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Binding and folding of natively disordered SNAP‐25 to the protease domain of botulinum neurotoxin A by fluorescence resonance energy transfer and circular dichorism
Author(s) -
Lai Huiguo,
Feng Minghao,
Yang David ChihHsin
Publication year - 2011
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.25.1_supplement.707.7
Botulinum neurotoxin A light chain (BoNT/A LC) is the metalloprotease domain of the neurotoxin. BoNT/A LC is extremely specific. The only known natural substrate of BoNT/A LC is SNAP‐25, which plays key roles in the vesicle fusion cycle in the secretion of neurotransmitters. The tertiary and secondary structures of SNAP‐25 were analyzed by fluorescence resonance energy transfer (FRET) and circular dichroism (CD), respectively. Full length SNAP‐25, and its N‐ and C‐domains were fluorescently double labeled with genetically encoded EGFP and tetracysteine‐ReAsH. FRET of the double labeled proteins provided conformational constraints of SNAP‐25. The secondary structures were examined by CD. SNAP‐25 is evidently an intrinsically disordered protein as examined by FRET and CD. Upon binding of zinc‐depleted BoNT/A LC, major conformational changes of SNAP‐25 were observed using FRET and CD. Both N‐ and C‐domains of SNAP‐25 bound to BoNT/A LC with appreciable changes in the tertiary and secondary structures. Time courses and the equilibrium constants of the conformational changes of SNAP‐25 and subdomains revealed the binding and folding process in the unusual substrate binding strategy.