Physicochemical properties of cloned nucleocapsid parotein from HIV. Interactions with metal ions
- Yale Univ., New Haven, CT (United States)
The nucleocapsid (NC) protein (p15) of the human immunodeficiency virus (HIV) has been cloned and overproduced (under the control of a phage T7 promoter) in soluble form in an Escherichia coli host. The soluble NC protein is a fusion protein containing 15 amino acids from the T7 gene 10 and 7 amino acids from the HIV p24 protein at the N-terminus to make a protein of 171 amino acids. The plasmid containing the fusion gene is designated p15DF. A homogeneous product has been isolated from the induced cells and, when isolated under aerobic conditions, contains 0.3-0.5 mol of Zn/mol of protein and has only 2 titratable SH groups. Reduction and refolding in the presence of Zn(II) yields a protein containing 2.0 mol of Zn/mol of protein and 6 titratable SH groups. On the other hand, if the cells are sonicated in 2 mM CdCl{sub 2} and purified at pH 5.0, an unoxidized protein containing 2 mol of Cd/mol of protein is obtained. The Cd(II) ions can be exchanged with Zn(II), Co(II), or {sup 113}Cd(II). {sup 113}CdNMR of the {sup 113}Cd(II){sub 2}NC protein shows two {sup 113}Cd NMR signals at 659 and 640 ppm, respectively, each integrating to {approximately} Cd(II) ion. The downfield chemical shifts suggest coordination of each {sup 113}Cd(II) ion to 3 sulfur donor atoms. The spectroscopic data fully support the prediction that the NC protein binds metal ions to each of the tandem repeats of the -Cys-X{sub 2}-Cys-X{sub 4}-His-X{sub 4}-Cys-sequence contained in the N-terminal half of the molecule. {sup 113}Cd NMR shows, however, that the sites are not identical.
- OSTI ID:
- 5081318
- Journal Information:
- Biochemistry; (United States), Vol. 30:21; ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
NUCLEOPROTEINS
NUCLEAR MAGNETIC RESONANCE
AIDS VIRUS
AMINO ACID SEQUENCE
CADMIUM 113
CADMIUM CHLORIDES
NMR SPECTRA
PURIFICATION
ULTRAVIOLET SPECTRA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CADMIUM COMPOUNDS
CADMIUM HALIDES
CADMIUM ISOTOPES
CHLORIDES
CHLORINE COMPOUNDS
EVEN-ODD NUCLEI
HALIDES
HALOGEN COMPOUNDS
INTERMEDIATE MASS NUCLEI
INTERNAL CONVERSION RADIOISOTOPES
ISOMERIC TRANSITION ISOTOPES
ISOTOPES
MAGNETIC RESONANCE
MICROORGANISMS
MOLECULAR STRUCTURE
NUCLEI
ORGANIC COMPOUNDS
PARASITES
PROTEINS
RADIOISOTOPES
RESONANCE
SPECTRA
STABLE ISOTOPES
VIRUSES
YEARS LIVING RADIOISOTOPES
550201* - Biochemistry- Tracer Techniques