The role of the C-terminal region of pulchellin A-chain in the interaction with membrane model systems

• Published in: Biochimica et biophysica acta 2012-01, Vol.1818 (1), p.82-89
• Main Authors: Reyes, Luis Fernando, Nobre, Thatyane M., Pavinatto, Felippe J., Zaniquelli, Maria E.D., Caseli, Luciano, Oliveira, Osvaldo N., Araújo, Ana Paula U.
• Format: Article
• Language: English
• Subjects: Abrus - chemistry; Amino Acid Sequence; Cloning, Molecular; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Hydrophobic and Hydrophilic Interactions; Langmuir monolayers; Membrane interaction; Models, Molecular; Molecular Sequence Data; Phosphatidylglycerols - chemistry; Phosphatidylglycerols - metabolism; Plant Proteins - chemistry; Plant Proteins - genetics; Plant Proteins - metabolism; Plasmids; PM-IRRAS; Protein Structure, Tertiary; Protein Subunits - chemistry; Protein Subunits - genetics; Protein Subunits - metabolism; Pulchellin; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Ribosome inactivating protein; Ribosome Inactivating Proteins - chemistry; Ribosome Inactivating Proteins - genetics; Ribosome Inactivating Proteins - metabolism; Spectrophotometry, Infrared; Surface Properties; Toxins, Biological - chemistry; Toxins, Biological - genetics; Toxins, Biological - metabolism; Unilamellar Liposomes - chemistry; Unilamellar Liposomes - metabolism
• ISSN: 0005-2736; 0006-3002; 1879-2642
• DOI: 10.1016/j.bbamem.2011.10.002

Pulchellin is a Ribosome Inactivating Protein containing an A-chain (PAC), whose toxic activity requires crossing the endoplasmic reticulum (ER) membrane. In this paper, we investigate the interaction between recombinant PAC (rPAC) and Langmuir monolayers of dipalmitoyl phosphatidyl glycerol (DPPG), which served as membrane model. Three catalytically active, truncated PACs with increasing deletion of the C-terminal region, possessing 244, 239 and 236 residues (rPAC244, rPAC239 and rPAC236), were studied. rPAC had the strongest interaction with the DPPG monolayer, inducing a large expansion in its surface pressure–area isotherm. The affinity to DPPG decreased with increased deletion of the C-terminal region. When the C-terminal region was deleted completely (rPAC236), the interaction was recovered, probably because other hydrophobic regions were exposed to the membrane. Using Polarization Modulated-Infrared Reflection Absorption Spectroscopy (PM-IRRAS) we observed that at a bare air/water interface rPAC comprised mainly α-helix structures, the C-terminal region had unordered structures when interacting with DPPG. For rPAC236 the α-helices were preserved even in the presence of DPPG. These results confirm the importance of the C-terminal region for PAC-ER membrane interaction. The partial unfolding only with preserved C-terminal appears a key step for the protein to reach the cytosol and develop its toxic activity. ► We investigated the interaction between recombinant PAC and Langmuir monolayers. ► Three catalytically active, truncated PACs with increasing deletion of the C-terminal region were studied. ► The affinity to DPPG decreased with increased deletion of the C-terminal region. ► Using PM-IRRAS we confirm the importance of the C-terminal region for PAC-membrane interaction. ► We consider this interaction fundamental for the protein to reach the cytosol and develop its toxic activity.