Assignment of the contribution of the tryptophan residues to the spectroscopic and functional properties of the ribotoxin alpha-sarcin.
alpha-Sarcin, a potent cytotoxic protein from Aspergillus giganteus, contains two tryptophan residues at positions 4 and 51. Two single, W4F and W51F, and the double mutant, W4/51F, have been produced and purified to homogeneity. These two residues are neither required for the highly specific ribonucleolytic activity of the protein on the ribosomes (production of the so called alpha-fragment) nor for its interaction with lipid membranes (aggregation and fusion of vesicles), although the mutant forms involving Trp-51 show a decreased ribonuclease activity. Proton NMR data reveal that no significant changes in the global structure of the enzyme occur upon replacement of Trp-51 by Phe. Substitution of each Trp residue results in a 4 degrees C drop in the thermal denaturation midpoint, and the double mutant's midpoint is 9 degrees C lower. Trp-51 is responsible for most of the near-UV circular dichroism of the protein and also contributes to the overall ellipticity of the protein in the peptide bond region. Trp-51 does not show fluorescence emission. The membrane-bound proteins undergo a thermal denaturation at a lower temperature than the corresponding free forms. The interaction of the protein with phospholipid bilayers promotes a large increase of the quantum yield of Trp-51 and its fluorescence emission is quenched by anthracene incorporated into the hydrophobic region of such bilayers. This indicates that the region around this residue is located in the hydrophobic core of the bilayer following protein-vesicle interaction.
de Antonio
C
C
Departamento de Bioquímica y Biología Molecular, Facultad de Química, Universidad Complutense, Madrid, Spain.
Martínez del Pozo
A
A
Mancheño
J M
JM
Oñaderra
M
M
Lacadena
J
J
Martínez-Ruiz
A
A
Pérez-Cañadillas
J M
JM
Bruix
M
M
Gavilanes
J G
JG
D000873:Anthracenes / Q000494:pharmacology; D002942:Circular Dichroism; D003603:Cytotoxins / Q000737:chemistry* / Q000235:genetics; D004722:Endoribonucleases / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D005454:Fluorescence Polarization; D005656:Fungal Proteins; D006358:Hot Temperature; D008958:Models, Molecular; D015335:Molecular Probes; D009154:Mutation; D009183:Mycotoxins / Q000737:chemistry / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D010715:Phosphatidylglycerols; D011487:Protein Conformation; D011489:Protein Denaturation; D011500:Protein Synthesis Inhibitors / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan / Q000737:chemistry*
D000873:Anthracenes; D003603:Cytotoxins; D005656:Fungal Proteins; D015335:Molecular Probes; D009183:Mycotoxins; D010715:Phosphatidylglycerols; D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins; C014801:alpha-sarcin; D014364:Tryptophan; C002773:dimyristoylphosphatidylglycerol; D004722:Endoribonucleases; C034020:anthracene