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Core‐spanning hydrophilic residues lend stability and preserve functionality in β‐helical protein HpmA265 (752.5)
Author(s) -
Glasgow Evan
Publication year - 2014
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.28.1_supplement.752.5
Subject(s) - circular dichroism , guanidine , chemistry , folding (dsp implementation) , protein folding , peptide , crystallography , hydrogen bond , molecule , mutant , stereochemistry , biophysics , biochemistry , biology , organic chemistry , electrical engineering , gene , engineering
In the present work, the bacterial β helical hemotoxin HpmA has been cloned and expressed in truncated form (HpmA265) and subject to several structural experiments. Folded (active) HpmA265 can rescue the hemolytic function of denatured, full‐length HpmA in vitro by direct interaction, and this phenomenon is quantified with a hemolytic assay. In attempt to correlate functionality with structure, circular dichroism spectroscopy has been used in conjunction with guanidine hydrochloride titrations to assess the structural/functional contributions of key hydrophilic residues in the protein’s core. Native HpmA265 was found to have a folding energy of ∆Gº = ‐50.2 ± 2.7 kJ/mol. Ala or Phe substitutions were made either at Q125 or Y134, and mutant folding energies were ‐25.0 ± 2.4 kJ/mol (Q125A), ‐48.0 ± 6.5 kJ/mol (Y134A), ‐32.4 kJ/mol (Q125F), and ‐45.4 ± 4.5 kJ/mol (Y134F). The data suggest more substantial perturbation by mutation of Q125 than of Y134. This idea complements the graphically‐predicted contribution of Q125 versus Y134, in that Q125 coordinates with an embedded water molecule in a core‐spanning hydrogen bond network and appears to be held in the proper orientation by the Y134 hydroxyl group. Additional networked residues will be assessed in this way. A greater understanding of the β helix fold may contribute to understanding of similar proteins, such as β amyloid and bacterial adhesion factors. Grant Funding Source : Supported by NSF‐RUI 105435 (to TW)