A binding shift assay for the zinc-bound and zinc-free HIV-1 nucleocapsid protein by capillary electrophoresis.
Affinity capillary electrophoresis was used to detect a shift in mobility when a zinc ion binds to the highly basic nucleocapsid protein (NCp7) of HIV-1. NCp7 contains two Cys-X2- Cys-X4-His-X4-Cys zinc fingers. With constant concentrations of NCp7 as a receptor and various concentrations of zinc as a ligand in the sample buffer and the electrophoresis buffer, we observed changes in electrophoretic mobilities of NCp7 protein when complexes were formed with zinc. Scatchard analysis of the mobility indicates the presence of at least two types of binding sites for zinc. At pH 6.0, one site is shown to bind zinc strongly with a binding constant Kb = 3.25 x 10(5) M-1 and the second site has a Kb = 1.8 x 10(5) M-1. The binding of zinc to the first zinc finger decreased the affinity of zinc for the second zinc finger approximately twofold. The Hill coefficient for this negative cooperativity is 0.9. A series of NCp7 mutants were also examined in the assay to determine their ability to bind zinc. This assay affords a quick method to observe a zinc ion binding to NCp7 and to calculate binding constants.
Guszczynski
T
T
ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland, 21702-1201, USA.
Copeland
T D
TD
D000595:Amino Acid Sequence; D002213:Capsid / Q000737:chemistry* / Q000378:metabolism; D036022:Capsid Proteins; D019075:Electrophoresis, Capillary / Q000379:methods; D015683:Gene Products, gag / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D015497:HIV-1 / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D019251:Nucleocapsid / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D017354:Point Mutation; D014764:Viral Proteins; D015032:Zinc / Q000378:metabolism; D016335:Zinc Fingers; D054301:gag Gene Products, Human Immunodeficiency Virus
D036022:Capsid Proteins; D015683:Gene Products, gag; C071884:NCP7 protein, Human immunodeficiency virus 1; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; D015032:Zinc