Anisotropic circular dichroism signatures of oriented thylakoid membranes and lamellar aggregates of LHCII

Abstract

In photosynthesis research, circular dichroism (CD) spectroscopy is an indispensable tool to probe molecular architecture at virtually all levels of structural complexity. At the molecular level, the chirality of the molecule results in intrinsic CD; pigment-pigment interactions in protein complexes and small aggregates can give rise to excitonic CD bands, while "psi-type" CD signals originate from large, densely packed chiral aggregates. It has been well established that anisotropic CD (ACD), measured on samples with defined non-random orientation relative to the propagation of the measuring beam, carries specific information on the architecture of molecules or molecular macroassemblies. However, ACD is usually combined with linear dichroism and can be distorted by instrumental imperfections, which given the strong anisotropic nature of photosynthetic membranes and complexes, might be the reason why ACD is rarely studied in photosynthesis research. In this study, we present ACD spectra, corrected for linear dichroism, of isolated intact thylakoid membranes of granal chloroplasts, washed unstacked thylakoid membranes, photosystem II (PSII) membranes (BBY particles), grana patches, and tightly stacked lamellar macroaggregates of the main light-harvesting complex of PSII (LHCII). We show that the ACD spectra of face- and edge-aligned stacked thylakoid membranes and LHCII lamellae exhibit profound differences in their psi-type CD bands. Marked differences are also seen in the excitonic CD of BBY and washed thylakoid membranes. Magnetic CD (MCD) spectra on random and aligned samples, and the largely invariable nature of the MCD spectra, despite dramatic variations in the measured isotropic and anisotropic CD, testify that ACD can be measured without substantial distortions and thus employed to extract detailed information on the (supra)molecular organization of photosynthetic complexes. An example is provided showing the ability of CD data to indicate such an organization, leading to the discovery of a novel crystalline structure in macroaggregates of LHCII.

Low pH Modulates the Macroorganization and Thermal Stability of PSII Supercomplexes in Grana Membranes

“…For the case of isolated granal membranes, where the extensive vertical stacking of the membranes is lost, the observed characteristic excitonic CD signals are very similar to those found for unstacked thylakoid membranes (46). In this highly complex system it is difficult to discriminate the contribution of the different components, because the CD spectra of BBY membranes are largely dominated by the CD features of the LHCII molecules (46). The signal appearing at 650 nm is attributable to short-range chl b related interactions within LHCII complexes either in isolated state or in both BBY and intact thylakoid membranes (47)(48)(49); therefore, the 610/646 nm band in our BBY membrane preparations can be used as a fingerprint of LHCII stability.…”

“…The short-and long-range pigment-pigment interactions serve as fingerprints of the supramolecular organization and conformational stability of the complexes in photosynthetic membranes (45). For the case of isolated granal membranes, where the extensive vertical stacking of the membranes is lost, the observed characteristic excitonic CD signals are very similar to those found for unstacked thylakoid membranes (46). In this highly complex system it is difficult to discriminate the contribution of the different components, because the CD spectra of BBY membranes are largely dominated by the CD features of the LHCII molecules (46).…”

Structural Changes and Non-Photochemical Quenching of Chlorophyll a Fluorescence in Oxygenic Photosynthetic Organisms

“…On the other hand, it is known that the magnitude of psi-type CD in granal thylakoids is considerably weaker than in microcrystalline LHCII ( Fig. 16.2 ; Simidjiev et al 1997 ;Miloslavina et al 2012 ). Taken together, the available electron microcopy and CD data strongly suggest that the stacked regions contain large ordered arrays of ( super -) complexes , but the overall long -range order is probably weaker than in the semicrystalline domains .…”

Excitonic States and Excitation Energy Transfer in Plant Light-Harvesting Complexes in Different Molecular Environments

“…This section compares the CD and ACD spectra of isolated LHCII in aggregated state and in reconstituted membranes, oriented by different methods. Whereas the psi-type ACD of lamellar macroaggregates in compressed gels has been reported earlier [69,162], purely excitonic ACD spectra of LHCII have not been published yet.…”

“…the CD of molecules with fixed macroscopic space orientation [67,68]. For example, in an ideal case, by measuring the CD of the dimer separately with light propagating along the x and y coordinate axes, one could extract the individual exciton CD bands [69] and hence obtain direct physical parameters, such as the orientation of the excitonic transition dipole moments or the coupling energy (from the difference in the bands' central frequencies), which are not accessible from ordinary CD measurements.…”