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Protein structure/function studies: The avian myeloblastosis virus nucleocapsid protein

Smith, Lisa Marie

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1993, Doctor of Philosophy, Case Western Reserve University, Biochemistry.

Biophysical techniques, including plasma emission spectroscopy, fluorescence, circular dichroism, and nuclear magnetic resonance, were applied to the investigation of the structure and the histone-like nucleic acid binding function of the nucleocapsid protein from avian myeloblastosis virus. Nucleocapsid proteins are vital components of retroviruses, and the only known sequence homology among these proteins is the “Cys-His motif”: Cys-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Gly-His-Xaa-Xaa-Xaa-Xaa-Cys. The nucleocapsid protein from avian myeloblastosis virus contains two Cys-His motifs. The hypothesized role of the conserved cysteines and histidines as zinc ligands was tested by plasma emission spectroscopy. Sodium and phosphorus were the only metal ions detected in stoichiometric amounts, and the molar ratio of zinc to nucleocapsid protein was approximately 1:50 in isolated virions. Circular dichroism was used to determine the secondary structural content of the nucleocapsid protein. Addition of zinc had no effect on the circular dichroism spectrum. A fluorescence binding assay was developed and the binding constant for nucleocapsid protein binding poly(1,N6-ethenoadenylic acid) was determined to be approximately micromolar at pH 5.5 and 7.0. Slight positive cooperativity was also measured upon polynucleotide binding. Also, the addition of zinc had no effect on binding.

The nucleocapsid protein from avian myeloblastosis virus is a phosphoprotein. 1H and 31P nuclear magnetic resonance, and fluorescence polarization anisotropy, were used to investigate the impact of phosphorylation on its structure and function. Dephosphorylated nucleocapsid was structurally very similar to the native protein, and was functionally indistinguishable. Nuclear magnetic resonance was used to study the interaction of nucleocapsid protein with oligo- and mononucleotides. Subtle changes in the spectra of the protein and of the nucleotides were noted.

The correlation of proton nuclear magnetic resonance chemical shifts with secondary structure in proteins was also analyzed. Statistical analysis of a database of proton chemical shifts from proteins of known structure yielded twenty-six chemical shift windows within the 1H nuclear magnetic resonance spectrum in which the distribution of secondary structural elements is significantly skewed. Fourteen windows were populated by side chain protons. Correlation of secondary structure and chemical shift for side chain protons suggests that secondary structure imposes constraints upon side chains that are evident in their proton chemical shifts.

n/a n/a (Advisor)
198 p.

Recommended Citations

Citations

  • Smith, L. M. (1993). Protein structure/function studies: The avian myeloblastosis virus nucleocapsid protein [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1056737167

    APA Style (7th edition)

  • Smith, Lisa. Protein structure/function studies: The avian myeloblastosis virus nucleocapsid protein. 1993. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1056737167.

    MLA Style (8th edition)

  • Smith, Lisa. "Protein structure/function studies: The avian myeloblastosis virus nucleocapsid protein." Doctoral dissertation, Case Western Reserve University, 1993. http://rave.ohiolink.edu/etdc/view?acc_num=case1056737167

    Chicago Manual of Style (17th edition)