Figure 4

Topology and secondary structure of Tic20. (A) PEG-Mal labelling of Pisum sativum (Ps) inner envelope (IE) vesicles in the presence or absence of 1% SDS for the indicated times using a specific antibody against PsTic20 for immunodetection. Asterisks indicate a weak band most likely representing Tic20 with one labelled cystein (Cys) within the transmembrane region. A representative result of three repetitions is shown. (B) Topological model of Tic20 - indicating the position of Cys residues in PsTic20 - considering the PEGylation assay in (A) (based on structural prediction of TMHMM Server [23] and [25]). Boxes symbolise α-helical transmembrane domains (TM 1-4). IMS - intermembrane space. (C) The mature parts of Tic20 from Pisum sativum (PsTic20, amino acids 83-253) and Arabidopsis thaliana (AtTic20 amino acids 59-274) were overexpressed in an E.coli cell lysate system and in E.coli BL21 cells, respectively. Both proteins were purified by Ni²⁺-affinity chromatography. Coomassie-stained gels of representative purifications are shown. (D) Circular dichroism spectrum of overexpressed and purified PsTic20 in 20 mM Na-phosphate buffer (pH 8.0), 150 mM NaF, 0.8% Brij-35. The presented chromatogram is the average of three independent experiments. Secondary structure elements were quantified using the CDSSTR method from the DichroWeb server and results are presented in the inset.