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Template‐assisted hemolytic activity of carboxy‐terminal mutants of truncated hemolysin A
Author(s) -
Hocking Jason Mark,
Weaver Todd Michael
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.529.2
Subject(s) - hemolysin , mutant , periplasmic space , hemolysis , secretion , chemistry , biology , virulence , escherichia coli , biochemistry , gene , immunology
Proteus species, a common urinary tract infectious gram‐negative bacterium, contains the virulence factor hemolysin A. Hemolysin A is one member of the two‐partner secretion (TPS) pathway. In the TPS pathway hemolysin B (HpmB) secretes and activates hemolysin A from the periplasm into the external environment. Additional studies have reported the activation of inactive hemolysin A after mixing with an exogenous pool of truncated and post‐HpmB secreted hemolysin A. This so‐called template‐assisted hemolytic activity has been monitored under a number of different conditions. More recently, a 3‐dimensional structure of truncated hemolysin A, dubbed HpmA265, has been determined. In this crystal structure one of the important interactions occurs between the carboxy‐terminal ends of two beta‐helix monomers, thereby forming a cross beta‐stranded, dry dimer interface. In an effort to understand the template‐assisted activity of HpmA265, two types of mutants were constructed. First, a series of carboxy‐terminal mutants were constructed where a lysine residue was substituted within the normally hydrophobic core. Additionally, a truncated form of hemolysin A was constructed to contain only the first 141 residues and has been termed HpmA141. Each of these mutants were purified and analyzed for template‐assisted activity and temperature stability. The lysine replacements engineered at the carboxy‐terminus of HpmA265 did not inhibit activity. However, temperature stability differences have been observed between these carboxy‐terminal mutants and HpmA265. Furthermore, HpmA141 showed some residual template‐assisted activity even though it only harbors the first 141 residues. The later results are supportive of a model whereby the carboxy‐terminal end of truncated HpmA is an important interface during template‐assisted activity. Research supported by NSF‐RUI (MCB 0744754)