Controlled Nucleation of His-tag Protein Assemblies Using Rigid Symmetric Multivalent Nitrilotriacetic acid Chelating Ligands
Abstract
In spite of recent achievements focused on accelerating the rate of protein structure determination, the rapid production of high quality crystals for X-ray crystallographic analysis is still a major impediment to the elucidation of atomically resolved protein structures. Since nucleation is an early step in protein crystallization, the development of generalized methods to control nucleation could help reduce the bottleneck in the protein crystallization process. The goal of this thesis is to develop a new approach for the crystallization of Histag soluble proteins and membrane proteins. This newly developed approach involves using rigid symmetric multivalent nitrilotriacetic acid (NTA) chelating ligands for the nucleation and speciation of His-tag proteins in solution. A library of symmetric NTAchelating ligands had been developed by Thomson and coworkers was used to speciate His-tag GFP, His-tag CPF and His-tag mCherry, in a stoichiometric manner in the presence of Ni2+. The CFP and mCherry are two mutants of GFP designed to reduce the tendency for dimerization. These protein complexes formed were characterized by blue native polyacrylamide gel electrophoresis (BN-PAGE) and analytical ultracentrifugation (AUC), and transmission electron microscopy analysis. It was anticipated that these ligands would be able to bind polyhistidine-tagged proteins in the presence of Ni 2+, control protein:ligand speciation and promote self-assembly of the protein:ligand complexes in a manner that promotes protein crystal nucleation. These studies showed that the rigid symmetric multivalent NTA chelating ligands method is a promising approach for the crystallization of His-tag proteins. Initial studies with the expression and purification of the membrane protein OmpF, and the crystallization of GFP are also described.
Degree
M.S.
Advisors
Thompson, Purdue University.
Subject Area
Biochemistry|Bioinformatics
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