Date of Award

Spring 1-1-2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology, Molecular Biology and Biochemistry

First Advisor

Mark Winey

Second Advisor

Andreas Hoenger

Third Advisor

Jennifer M. Martin

Fourth Advisor

Michael H. Stowell

Fifth Advisor

Dylan J. Taatjes

Abstract

Structural biology is a discipline in biology that focuses on the relationship between structure and function in macromolecules, nucleic acids and lipids. In the studies presented in this thesis, the structure and function of three different proteins were investigated by using techniques from molecular biology, biochemistry, and biophysics: bacterial aspartate chemoreceptor (Tar), EBV's latent membrane protein-1 (LMP-1), and the kinesin-14 Kar3Cik1.

In the first part I was using molecular modeling to identify residues that are involved in coiled-coil interactions of the 4-helix bundle of the Tar chemoreceptor in S. typhimurium. The receptor was mutated at distinct residues that perturb the helix-helix interactions and analyzed for function. My studies found that by weakening the helical interactions in the so-called protein interaction region of the receptor, the receptor remained mostly in its on-state. Whereas weakening helical interactions in the so-called adaptation region of the chemoreceptor favored the receptor's off-state. These results may be evidence for a Ying-Yang relationship between the chemoreceptor's adaptation and protein interaction region. Signal transduction through the chemoreceptor may be a result of changes in the helical interactions caused by the binding of ligand.

The second part of my thesis dealt with the oncogenic Barr-Epstein virus membrane protein LMP-1 that affects B-cells in humans and is associated with several malignancies. LMP-1's main role is to mimic the signal produced by CD40, but LMP-1 is also involved in several other signaling pathways. It is unknown whether LMP-1 functions in a signaling complex with multiple outputs or in multiple complexes with unique signaling outputs. To probe this question, size exclusion chromatography was used to fractionate cellular lysates of EBV positive cells and they were co- immunoprecipitated for LMP-1. The Co-IP samples were sent for Multidimensional Protein Identification Technology (MudPIT) analysis to identify LMP-1 associated signaling partners.

Kinesin motors are involved in many cellular functions in eukaryotes. In a simple eukaryote like budding yeast, kinesins are involved in 3 main functions: nuclear migration, chromosomal segregation, and karyogamy. Here we investigated Kar3Cik1, a heterodimeric member of the kinesin-14 family. Kar3Cik1 is involved in mating and mitotic cells, but is not essential for mitosis. Using cryo-EM and helical 3-D image reconstruction techniques I was able to determine Kar3Cik1's binding configurations in complex with microtubules in the ADP, nucleotide-free, and ATP states. This provides information as to Kar3Cik1's mechanism of movement along microtubules.

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