Additional file 5: of Two-component cyclase opsins of green algae are ATP-dependent and light-inhibited guanylyl cyclases

Figure S5. Comparison of different Cr2c-Cyclop1 constructs. A. Schematic models of Cr2c-Cyclop1. Cr2c-Cyclop1.fl is the full length sequence from JGI database. Cr2c-Cyclop1.s is the sequence cloned from cDNA with deletions of a short middle part and C-terminal. Cr2c-Cyclop1.sc is an artificial sequence with a deletion of the C-terminal. Cr2c-Cyclop1.sm sc is an artificial sequence with a deletion of the short middle sequence. The gray dashed lines indicate the deleted regions. Four conserved domains are labeled with different colors. Blue, opsin domain; red (His-Kinase), histidine kinase domain; green (RR), response regulator domain; purple (GC), guanylyl cyclase domain. B. Comparison of dark and light (532 nm, ~ 20 μW/mm2) activities of four different constructs with different lengths; activities in the dark and light came from one oocyte membrane. Approximately thirty nanograms of cRNA were injected for all constructs, 3 dpi. n = 3–6, error bar = SD. Reaction buffer: 75 mM Tris-Cl, 10 mM NaCl, 5 mM MgCl2, 0.2 mM GTP, 0.25 mM ATP, 5 mM DTT, pH 7.3. C. The fluorescence emission values were measured for four constructs individually, 12 oocytes membrane expressing individual construct was extracted and applied for each measurement. Control values were subtracted for different samples. n = 3, error bar = SD. D. Cr2c-Cyclop1.s activity under different NaCl concentrations. Other components in the reactions are as follows: 75 mM Tris-Cl, 5 mM MgCl2, 0.2 mM GTP, 0.25 mM ATP, 5 mM DTT, pH 7.3. n = 3, error bar = SD. Samples were from two batches of oocytes. Illumination condition, 532 nm, ~ 20 μW/mm2 light. (PDF 63 kb)