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Molecular dissection of hemolysin A template‐assisted activity
Author(s) -
Weaver Todd Michael,
McDermott James Joyce
Publication year - 2012
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.26.1_supplement.545.2
Subject(s) - circular dichroism , chemistry , monomer , dimer , size exclusion chromatography , hemolysis , biophysics , chromatography , biochemistry , polymer , enzyme , biology , organic chemistry , immunology
In this study, the role of molecular organization was investigated as related to hemolysin A (HpmA265) template‐assisted behavior. Previous studies have shown that full‐length HpmA can be activated through interaction with purified HpmA265 in template‐assisted fashion. HpmA265 was analyzed via template‐assisted hemolysis, circular‐dichroism, size‐exclusion chromatography and multi‐angle light scattering (SEC‐MALS) before and after tryptic digestion. The results from these investigations were utilized to determine the absolute molecular weight of the template‐assisted active species. The pre‐tryptic digestion data reported a heterogeneous mixture of HpmA265 monomer and dimer. Quantitative hemolytic assays have determined the monomer to be the molecularly active species. Subsequent tryptic digestion, followed by SEC‐MALS, generated a homogenous dimeric species with enhanced template‐assisted behavior and thermal stability. From these data, a new model for template‐assisted activity has been developed where exposed, homogenous, and monomeric on‐edge beta‐strands of HpmA265 facilitate the activation of full‐length HpmA. These findings are significant and may apply to other beta‐helix protein dependent disease states like Alzheimer's Parkinson's and transmittable prion disorders. National Science Foundation Grant (MCB0744754) and (MCB1050435) supported the work.