A biophysical investigation into the self-assembly of α-helix - polyproline II helix oligomers

  • Timothy J Heal

Student thesis: Doctoral ThesisDoctor of Philosophy (PhD)

Abstract

Ag I/II polypeptides are a family of cell wall anchored proteins expressed by diverse bacterial species across the Lactobacillales. They mediate a wide range of interactions with other biomolecules which facilitate adherence to epithelial cells, tooth surfaces and other microorganisms, so playing a significant role in the bacterial colonisation of mucosal membranes. This feature strongly implicates Ag I/II polypeptides in pathogenic Streptococcus infections in both mammals and other animals. Proteins of this family are characterised by two globular domains separated by an extended stalk. Previous work on the Streptococcus mutans Ag I/II polypeptide SpaP, shows that this stalk is composed of the association into an anti-parallel "coiled coil'' of an α-helix and a polyproline II helix, each composed of a non-contiguous domain of the protein.

Work presented in this thesis verifies the presence of this coiled coil fold in the sequentially divergent Ag I/II polypeptides of Streptococcus agalactiae (BspA) and Streptococcus pyogenes (AspA) by biophysical methods, establishing the universality of the fold across the family. Mutagenesis was carried out to explore the basis for the α-helix - PPII helix association, establishing that hydrogen bonding to the backbone of the PPII helix from asparagine residues on the α-helix is essential to the formation of the fold in AspA and BspA. Parallel experiments established the stabilising effect of tyrosine residues to the interaction. The specificity of binding was investigated by examining the mixing of the α-helical domain of AspA and PPII-helical domain of BspA, and conversely the mixing of the α-helical domain of BspA and PPII-helical domain of AspA. These experiments showed that while binding did occur in both cases, the strength of the binding was thermodynamically weaker, indicating 'comparative' specificity, although not specificity to a degree where α helices from AspA and BspA bind only to their naturally occurring PPII helix binding partners.

Two fusion proteins were produced, one composed from the α-helical domain of AspA and PPII-helical domain of BspA, and a second from the α-helical domain of BspA and PPII-helical domain of AspA. Mixing of the fusion proteins was shown to result in the formation of kinetically stable products with a wide range of hydrodynamic shapes and sizes, indicating that α-helix - PPII helix interactions have high enough potential binding specificity to engineer modular interactions akin to those seen in other areas of protein design, such as α-helix - α-helix coiled coil designs. Mixtures of two of the fusion proteins were shown by TEM to produce macromolecular fibrils with a 'rope-like' structure. This work primarily contributes to opening the possibility of adding the α-helix - PPII helix coiled coil structure to the tool kit of protein design.
Date of Award28 Nov 2019
Original languageEnglish
Awarding Institution
  • The University of Bristol
SupervisorPaul R Race (Supervisor) & Dek N Woolfson (Supervisor)

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