Date of Award
Spring 2015
Degree Type
Thesis
Degree Name
Master of Science (MS)
Department
Chemistry
First Advisor
David H. Thompson
Committee Chair
David H. Thompson
Committee Member 1
Chengde Mao
Committee Member 2
Mingji Dai
Abstract
Single particle analysis (SPA) by cryogenic electron microscopy (Cryo-EM) is one of the powerful tools for determining biological macromolecule structure, particularly when combined with computational methods or X-ray crystallography. However, the SPA is challenged by sample processing efficiency. In this work, polyrotaxane (PRTx) was designed and synthesized such that laterally and rotationally mobile lysine nitrolicacetic acid (Lys-NTA) modified α-cyclodextrins (α-CD) were threaded onto a polyethylene glycol (PEG) core. Polyrotaxanes threaded with amine modified α-CD were also designed and synthesized for stoichiometric control of His-tagged protein capture by PRTx. For higher efficiency of single particle analysis along a single strand of the polyrotaxane, gold nanoparticles were used to cross-link the polyrotaxanes into a network using an interfacial approach. The gold nanoparticle distribution in TEM images and the UV-Vis spectroscopic changes suggested that a polyrotaxane network was formed. In addition, to better control the ligands on the network and to make the reaction on the grid facile, α-CD was functionalized with mono-6-azido moieties prior to network formation. NMR evidence suggested that mono-6-azido CD (N3-α-CD) was successfully threaded onto 10K PEG to form a polyrotaxane. This polyrotaxane was combined with Lys-NTA via a bio-orthogonal copper-free click reaction with N3-α-CD to produce a polymer network for specific binding with his-tag T7 phage.
Recommended Citation
Ha, Minji, "Non-covalent affinity materials for protein structure determination via single particle reconstruction analysis cryo-electron microscopy" (2015). Open Access Theses. 510.
https://docs.lib.purdue.edu/open_access_theses/510
