Structure and Structural Flexibility of Chloroplast Thylakoid Membranes

Chloroplast thylakoid membranes isolated from plants form a highly organized stackedmulti-lamellar system, which is di®erentiated into grana and stroma lamellar regions andhighly enriched in membrane bound protein complexes constituting the photosyntheticmachinery.The present PhD project addresses the structure of the thylakoid membrane system ona length scale of 100 ºAand the question of structural °exibility upon illumination andchanges in the physico-chemical environment, such as osmolarity and ionic strength. Thestructure and structural °exibility of the thylakoid membrane system have been studiedwith a combination of small-angle scattering techniques, microscopy and spectroscopy.A model is presented for analyzing the SAXS data as two di®erent stacks of lipid bilayerscorresponding to the grana and stroma lamellae, respectively. Each stack is described as aone-dimensional lattice showing disorder of the second kind, i.e. without crystalline long-range order and allowing for bending °uctuations of the bilayers. The model is ¯tted tothe data in the full q-range in order to take into account the di®use scattering appearingbetween the Bragg peaks.Small-angle X-ray scattering reveals two scattering bands in the region 0.05 ºA¡1 - 0.2ºA¡1. Due to the small coherent scattering length of the thylakoid membrane stacks, theoverall shape of the scattering bands is mainly determined by the form factors, which inturn depend on the electron density distribution of the unit cells constituting the one-dimensional lattices. The analysis shows that application of osmotic pressure results in adecrease of the lumen distance in particular, along with a decrease in the inter-thylakoidaldistance and a minor increase in the membrane thickness.Prolonged, 15 min pre-illumination in strong light is shown only to have minor e®ectson the characteristic distances, though there seems to be a tendency towards 5 - 10 %increased repeat distances. The increase in the repeat distances is shown to be a jointe®ect of increased lumen and inter-thylakoidal spaces. However, the changes show an ir-iregular dependency on the osmolarity and signi¯cant variations among di®erent isolations.The SANS data allow a direct observation of the ¯rst order Bragg peak of the stromalamellae. Raising the osmotic pressure of the medium results in a decrease of the repeatdistance of the stroma lamellae. The depletion of salts from the medium leads to increasedequatorial scattering, corresponding to a more °exible membrane alignment. Moreover,the meridional peaks are slightly displaced towards smaller q-values, indicating that thedepletion is followed by increased repeat distances of the stroma lamellar stacks.Under the in°uence of prolonged, 15 min pre-illumination in strong light there is an irre-versible loosening of the stroma lamellae with a relative increase in the repeat distance of4 - 8 %, depending on the osmolarity of the medium. In contrast, short periods of on-lineillumination leads to a decrease in the stroma lamellar repeat distance. Dark adaptationfollowing the short light treatments shows that the structural changes are reversible.Parallel studies using circular dichroism spectroscopy show that the structure and struc-tural °exibility of chirally organized macroaggregates of protein complexes found in thegrana are intimately coupled to the stacking and °exibility of the grana thylakoid mem-branes. In particular, the main chlorophyll a/b light harvesting complex, LHCII, is shownto play an important role in the structural dynamics of the grana thylakoid membranes.In contrast to the indirect structural information obtained from the scattering and spec-troscopy studies, the techniques of X-ray microscopy and light microscopy provide theopportunity to obtain real-space images of thylakoid membranes. These images providefurther insight into the morphological changes induced under di®erent environmental con-ditions.