Development of NTA-modified ligands and non-covalent cyclodextrin-based templates for crystallization of histidine-tagged proteins
Abstract
The goal of this dissertation is the development of two new approaches for the crystallization of histidine-tagged proteins. The first approach involves using novel NTA-based ligands with cyclodextrin (CD) templates to crystallize his8-GFP at the solid-liquid interface via a site-hopping mechanism. These ligands were used in a microwell format to crystallize his 8-GFP on both α- and β-cyclodextrin. The GFP crystals grown on the CD templates were characterized by fluorescence microscopy. The binding kinetics of the NTA-based ligands were also characterized using both fluorescence recovery after photobleaching (FRAP) and isothermal titration calorimetry (ITC) measurements. It was anticipated that this approach to two-dimensional crystallization would allow the protein to govern the protein-protein interactions so that the protein was not restricted to lateral movement along the template. This is a major improvement over current two-dimensional crystallization approaches. The second approach involves using water-soluble NTA-based nucleating agents for the speciation of histidine-tagged proteins in solution. A library of symmetric NTA-based ligands that had been previously synthesized by the Thompson group was used to speciate his8-GFP in a stoichiometric manner in the presence of nickel. The protein complexes formed were characterized by blue native polyacrylamide gel electrophoresis (BN-PAGE), high performance size exclusion chromatography, sedimentation velocity analytical ultracentrifugation, and transmission electron microscopy analyses. It was anticipated that these ligands would be able to speciate histidine-tagged proteins in a predictable stoichiometric manner. These studies showed that the non-covalent cyclodextrin template and water soluble nucleating agents are two promising approaches for the crystallization of histidine-tagged proteins.
Degree
Ph.D.
Advisors
Thompson, Purdue University.
Subject Area
Analytical chemistry|Biochemistry
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