The experiments were repeated five times and resulted in very similar differences in the CD spectra and their thermal behavior The thermal destabilization of different protein complexes was monitored via the amplitudes of their corresponding CD bands. The (−)650 nm band exhibited

the same temperature dependence for WT and dgd1 and INK1197 cell line displayed essentially identical transition temperatures (T m) at ~60°C (Table 1). On the other hand, the mutation substantially affected the thermal stability of the Chl a excitonic bands at around 450 nm, determined either as CD(448–438) (not shown) or CD(448–459) (Fig. 1b). The T m values were lower by ~6°C for the mutant than for the WT (Table 1). The Ψ-type signal (CD(685–730)) also exhibited different temperature dependencies for WT and dgd1 (Fig. 1c). The transition temperature for this band was 54 ± 2°C for the WT, whereas for dgd1 it was found at 48 ± 1°C (Table 1). Table 1 Transition temperatures (T m) of selected CD bands or band pairs for WT and dgd1 thylakoid membranes

CD signal (nm) Assignment T m′ °C (WT) T m′ °C (dgd1) 685–730 Ψ-type 54 ± 2 48 ± 1 685–671 Ψ-type 54 ± 1 49 ± 1 505–550 Ψ-type 56 ± 1 51 ± 1 610–650 Excitonic (Chl b, LHCII) 61 ± 2 58 ± 2 448–459 Excitonic (Chl a) 59 ± 2 54 ± 1 448–438 Excitonic (Chl a) 57 ± 1 50 ± 1 The membranes were thermostated for 10 min at different temperatures in the range between 5 and 80°C before Tryptophan synthase recording the CD spectra at the given temperature; Survivin inhibitor the amplitudes for the individual bands were calculated from the difference in the intensity at see more specific

wavelengths (see also the text). T m is defined as the temperature at which the intensity of the CD band is decreased to 50% of its value at 25°C. The values for T m and their standard errors are determined from five independent experiments Green (native) gel electrophoresis In order to discriminate between the thermal behavior of the different photosynthetic complexes, green gel electrophoresis of heat-treated thylakoid membranes from WT and dgd1 was performed (Fig. 2a) and analyzed for the contents of PSI supercomplexes (Fig. 2b) and LHCII trimers (Fig. 2c). The data show that the PSI supercomplex in dgd1 is less stable upon heat treatment than the WT—the intensity of the corresponding green gel band decreases by 50% at 57°C for dgd1 and at 61°C for WT, respectively (Fig. 2b). In contrast, the destabilization of LHCII trimers follows the same pattern in both the WT and dgd1 up to 65°C (Fig. 2c). Fig. 2 a Native green gel analysis of heat-treated WT and dgd1 thylakoid membranes at different temperatures. The samples are treated for 10 min before loading on the gel. The main bands denoted as I and II represent PSI supercomplex and LHCII trimers, respectively.