Conformational and nucleic acid binding studies on the synthetic nucleocapsid protein of HIV-1.
A 55 residue peptide corresponding to the nucleocapsid protein of HIV-1 (NCp7) containing two zinc binding domains as well as three truncated peptides were synthesized by Fmoc-based solid phase synthesis using the fragment condensation approach. Circular dichroism (CD) data support a conformational model in trifluoroethanol/buffer solution consisting of two helical segments at the chain ends with two Zn-modules in the center of the molecule. CD titration experiments show that the synthetic protein binds two equivalents of Zn2+ stoichiometrically, and the Zn2+ induced conformational changes are completely reversible by addition of EDTA. NCp7 and its S-acetamidomethylated analog (NCp7-Acm), devoid of the zinc co-ordination centers, exhibit preferential binding to RNA with a Kd = approximately 10(-9) M irrespective of the cysteine modification as determined by filter binding assays. The binding affinity of the NCp7 protein to single-stranded DNA is lower than to RNA. Binding to double-stranded DNA is lower than to ssDNA. The NCp7-Acm protein exhibits reduced single-stranded DNA binding affinity compared to the unmodified protein. Nucleic acid binding analyses with the fragments of NCp7 protein suggest that two basic amino acid stretches are involved in RNA binding of the NCp7.
Surovoy
A
A
Shemyakin Institute of Bioorganic Chemistry, Moscow, Russia.
Dannull
J
J
Moelling
K
K
Jung
G
G
D000595:Amino Acid Sequence; D036022:Capsid Proteins; D002942:Circular Dichroism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D015683:Gene Products, gag / Q000737:chemistry / Q000378:metabolism*; D015497:HIV-1 / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D011487:Protein Conformation; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D014764:Viral Proteins; D015032:Zinc / Q000378:metabolism; D016335:Zinc Fingers / Q000502:physiology; D054301:gag Gene Products, Human Immunodeficiency Virus
D036022:Capsid Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D015683:Gene Products, gag; C071884:NCP7 protein, Human immunodeficiency virus 1; D016601:RNA-Binding Proteins; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; D015032:Zinc