Circular dichroism and fluorescence spectroscopic properties of the major core protein of feline immunodeficiency virus and its tryptophan mutants - Assignment of the individual contribution of the aromatic side chains

The gene coding for the major capsid protein of feline immunodeficiency vir us (FIV) has been cloned into the expression vector pQE60, which allows pro tein purification by affinity chromatography on a nitrilotriacetic acid/Ni/ agarose column. The gene was expressed in Escherichia coli and the resultan t soluble protein (FIV-rp24) purified to electrophoretic homogeneity. The a mino-acid composition of the recombinant protein is almost identical to tha t predicted from the DNA sequence. This protein has two tryptophan residues at positions 40 and 126 that have been replaced by phenylalanine by site-d irected mutagenesis to obtain two single mutants and a double mutant. Circu lar dichroism and fluorescence spectroscopy were employed to study the stru ctural features of FIV-rp24 protein and its tryptophan mutants. The analysi s of the CD spectra indicated that or-helix is the major secondary structur al element (48-52%) and that the overall three-dimensional structure is not modified by the mutations. The fluorescence emission spectra showed that b oth tryptophan residues occupy a highly hydrophobic environment. Moreover, the different tyrosine fluorescence intensities of wild-type and mutant pro teins are indicative of the existence of resonance energy transfer processe s to nearby tryptophan. The individual contributions of each tryptophan res idue to the spectroscopic properties of the wild-type protein were obtained from the spectra of all these proteins. Thermal denaturation studies indic ate that the two tryptophan residues do not contribute equally to the stabi lization of the three-dimensional structure.