Biophysical characterization of the HMG-1 Box domain of the lymphoid enhancer binding factor-1

The primary focus of this work is the biophysical characterization of the HMG-1 Box DNA binding domain of the Lymphoid Enhancer Binding Factor-1 (LEF-1). NMR was the primary tool used in the elucidation of the structure of the LEF-1 HMG-1 Box domain in complex with a 15 base-pair molecule of DNA.;Chapter 1 provides (1) an overview of the HMG family of proteins focusing on their common functionality as architectural components of chromatin, (2) background on the components of the system of interest, namely LEF-1 and its HMG-1 Box DNA binding domain and (3) illustrative descriptions and examples of how nature has molded the versatile HMG-1 Box domain to create a repertoire of proteins which possess distinct and unique transcriptional activation capabilities.;Chapter II provides a description of the molecular biology experiments used to delineate the minimal LEF-1 DNA binding site. It also contains a description of the means by which the LEF-1 domain and the various DNA molecules were expressed, synthesized and purified.;Chapter III describes the NMR experiments used to (1) titrate the LEF-1 domain onto DNA, (2) obtain all the protein and DNA resonance assignments and (3) obtain the intra- and inter-molecular restraints. Also described are the means by which these restraints were used to independently calculate the structures of the protein and DNA and the computational methods used to dock the protein onto the DNA.;Chapter IV provides images and detailed physical descriptions of the LEF-1 domain/DNA structures. Comparison to structures of other BMG-1 box domains enabled us to determine the origins of LEF-1 sequence specificity. The structures also illustrate how the LEF-1 domain induces gross perturbations in the DNA structure.;Finally, chapter V provides descriptions of the biophysical characterization of the LEF-1 domain free in solution. Circular dichroism was used to obtain global secondary structure information on the free domain while NMR was used to obtain residue specific structural information. The results indicate that, prior to binding and severely bending DNA, the LEF-1 domain exists as a dynamic and disordered molecule.