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Structural and functional importance of third‐circuit hydrogen bonds within hemolysin A
Author(s) -
Apolinario Michael Angelo,
Weaver Todd Michael
Publication year - 2013
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.27.1_supplement.996.5
Subject(s) - antiparallel (mathematics) , hydrogen bond , chemistry , phenylalanine , alanine , circular dichroism , crystallography , molecule , helix (gastropod) , stereochemistry , biophysics , amino acid , biochemistry , biology , organic chemistry , ecology , physics , quantum mechanics , snail , magnetic field
In this study, hemolysin A was used as a model to investigate the β‐helix structure‐function (SF) paradigm. The research implements a truncated form of hemolysin A termed HpmA265. HpmA265 facilitates uni‐directional, concentration‐dependent and biphasic template‐plate assisted hemolytic activation of full length HpmA. Prior x‐ray crystallographic results reports that the HpmA265 structure consists of a three‐sided, right‐hand parallel β‐helix, adjoining two and four‐stranded antiparallel sheets and four inner core solvent molecules. The specific objective of research examined a complex bonding network surrounding the third circuit of HpmA265 and explored its contributions to thermal stability and hemolytic activity. A series of phenylalanine and alanine site‐directed mutants (SDMs) were used to investigate the importance of an inner core hydrogen‐bonding network between Gln125, Tyr134, and an embedded solvent. Additionally, a cross‐strand carboxyamide ladder between circuits 2 and 4 was targeted by both alanine and lysine replacements. Circular dichroism spectroscopy and template‐assisted hemolytic assays were conducted to ascertain thermodynamic and functional differences between these HpmA265 SDMs. Collectively, the alanine SDMs reported a 6 – 12 °C lowering of the melting point (Tm) with minimal change in hemolytic activity. However, the phenylalanine SDMS led to a lower Tm and a decrease in hemolytic activity. The difference in alanine and phenylalanine hydrophobicity appears to play a crucial role in the β‐helix SF paradigm. The research supported by NSF‐RUI 1050435 (to TW).