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, conta ins 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 homogen eity, These two residues are neither required for the highly specific ribon ucleolytic activity of the protein on the ribosomes (production of the so c alled alpha -fragment) nor for its interaction with lipid membranes (aggreg ation and fusion of vesicles), although the mutant forms involving Trp-51 s how a decreased ribonuclease activity. Proton NMR data reveal that no signi ficant 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 degreesC drop in the thermal denaturation midpoint, and the double mutant's midpoin t is 9 degreesC lower. Trp-51 is responsible for most of the near-UV circul ar dichroism of the protein and also contributes to the overall ellipticity of the protein in the peptide bond region. Trp-51 does not show fluorescen ce emission,The membrane-bound proteins undergo a thermal denaturation at a lower temperature than the corresponding free forms. The interaction of th e protein with phospholipid bilayers promotes a large increase of the quant um yield of Trp-51 and its fluorescence emission is quenched by anthracene incorporated into the hydrophobic region of such bilayers, This indicates t hat the region around this residue is located in the hydrophobic core of th e bilayer following protein-vesicle interaction, (C) 2000 Wiley-Liss, Inc.