Slow Folding of Three-Fingered Toxins Is Associated with the Accumulation of Native Disulfide-Bonded Intermediates
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- 作者：Margherita Ruoppolo ; Fabio Talamo ; Piero Pucci ; Mireille Moutiez ; Eric Què ; mè ; neur ; Andrè ; Mè ; nez ; and Gennaro Marino
- 刊名：Biochemistry
- 出版年：2001
- 出版时间：December 18, 2001
- 年：2001
- 卷：40
- 期：50
- 页码：15257 - 15266
- 全文大小：149K
- 年卷期：v.40,no.50(December 18, 2001)
- ISSN：1520-4995
文摘
Snake neurotoxins are short all-
proteins that display a complex organization of the disulfidebonds: two bonds connect consecutive cysteine residues (C43-C54, C55-C60), and two bonds intersectwhen bridging (C3-C24, C17-C41) to form a particular structure classified as "disulfide -cross". Weinvestigated the oxidative folding of a neurotoxin variant, named 
62, to define the chemical nature ofthe three-disulfide intermediates that accumulate during the process in order to describe in detail its foldingpathway. These folding intermediates were separated by reverse-phase HPLC, and their disulfide bondswere identified using a combination of tryptic hydrolysis, manual Edman degradation, and massspectrometry. Two dominant intermediates containing three native disulfide bonds were identified, lackingthe 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 beforeforming the native protein. Conversely, the des-[43-54] intermediate appeared to be the immediateprecursor of the oxidized neurotoxin. A kinetic model for the folding of neurotoxin 62 which fits withthe observed time-course accumulation of des-[17-41] and des-[43-54] is proposed. The effect of turn2, located between residues 17 and 24, on the overall kinetics is discussed in view of this model.
proteins that display a complex organization of the disulfidebonds: two bonds connect consecutive cysteine residues (C43-C54, C55-C60), and two bonds intersectwhen bridging (C3-C24, C17-C41) to form a particular structure classified as "disulfide -cross". Weinvestigated the oxidative folding of a neurotoxin variant, named 62, to define the chemical nature ofthe three-disulfide intermediates that accumulate during the process in order to describe in detail its foldingpathway. These folding intermediates were separated by reverse-phase HPLC, and their disulfide bondswere identified using a combination of tryptic hydrolysis, manual Edman degradation, and massspectrometry. Two dominant intermediates containing three native disulfide bonds were identified, lackingthe 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 beforeforming the native protein. Conversely, the des-[43-54] intermediate appeared to be the immediateprecursor of the oxidized neurotoxin. A kinetic model for the folding of neurotoxin 62 which fits withthe observed time-course accumulation of des-[17-41] and des-[43-54] is proposed. The effect of turn2, located between residues 17 and 24, on the overall kinetics is discussed in view of this model.