Purified promyelocytic leukemia coiled-coil aggregates as a tetramer displaying low alpha-helical content.
The promyelocytic leukemia (PML) gene is involved in the 15/17 chromosomal translocation of acute promyelocytic leukemia (APL). It encodes a nuclear phosphoprotein containing an alpha-helical coiled-coil domain with four heptad repeats. The heptad repeats consist of four clusters of hydrophobic amino acids that mediate in vivo the complex formation between PML and other PML molecules or PML-RARalpha mutant protein. In this report, we show the production of PML coiled-coil (fragment 223-360) as a fusion protein, its solubilization by the combined action of two different detergents, and its purification with affinity chromatography after column proteolytic cleavage. The FPLC chromatograms of the purified coiled-coils, carried out under non-denaturing conditions, show that the peptide elutes only in the presence of Sarkosyl detergent (conc. 0.1%) and, under these conditions, elutes as a tetrameric complex. This confirms the evidence from in vivo experiments that this region is responsible for protein complex formation. The HPLC analyses show the presence of a single peak eluting under highly hydrophobic conditions, indicating the high hydrophobicity of the peptide in accordance with the primary sequence analysis. Finally, the purified peptide was structurally characterized by means of circular dichroism (CD) measurements that were carried out with low Sarkosyl concentration (0.003%). The CD spectra indicate a low alpha-helical content (13.5%) with respect to predictions based on the primary sequence analysis (PSI-PRED, SS-PRO, and J-PRED), suggesting that the alpha-helix content could be modulated by coiled-coil surrounding domains and/or by other post-translational modifications, even if the effect of the Sarkosyl on the peptide secondary structure cannot be excluded.
Department of Internal Medicine, Applied Biochemistry and Clinical Chemistry section, Via del Giochetto s.n.c., 06100 Perugia, Italy. francesco.antolini@brindisi.enea.it
Francesco
F
Antolini
Mario
M
Lo Bello
Marco
M
Sette
D000595:Amino Acid Sequence; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D009687:Nuclear Proteins; D010455:Peptides / Q000737:chemistry; D000071156:Promyelocytic Leukemia Protein; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D013997:Time Factors; D014157:Transcription Factors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D025521:Tumor Suppressor Proteins
D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D010455:Peptides; D000071156:Promyelocytic Leukemia Protein; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C070576:PML protein, human; D005982:Glutathione Transferase