Effects of lysine mutations on the structure and function of hemolysin A from Proteus mirabilis (755.4)

Abstract

Proteus mirabilis (HpmA), a Gram‐negative bacterium, implements the two‐partner secretion pathway to produce a hemolytically active exoprotein. The full‐length HpmA was cloned and truncated to contain the first 265 amino acids (HpmA265) in order analyze the structure and function more readily. In the native crystal structure, a dimer was observed to form via on‐edge stacking of carboxy‐terminal parallel beta‐strands. A series of carboxy‐terminal lysine mutants were investigated in this study. Specifically, Phe241, Met245 and Leu263 were altered with the polar charged lysine residue to disrupt the non‐polar carboxy‐terminal dimer and thereby, induce monomer formation. The resultant HpmA265 mutants were analyzed via template‐assisted hemolysis assay, dynamic light scattering and circular dichroism. The results indicate that the loss of hemolytic activity can be correlated with replacement of the most carboxy‐terminal amino acid, Leu263. Additionally, there appears to be less well‐defined beta‐sheet structure in each of the HpmA265 carboxy‐terminal mutants. Further study will aim to conclude whether the HpmA265 monomer or dimer acts as the functional template during hemolysis.Grant Funding Source: Supported by NSF‐RUI 1050435