Deconstruction and reconstruction of a protein capsid

Update item information
Publication Type dissertation
School or College College of Science
Department Chemistry
Author Chen, Hsiao-Nung
Title Deconstruction and reconstruction of a protein capsid
Date 2014-08
Description The self-assembly of multiple protein subunits via noncovalent interactions provides a diverse collection of nanoarchitectures. Polyhedral capsids represent a particularly interesting type of structure in which the protein forms a closed three-dimensional surface that can function as a molecular container. Understanding capsid self-assembly could benefit further development of nanomaterials for many applications, such as drug delivery and biocatalysis. In this thesis, Aquifex aeolicus lumazine synthase (AaLS) is used as a model for investigating capsid self-assembly. The 60-subunit capsids formed by AaLS in vivo can be viewed as dodecamers-of-pentamers. Currently, methods are lacking for controlling AaLS assembly in vitro, which imposes important limitations on cargo loading. Interestingly, the diverse quaternary structures in the lumazine synthase family, which includes capsids and pentamers, imply the possibility of exchanging assembly states in vitro. To better understand AaLS capsid assembly, the dodecahedral capsid was converted to pentamers via a strategy involving rational design and sitedirected mutagenesis. Biophysical characterizations confirm that simultaneous substitution of three interfacial residues can yield stable pentamers. A pentameric AaLS variant that possesses a unique, strategically placed cysteine residue was engineered to enable capsid formation in vitro. This cysteine was modified with a thiophenol group by sequential thiol-disulfide exchange reactions. The increase in nonpolar surface area upon modification of this cysteine allows for assembly of the pentamers into capsids that resemble wild-type AaLS, presumably by recapitulating hydrophobic interactions present at the pentamer-pentamer interface. In an alternative approach, a pH-dependent switch for AaLS capsid disassembly was developed. For this switch, the ability to change assembly state relies on the presence of three engineered histidine residues per subunit, located near the three-fold symmetric interface of the capsid. These histidines minimally interfere with the capsid structure at high pH where their side chains are neutral. However, at lower pH, histidine protonation can trigger the dissociation of the capsid into pentamers, presumably due to charge repulsion. Further, this switch is reversible, as 60-subunit capsids can reform upon raising the pH. These studies of interconverting AaLS quaternary structures open the door to the development of improved encapsulation systems for use in medicine or nanobiomaterials.
Type Text
Publisher University of Utah
Subject Capsid; Protein engineering; Self-assembly
Dissertation Institution University of Utah
Dissertation Name Doctor of Philosophy
Language eng
Rights Management Copyright © Hsiao-Nung Chen 2014
Format Medium application/pdf
Format Extent 2,269,282 bytes
Identifier etd3/id/3139
ARK ark:/87278/s67m3h7c
Setname ir_etd
Date Created 2014-10-08
Date Modified 2017-06-12
ID 196706
Reference URL https://collections.lib.utah.edu/ark:/87278/s67m3h7c