisms). Disadvantages of biomass production are (i) competition with food production; (ii) requirement of fresh water, which becomes increasingly scarce; and (iii) requirement of large areas of arable land. (Note that the use of algae can overcome some of these disadvantages, but that requires a more complex capital intensive production than the use of plants.)
Most biomass is produced by photosynthesis46. Improving the efficiency of photosynthesis may overcome some of the disadvantages of biomass production. Three paths may lead to such an increase of efficiency: (i) reduction of losses in light-to-fuel conversion; (ii) conversion of solar energy directly into fuel; (iii) realization of artificial devices that perform better than the natural system. Future photosynthesis research should aim at the knowledge required to follow these paths.

Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
  • van Amerongen, Herbert, Promotor
  • Ruban, A.V., Co-promotor, External person
Award date28 Oct 2008
Place of Publication[S.l.]
Print ISBNs9789085852056
Publication statusPublished - 2008


  • fluorescence
  • fluorescence emission spectroscopy
  • photosynthesis
  • proteins

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