Design, synthesis and performance of cyclodextrin-based noncovalent templates for crystallization of histidine -tagged proteins

Mingkang Zhou, Purdue University

Abstract

The goal of this thesis is the development of a potential solution to 2-D crystallization of proteins using non-covalent NTA-based host:guest amphiphiles. These novel amphiphiles were used at the solid-liquid interface to crystallize his6-GFP via a site hopping mechanism instead of the previous methods that use covalently bound affinity ligands which function solely through lipid lateral diffusion. By creating highly symmetric scaffolds based on reversible protein-scaffold interactions, it was anticipated that protein-protein interactions would govern assembly without imposing lateral diffusion kinetic limitations of the protein-lipid complex. A family of amphiphilic chelating lipids possessing true or approximate C3-, C4-, C6- and C8-fold symmetric cores were developed to test this concept. β-Cyclodextrins peracylated on the 1° hydroxymethyl rim of the molecule and novel nitrilotriacetic acid-modified guest ligands were designed and synthesized to promote two-dimensional crystallization of proteins at the lipid-water interface. The self-assembly properties of these non-covalent amphiphiles were characterized. Different non-covalent amphiphile pairs were then tested for their ability to promote his6-tag GFP crystallization in the presence of Ni2+ due to interfacial templating effects using pressure-area isotherm, AFM, SPR, FRAP, confocal microscopy and FESEM methods. These studies show that the non-covalent template strategy for 2-D protein crystallization of his6-tagged proteins is a promising approach that offers great flexibility in designing interfaces that can promote protein crystallization.

Degree

Ph.D.

Advisors

Thompson, Purdue University.

Subject Area

Organic chemistry

COinS