Date of Completion

5-28-2019

Embargo Period

5-28-2019

Keywords

dsDNA virus, I-domain, protein evolution, P22 bacteriophage, CUS-3 bacteriophage, Sf6 bacteriophage

Major Advisor

Dr. Carolyn Teschke

Associate Advisor

Dr. Andrei Alexandrescu

Associate Advisor

Dr. Nathan Alder

Associate Advisor

Dr. Eric May

Associate Advisor

Dr. Simon White

Field of Study

Molecular and Cell Biology

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

Conservation and Divergence Between P22-like Bacteriophages Coat Protein’s

I-domains and Procapsid-like Particles

Therese N. Tripler, PhD

University of Connecticut, 2019

P22, CUS-3 and Sf6 are distantly related bacteriophages that belong to the P22-like group based on capsid protein sequence similarity. Despite being distantly related they share important similarities. They form T=7 icosahedral capsids to protect their dsDNA, have capsid proteins that adopt the HK97 fold with an inserted domain, share similar arrangement of their structural genes in their genomes, and infect human enteric pathogens. P22, CUS-3and Sf6 infect Salmonella enterica, Escherichia coli, and Shigella flexneri, respectively. Here, we explored structural differences between the inserted domains (I-domains), differences in CUS-3 and Sf6 bacteriophage procapsid-like particle assembly and stability, and the effects of Sf6 scaffolding protein truncations on assembly.

The P22, CUS-3 and Sf6 I-domains adopt a six-stranded anti-parallel b-barrel with important differences. Differences are found between loop regions, electrostatic surfaces that are found at the interface between the I-domain and the protein core, and surface TUT (threonine-hydrophobic-threonine) motifs possibly important for carbohydrate interactions. Thus, the insertion domains may have evolved to fit different functional needs in capsid stability, assembly, and coat protein folding.

Like P22, CUS-3 and Sf6 coat and scaffolding proteins are shown here to assemble into procapsid-like particles in the absence of all other structural proteins. CUS-3 and Sf6 procapsid-like particles were found to be less stable compared with P22. This provides evidence that these assembly systems are governed by differences in their electrostatic and hydrophobic interactions.

For P22 bacteriophage, scaffolding protein nucleates and directs capsid assembly. In P22 assembly, the C-terminal is necessary to form procapsids. Sf6 truncated scaffolding protein containing residues 1-155 formed mostly T=7 procapsid-like particles similar to those particles formed by full-length scaffolding protein. Unlike P22, the C-terminal of scaffolding protein is not necessary for assembly.

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