Adeno-associated virus type-2 (AAV-2) is a leading candidate vector for gene therapy. My PhD is a part of the ongoing research project towards the long-term goals of engineering AAV to evade immune neutralization when repeated doses are required, and of modulating the specificity of cellular targeting. The specific aim of my PhD research is the structural analysis of AAV-2 using X-ray crystallography and cryo-Electron Microscopy (cryo-EM). The two parts of this thesis follow my participation in the crystallographic structure determination of AAV-2 at 3Å resolution. In the first part, cryo-EM has been used to visualize the wild type AAV-2 (wt-AAV2) and its structure determined using the principles of icosahedral three-dimensional reconstruction. A difference map has been calculated by subtracting the atomic structure of the major component, viral protein 3 (VP3), from theEM density of the wt-AAV2. The results indicate the probable location of the viral nucleic acid and the other minor viral proteins, VP1 and VP2, not imaged in the crystallographic structure. This research lays the foundation for the future studies of AAV-2-antibody complexes that will be studied by the same techniques. The second part of the research involves the analysis of crystal contacts of the virus in the crystallographic unit cell. Surface features such as molecular shape and chemical character determine interactions of a molecule with its surroundings in vivo or in crystalline forms. Amino acids involved in particle interactions have been identified from buried surface area calculations. This research addresses the question of what drives a virus to choose one packing scheme over another and whether the crystal contacts carry any biological information. The residues buried in inter-particle contacts play a stabilizing role in the packing and are often a part of a surface with demonstrated biological function such as immunogenic stimulation and receptor recognition. The results of this research will form the groundwork for the study of virus-receptor and virus-antibody complexes. / A Dissertation Submitted to the Institute of Molecular Biophysics in Partial
FulfiLlment of the Requirements for the Degree of Doctor of Philosophy. / Fall Semester, 2003. / July 15, 2003. / Molecular Packing, 3D Reconstruction, Virus Structure, Cryo-Em, AAV-2 / Includes bibliographical references. / Michael S. Chapman, Professor Directing Dissertation; Robert H. Reeves, Outside Committee Member; Kenneth A. Taylor, Committee Member; Timothy A. Cross, Committee Member; Nancy L. Greenbaum, Committee Member.
| Identifer | oai:union.ndltd.org:fsu.edu/oai:fsu.digital.flvc.org:fsu_175930 |
| Contributors | Bhatia, Smita (authoraut), Chapman, Michael S. (professor directing dissertation), Reeves, Robert H. (outside committee member), Taylor, Kenneth A. (committee member), Cross, Timothy A. (committee member), Greenbaum, Nancy L. (committee member), Program in Molecular Biophysics (degree granting department), Florida State University (degree granting institution) |
| Publisher | Florida State University, Florida State University |
| Source Sets | Florida State University |
| Language | English, English |
| Detected Language | English |
| Type | Text, text |
| Format | 1 online resource, computer, application/pdf |
| Rights | This Item is protected by copyright and/or related rights. You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s). The copyright in theses and dissertations completed at Florida State University is held by the students who author them. |
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