All ETDs from UAB

Advisory Committee Chair

Peter E Prevelige

Advisory Committee Members

William J Britt

Terje Dokland

Michael Niederweis

Document Type

Dissertation

Date of Award

2016

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

The virus capsids of many dsDNA bacteriophage are finely tuned macromolecular machines. Their functionality begins at assembly, when their capsids are built under the guidance of a class of proteins referred to as scaffolding proteins. The scaffolding protein of bacteriophage P22 is a flexible, highly elongated protein that binds to the interior surface of the P22 procapsid via its C-terminal domain. The finding that N-terminus is dispensable for procapsid binding prompted the development of chimeric scaffolding protein molecules, wherein N-terminal residues of scaffold are replaced with a variety of different peptide sequences or functional proteins that are then encapsidated within the procapsid. These studies established P22 as a functional nano-reactor platform. We have expanded on these previous studies with P22 and developed the use of the P22 nano-reactor system to selectively synthesize two distinct forms of the photoactive semiconductor TiO2. Form selectivity comes from the use of two unique peptide sequences on the N- terminus of the scaffolding protein. Additionally, in contrast with other protein cage-based systems, TiO2 synthesized with P22 leads to confinement of the mineral within the capsid lattice and a small average nanoparticle size. These features result in the effective solubilization of an otherwise insoluble inorganic material and facilitate its use for photocatalysis in aqueous solution. The use of P22 as a scaffold-based reactor system is not its only functionality as a nano-machine. In its biological context, the virus relies on the dodecameric portal protein to sense and respond to the density of DNA inside of its head. This mechanism underlies termination of genome packaging. Understanding how the portal protein functions as a molecular sensor could facilitate novel applications of P22 as a molecular machine. In an effort to understand signal transmission through the portal, we constructed and characterized mutants of the protein that result in altered packaging phenotypes. Characterization of these packaging phenotypes, as well as biophysical characterization of the portal mutants in vitro, point towards a mechanism wherein portal acts as a pressure-sensitive switch. Alterations in side chain re-packing upon compression of the protein appear to be the origin of the observed packaging phenotypes.

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