The role of the C-terminal region of pulchellin A-chain in the interaction with membrane model systems.
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 (rPAC(244), rPAC(239) and rPAC(236)), 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 (rPAC(236)), 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 rPAC(236) 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.
Centro de Biotecnologia Molecular Estrutural, Instituto de Física de São Carlos, Universidade de São Paulo, PO Box 369, 13560-970 São Carlos, SP, Brazil.
Luis Fernando
LF
Reyes
Thatyane M
TM
Nobre
Felippe J
FJ
Pavinatto
Maria E D
ME
Zaniquelli
Luciano
L
Caseli
Osvaldo N
ON
Oliveira
Ana Paula U
AP
Araújo
D029912:Abrus / Q000737:chemistry; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010715:Phosphatidylglycerols / Q000737:chemistry / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054788:Ribosome Inactivating Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013055:Spectrophotometry, Infrared; D013499:Surface Properties; D014118:Toxins, Biological / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D053835:Unilamellar Liposomes / Q000737:chemistry / Q000378:metabolism
D010715:Phosphatidylglycerols; D010940:Plant Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D014118:Toxins, Biological; D053835:Unilamellar Liposomes; D054788:Ribosome Inactivating Proteins; C030345:1,2-dipalmitoylphosphatidylglycerol