All ETDs from UAB

Advisory Committee Chair

Terje Dokland

Advisory Committee Members

Asim K Bej

Gail E Christie

Peter E Prevelige

R Douglas Watson

Document Type

Dissertation

Date of Award

2009

Degree Name by School

Doctor of Philosophy (PhD) College of Arts and Sciences

Abstract

Bacteriophage P2 encodes a scaffolding protein (gpO), which is required for correct assembly of P2 procapsids from the major capsid protein (gpN). The 284-residue gpO also acts as a protease, cleaving itself into an N-terminal fragment, O*, which remains in the capsid following maturation. gpO is a classical serine protease, with a catalytic triad comprised of Asp 19, His 48 and Ser 107, located in its N-terminal domain. The C-terminal 90 amino acids of gpO, which contains a predicted &alpha-helical segment between residues 197 and 257, are required and sufficient for capsid assembly. This fragment exists as a multimer in solution, suggesting that oligomerization is required for scaffolding activity. Additionally, the C-terminal cysteine residue Cys 284 is required for capsid fidelity and is most likely involved in transient interactions with gpN. Our results suggest a model for gpO scaffolding action in which the N-terminal half of gpO binds strongly to gpN, while oligomerization of the C-terminal &alpha-helical domain of gpO and transient interactions between Cys 284 and gpN lead to capsid assembly. Although bacteriophage HK97 lacks a gene for a scaffolding protein, it encodes an N-terminal "delta" (&delta) domain comprised of the N-terminal residues 2-103 of its capsid protein (gp5), forming a predominantly &alpha-helical fold. Varying fragments of the C-terminal region of gpO were fused to gpN to test its functional similarity to the HK97 delta domain. The fusion of the gpO scaffolding domain to gpN promotes the assembly of small shells, suggesting that fusing gpO to gpN assists in shell closure but also restricts the conformational variability of the capsid protein. The Staphylococcus aureus pathogenicity island SaPI1 is mobilized by infection with the S. aureus helper phage 80&alpha. Compared to the 51-nm 80&alpha procapsids, SaPI1 forms a smaller 39-nm procapsid from 80&alpha structural proteins. The SaPI1 size determination factor has been putatively identified as the SaPI1 protein gp6, an internal scaffolding protein. The cleaved form of the 80&alpha scaffolding protein gp46 is retained in mature 80&alpha and SaPI1 virions, indicating that gp46 is required for proper assembly of 80&alpha and SaPI1 capsids.

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