Slow folding of three-fingered toxins is associated with the accumulation of native disulfide-bonded intermediates.
Snake neurotoxins are short all-beta proteins that display a complex organization of the disulfide bonds: two bonds connect consecutive cysteine residues (C43-C54, C55-C60), and two bonds intersect when bridging (C3-C24, C17-C41) to form a particular structure classified as "disulfide beta-cross". We investigated the oxidative folding of a neurotoxin variant, named alpha62, to define the chemical nature of the three-disulfide intermediates that accumulate during the process in order to describe in detail its folding pathway. These folding intermediates were separated by reverse-phase HPLC, and their disulfide bonds were identified using a combination of tryptic hydrolysis, manual Edman degradation, and mass spectrometry. Two dominant intermediates containing three native disulfide bonds were identified, lacking the C43-C54 and C17-C41 pairing and therefore named des-[43-54] and des-[17-41], respectively. Both species were individually allowed to reoxidize under folding conditions, showing that des-[17-41] was a fast-forming nonproductive intermediate that had to interconvert into the des-[43-54] isomer before forming the native protein. Conversely, the des-[43-54] intermediate appeared to be the immediate precursor of the oxidized neurotoxin. A kinetic model for the folding of neurotoxin alpha62 which fits with the observed time-course accumulation of des-[17-41] and des-[43-54] is proposed. The effect of turn 2, located between residues 17 and 24, on the overall kinetics is discussed in view of this model.
Ruoppolo
M
M
Dipartimento di Chimica, Università degli Studi di Salerno, Salerno, Italy. ruoppolo@mbox.chem.unisa.it
Talamo
F
F
Pucci
P
P
Moutiez
M
M
Quèmèneur
E
E
Mènez
A
A
Marino
G
G
D000595:Amino Acid Sequence; D000818:Animals; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009498:Neurotoxins / Q000737:chemistry* / Q000235:genetics; D010084:Oxidation-Reduction; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012910:Snake Venoms / Q000737:chemistry / Q000235:genetics; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D004220:Disulfides; D009498:Neurotoxins; D012910:Snake Venoms; D003545:Cysteine