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Structure‐function differentiation of beta‐edge negative‐design variants of hemolysin A using size‐exclusion chromatography
Author(s) -
McDermott James Joyce,
Weaver Todd Michael
Publication year - 2011
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.25.1_supplement.909.7
Subject(s) - hemolysis , size exclusion chromatography , chemistry , circular dichroism , mutant , dimer , hemolysin , monomer , secretion , biochemistry , stereochemistry , virulence , enzyme , biology , gene , polymer , organic chemistry , immunology
Hemolysin A (HpmA) from Proteus mirabilis is secreted via a Two‐Partner Secretion (TPS) pathway, which is utilized by Gram‐negative bacteria as an energy‐independent mechanism for secretion of virulence factors. Truncated HpmA (HpmA265) has been shown to activate full‐length HpmA in a template‐assisted fashion. Crystallographic analysis of HpmA265 revealed a right‐handed β‐helical structure and a dry dimeric interface between exposed carboxy‐terminal β‐edges. A series of carboxy‐terminal mutants of HpmA265 were engineered using negative‐design theory, where a positively charged lysine residue was substituted at the non‐polar dry, dimer interface. The replacements included phenylalanine 241 (F241K), methionine 245 (M245K), and leucine 263 (L263K). Recent circular dichroism and hemolysis studies reported decomposition of the classic beta signal and loss in hemolytic activity for M245K and L263K. To further investigate the relationship between HpmA265 structure and function, quantitative hemolytic activity was mapped to the resultant size‐exclusion chromatogram. Template‐assisted hemolysis of HpmA265 after size‐exclusion chromatography (SEC) showed hemolytic activity corresponding to a molecular weight of 27 kDa. This is close to the monomeric weight of 25 kDa for HpmA265, which suggest a monomeric form of HpmA265 initiates the folding of full‐length HpmA during hemolysis. Template‐assisted hemolytic activity of HpmA265 variants, F241K, L263K, and M245K, had activity concentrated around a molecular weight of 21 kDa, which again is indicative of monomeric activation of hemolysis. The following awards supported the research: NSF‐RUI (0744754) to TMW and ASBMB‐UAN Undergraduate Research Award to JJM.