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 sidechains.
The gene coding for the major capsid protein of feline immunodeficiency virus (FIV) has been cloned into the expression vector pQE60, which allows protein purification by affinity chromatography on a nitrilotriacetic acid/Ni/agarose column. The gene was expressed in Escherichia coli and the resultant soluble protein (FIV-rp24) purified to electrophoretic homogeneity. The amino-acid composition of the recombinant protein is almost identical to that predicted from the DNA sequence. This protein has two tryptophan residues at positions 40 and 126 that have been replaced by phenylalanine by site-directed mutagenesis to obtain two single mutants and a double mutant. Circular dichroism and fluorescence spectroscopy were employed to study the structural features of FIV-rp24 protein and its tryptophan mutants. The analysis of the CD spectra indicated that alpha-helix is the major secondary structural element (48-52%) and that the overall three-dimensional structure is not modified by the mutations. The fluorescence emission spectra showed that both tryptophan residues occupy a highly hydrophobic environment. Moreover, the different tyrosine fluorescence intensities of wild-type and mutant proteins are indicative of the existence of resonance energy transfer processes to nearby tryptophan. The individual contributions of each tryptophan residue to the spectroscopic properties of the wild-type protein were obtained from the spectra of all these proteins. Thermal denaturation studies indicate that the two tryptophan residues do not contribute equally to the stabilization of the three-dimensional structure.
Yélamos
B
B
Departamento de Bioquímica y Biología Molecular I, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, Spain.
Núñez
E
E
Gómez-Gutiérrez
J
J
Datta
M
M
Pacheco
B
B
Peterson
D L
DL
Gavilanes
F
F
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002415:Cats; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006358:Hot Temperature; D016089:Immunodeficiency Virus, Feline / Q000737:chemistry* / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics; D014758:Viral Core Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification
D017931:DNA Primers; D011994:Recombinant Proteins; D014758:Viral Core Proteins; D014364:Tryptophan