Non-covalent affinity materials for protein structure determination via single particle reconstruction analysis cryo-electron microscopy
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.
Degree
M.S.
Advisors
Thompson, Purdue University.
Subject Area
Organic chemistry
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