Structural Features of a Translocation-Competent Chloroplast Precursor Protein

Transit sequence‐dependent binding of the chloroplast precursor protein ferredoxin to lipid vesicles and its implications for membrane stability

“…The purity of the peptides was estimated to be over 98% as determined by analytical HPLC. The identity of the peptides was confirmed by N-terminal sequencing of 20 amino acids by Edman degradation according to [16], by quantitative amino acid analysis [17] and for the presequence also by mass spectroscopy. The peptides were stored as dry materials under nitrogen at -20°C.…”

“…The precursor was stored in small aliquots in 25 mM Tris-HC1, pH 7.5, 8 M urea and 0.02% (v/v) fl-mercaptoethanol at -20°C at concentrations ranging between 1-1.5 mg/ml. Apofd was prepared as described [17] and stored in 1 M Tris buffer at -20°C at a concentration of 1 mg/ml. Protein concentrations were determined according to Bradford [21] with BSA as reference.…”

The secondary structure of the ferredoxin transit sequence is modulated by its interaction with negatively charged lipids

“…At present, the physical properties of transit sequences that constitute the specific targeting signal and the mechanism by which the translocation machinery decodes these degenerated signals are not understood [11]. Recently it was shown that the ferredoxin transit sequence is unstructured in an aqueous solution but possesses a certain conformational flexibility depending on the environment [12]. By inducing specific structural changes upon interaction with the envelope lipids, defined secondary and tertiary structural elements might be formed which can serve as recognizable motifs for the import machinery.…”

“…As a model peptide we have chosen the transit sequence of the well studied ferredoxin precursor. This peptide can be obtained in a state competent for interaction with the import machinery [12].…”

“…The purified peptide was dried and stored under N2 at -20°C. Peptide was quantified as described by Pilon et al [12].…”