Structural studies of the saposins and PagP
By: by Victoria Elizabeth Ahn.
Published: 2004
Language: eng
Number of pages: 191
Description: This thesis comprises two parts. The first chapters describe structural studies of sphingolipid activator proteins, the saposins. This is followed by studies of the outer membrane protein PagP. The four saposins are derived from a common precurser protein; they are small, non-enzymatic glycoproteins required for the breakdown of glycosphingolipids (GSL) in the lysosome. Their general functions are to increase the accessibility of specific GSLs to their cognate acid hydrolases. Saposin B was the first characterized sphingolipid activator protein and saposin. While fairly well studied, further understanding into the nature of this protein required structural details. This thesis presents structural determination of saposin B, and further experiments towards elucidation of the mechanism of action. The saposin B dimer encompasses a large hydrophobic cavity for solubilization of lipids, and conformational changes appear to regulate accessibility to the interior. Saposin B laid the groundwork for additional crystallographic studies of the homologous saposins, A and C. These monomeric structures seem to corroborate the unique nature and mechanism of action of each individual saposin. Together, the three structures present new thoughts regarding other intracellular roles.PagP is an outer membrane enzyme present in Gram-negative bacteria that is upregulated to promote virulence. PagP palmitoylates lipid A of lipopolysaccharides in the outer membrane, resulting in increased resistance to host cationic antimicrobial peptides. One of only a few known outer membrane enzymes, the crystal structure of PagP was determined. Described in this thesis are the implications of the presence of a LDAO detergent molecule within the centre of the 8 stranded beta-barrel, for the mechanism of PagP acylation. The complexed structure, and subsequent site-directed mutagenesis led to determination of a structural basis for palmitate specificity, that involves a hydrocarbon ruler.
Notes: Adviser:  Gilbert G. Prive.

Thesis (Ph.D.)--University of Toronto, 2004.

Electronic version licensed for access by U. of T. users.

Source: Dissertation Abstracts International, Volume: 65-10, Section: B, page: 4981.