สืบค้นงานวิจัย
Functional contributions of positive charges in the pore‐lining helix 3 of the Bordetella pertussis CyaA- Hemolysin to hemolytic activity and ion-channel opening
Kurehong C. - ไม่ระบุหน่วยงาน
ชื่อเรื่อง (EN): Functional contributions of positive charges in the pore‐lining helix 3 of the Bordetella pertussis CyaA- Hemolysin to hemolytic activity and ion-channel opening
ผู้แต่ง / หัวหน้าโครงการ (EN): Kurehong C.
บทคัดย่อ (EN): The Bordetella pertussis CyaA‐hemolysin (CyaA‐Hly) domain was previously demonstrated to be an important determinant for hemolysis against target erythrocytes and ion‐channel formation in planar lipid bilayers (PLBs). Here, net‐charge variations in the pore‐lining helix of thirteen related RTX cytolysins including CyaA‐Hly were revealed by amino acid sequence alignments, reflecting their different degrees of hemolytic activity. To analyze possible functional effects of net‐charge alterations on hemolytic activity and channel formation of CyaA‐Hly, specific mutations were made at Gln574 or Glu581 in its pore‐lining α3 of which both residues are highly conserved Lys in the three highly active RTX cytolysins (i.e., Escherichia coli α‐hemolysin, Actinobacillus pleuropneumoniae toxin, and Aggregatibacter actinomycetemcomitans leukotoxin). All six constructed CyaA‐Hly mutants that were over‐expressed in E. coli as 126 kDa His‐tagged soluble proteins were successfully purified via immobilized Ni2+‐affinity chromatography. Both positive‐charge substitutions (Q574K, Q574R, E581K, E581R) and negative-charge elimination (E581Q) appeared to increase the kinetics of toxin‐induced hemolysis while the substitution with a negatively‐charged side‐chain (Q574E) completely abolished its hemolytic activity. When incorporated into PLBs under symmetrical conditions (1.0 M KCl, pH 7.4), all five mutant toxins with the increased hemolytic activity produced clearly‐resolved single channels with higher open probability and longer lifetime than the wild‐type toxin, albeit with a half decrease in their maximum conductance. Molecular dynamics simulations for 50 ns of a trimeric CyaA‐Hly pore model comprising three α2‐loop‐α3 transmembrane hairpins revealed a significant role of the positive charge at both target positions in the structural stability and enlarged diameter of the simulated pore. Altogether, our present data have disclosed functional contributions of positively‐charged side‐chains substituted at positions Gln574 and Glu581 in the pore‐lining α3 to the enhanced hemolytic activity and ion‐channel opening of CyaA‐Hly that actually mimics the highly‐active RTX (repeat‐in‐toxin) cytolysins. © 2017 by the authors. Licensee MDPI, Basel, Switzerland.
บทคัดย่อ: ไม่พบข้อมูลจากหน่วยงานต้นทาง
ภาษา (EN): en
เอกสารแนบ (EN): https://www.scopus.com/inward/record.uri?eid=2-s2.0-85015739334&doi=10.3390%2ftoxins9030109&partnerID=40&md5=41da5342f110faae38dde46e0e2c28b0
เผยแพร่โดย (EN): มหาวิทยาลัยมหิดล
คำสำคัญ (EN): Sheep
เจ้าของลิขสิทธิ์ (EN): มหาวิทยาลัยมหิดล
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Kurehong C.. "Functional contributions of positive charges in the pore‐lining helix 3 of the Bordetella pertussis CyaA- Hemolysin to hemolytic activity and ion-channel opening". ไม่ระบุหน่วยงาน. ม.ป.ป.

Kurehong C.. "Functional contributions of positive charges in the pore‐lining helix 3 of the Bordetella pertussis CyaA- Hemolysin to hemolytic activity and ion-channel opening". ไม่ระบุหน่วยงาน. ม.ป.ป.. Print.



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Functional contributions of positive charges in the pore‐lining helix 3 of the Bordetella pertussis CyaA- Hemolysin to hemolytic activity and ion-channel opening
Kurehong C.
มหาวิทยาลัยมหิดล
ไม่ระบุวันที่เผยแพร่
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