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Contribution of an inner core hydrogen‐bonding network to β‐helix stability within the two‐partner secretion exotoxin family
Author(s) -
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.996.13
Subject(s) - chemistry , circular dichroism , biophysics , helix (gastropod) , secretion , hydrogen bond , mutant , protein folding , stereochemistry , biochemistry , crystallography , biology , molecule , ecology , organic chemistry , snail , gene
TPS pathways, harboring both a membrane bound β‐barrel B‐component (TpsB) and a β‐helical A‐component (TpsA), can be found within bacterial, fungal and animal kingdoms. Bacterial TpsA components, signified by adhesions, proteases, and hemolysins, adopt a biogenic beta‐helical fold concomitant with TpsB‐dependent secretion. β‐helix proteins (βHPs) have become a prevalent structural fold being associated with antifreeze proteins, β‐amyloid, and prion proteins. The research implemented HpmA265 to investigate the βHP structure‐function (SF) paradigm. Specifically, a series of site‐directed mutants (SDMs) were constructed to target the inner core hydrogen‐bonding network shared between Q125 and Y134 at β‐arc 2 within first β‐circuit. Each of these two residues was replaced singly or doubly with either alanine, serine or phenylalanine residues. Additionally, these were combined with two other SDMs aimed at disrupting a carboxyamide ladder forming a bridge between non‐consecutive parallel β‐strands. The SF relationship attributed to these various site‐directed alterations was analyzed functionally within a template‐assisted assay and structurally using circular dichroism. The phenylalanine SDMs provided the greatest loss in hemolytic activity coupled to a weakened β‐signal and 10 °C decrease in Tm. These studies illustrate that the hemolytic activity of HpmA265 is linked directly to structural integrity of the β‐helix fold. In addition, the intricate inner core hydrogen‐bonding network within β‐arc 2 of β‐circuit 1 appears to be an important factor leading to successful TpsA activation.