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Biophysical characterization of gold nanocrystal protein conjugates: Formation, stoichiometry and function

By Michelle K. Calabretta

Abstract

The successful application of bio/nano materials in medicine, materials science, and molecular electronics is dependent on the development of functional, well-defined hybrid materials. In this thesis, I show that the overall structure and function of protein-gold nanocrystal conjugates is influenced by protein surface charge, stoichiometry, and orientation on the nanostructure. By comparing the non-specific conjugation behavior of the lac Repressor (LacI), lysozyme, and alpha-lactalbumin, we establish that basic regions are significantly involved in the assembly of bio/nano conjugates. Super structures, such as controlled nanocrystal aggregates, can result from non-specific protein conjugation depending on the number of basic regions on the protein surface. Moreover, proteins with basic functional domains, like the DNA binding domain of LacI, pose a challenge because non-specific conjugation through these regions adversely affects biofunction. This obstacle can be avoided by specifically conjugating proteins through regions not significantly involved in function. In order to prevent conjugation through the LacI DNA binding domain, we developed a mutant with solvent exposed cysteine residues to direct conjugation to gold nanocrystals through a gold-sulfur bond. The formation and stoichiometry of LacI- and T334C-gold nanocrystal conjugates was followed by protein radiolabeling and analytical ultracentrifugation, two solution techniques that circumvent the challenges associated with spectroscopic characterization of bio/nano conjugates. These techniques provided additional confirmation that LacI conjugates through a weaker, reversible electrostatic interaction, whereas T334C conjugates are more robust, in agreement with the prediction that T334C conjugates through a non-reversible gold-sulfur bond. Lastly, the operator DNA binding function of these conjugates was assessed with nitrocellulose filter binding, analytical ultracentrifugation, and electrophoretic mobility shift. Interestingly, the order of DNA-repressor-nanocrystal complex formation had an impact on operator binding. Regardless of the order of complex formation, LacI conjugates retain little to no DNA binding function. T334C, however, retains significant operator binding if the operator-repressor complex is formed prior to conjugation. These results demonstrate that specific conjugation through regions of low functional significance can greatly improve the biofunction of conjugated proteins. This thesis provides an improved understanding of the interaction between biomolecules and nanostructures, which will benefit the design of materials that are structurally and functionally sound

Topics: Biochemistry, Biophysics
Year: 2006
OAI identifier: oai:scholarship.rice.edu:1911/18878
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