Conformational μ-Conotoxin PIIIA Isomers Revisited: Impact of Cysteine Pairing on Disulfide-Bond Assignment and Structure Elucidation

Abstract

Peptides and proteins carrying high numbers of cysteines can adopt various 3D structures depending on their disulfide connectivities. The unambiguous verification of such conformational isomers with more than two disulfide bonds is extremely challenging, and experimental strategies for their unequivocal structural analysis are largely lacking. We synthesized all 15 possible isomers of the 22mer conopeptide μ-PIIIA and applied 2D NMR spectroscopy and MS/MS for the elucidation of its structure. This study provides intriguing insights in how the disulfide connectivity alters the global fold of a toxin. We also show that analysis procedures involving comprehensive combinations of conventional methods are required for the unambiguous assignment of disulfides in cysteine-rich peptides and proteins and that standard compounds are crucially needed for the structural analysis of such complex molecules.

Cysteines and Disulfide Bonds as Structure-Forming Units: Insights From Different Domains of Life and the Potential for Characterization by NMR

“…The pharmacological potential to modulate or block the ion channel activity and their synthetic availability make conotoxins promising candidates for new analgesics. However, Heimer et al recently showed on the example of µ-PIIIA (three disulfide bonds) the complexity of the synthesis, purification, and analytical characterization of one specific isomer in the multitude of different potentially formed disulfide-bridged isomers of those cysteine-rich peptides (Heimer et al, 2018). With respect to this, ionic liquids have proven to be a promising solvent for controlling the oxidative folding process (Miloslavina et al, 2010).…”

Revisiting the interaction of heme with hemopexin: Recommendations for the responsible use of an emerging drug

“…1) contain cysteine residues, which may be present in the oxidized form in the protein. In order to clarify the oxidation state of these cysteines, we incubated hemopexin with the alkylating agent 2-iodoacetamide and performed mass spectrometry measurements 35 . This analysis revealed that all cysteines are oxidized in wild-type hemopexin, which was also confirmed by a negative result from the Ellman’s test for free thiols 35 .…”

“…In order to clarify the oxidation state of these cysteines, we incubated hemopexin with the alkylating agent 2-iodoacetamide and performed mass spectrometry measurements 35 . This analysis revealed that all cysteines are oxidized in wild-type hemopexin, which was also confirmed by a negative result from the Ellman’s test for free thiols 35 . Thus, peptide H150 was synthesized with an intramolecular disulfide bond and in all other peptides, disulfide bonds were mimicked by insertion of a methylated cysteine during synthesis.…”

“…The molar mass of the derivatized protein was compared to the wild-type untreated hemopexin as determined by MALDI mass spectrometry using an ultrafleXtreme TOF/TOF mass spectrometer (Bruker Daltonics, Billerica, MA) with 2,5-dihydroxyacetophenone (Fluka, Buchs, Switzerland) as matrix. Additionally, Ellman’s test 35 was performed with hemopexin in comparison to cysteine (positive control) and cystine (negative control) by mixing 150 μl of the sample solutions with 100 μl Ellman’s reagent (1.76 mg/ml 5,5’-dithiobis-(2-nitrobenzoic acid) in 0.2 M sodium phosphate buffer, pH 8). Absorption was measured after 10 min incubation at 410 nm.…”

“…The intramolecular disulfide bond of peptide H150 (AVECHRGEC) was formed by air oxidation using a previously established protocol 62 . Complete oxidation was confirmed using Ellman’s test as described above 35 .…”