Abstract
Peptide toxins that adopt the inhibitory cystine knot (ICK) scaffold have very stable three-dimensional structures as a result of the conformational constraints imposed by the configuration of the three disulfide bonds that are the hallmark of this fold. Understanding the oxidative folding pathways of these complex peptides, many of which are important therapeutic leads, is important in order to devise reliable synthetic routes to correctly folded, biologically active peptides. Previous research on the ICK peptide ProTx-II has shown that in the absence of an equilibrating redox buffer, misfolded intermediates form that prevent the formation of the native disulfide bond configuration. In this paper, we used tandem mass spectrometry to examine these misfolded peptides, and identified two non-native singly bridged peptides, one with a Cys(III)-Cys(IV) linkage and one with a Cys(V)-Cys(VI) linkage. Based on these results, we propose that the C-terminus of ProTx-II has an important role in initiating the folding of this peptide. To test this hypothesis, we have also studied the folding pathways of analogs of ProTx-II containing the disulfide-bond directing group penicillamine (Pen) under the same conditions. We find that placing Pen residues at the C-terminus of the ProTx-II analogs directs the folding pathway away from the singly bridged misfolded intermediates that represent a kinetic trap for the native sequence, and allows a fully oxidized final product to be formed with three disulfide bridges. However, multiple two-disulfide peptides were also produced, indicating that further study is required to fully control the folding pathways of this modified scaffold.
Highlights
Disulfide bonds between Cys residues play a key role in stabilizing the conformational properties of peptides and proteins
Using HPLC, nanoelectrospray ionization mass spectrometry, and ion mobility mass spectrometry (IM-MS) we demonstrated that aerial oxidation of the linear precursor in water gave partial conversion to a mixture of incompletely oxidized isomers
Selection and optimization of the oxidative folding conditions is crucial to the successful synthesis of inhibitor cystine knot (ICK) peptides with the correct disulfide connectivities and bioactive fold
Summary
Disulfide bonds between Cys residues play a key role in stabilizing the conformational properties of peptides and proteins. Several families of peptide toxins with extensive networks of cystine bridges have been isolated and characterized from natural sources (Lavergne et al, 2015) These highly constrained peptides with multiple disulfide bridges adopt very stable threedimensional structures, and as a result have highly specific and potent interactions with biological targets such as ion channels and GPCRs (Ferrat and Darbon, 2005; Akondi et al, 2014; Cardoso and Lewis, 2019). One of the most common structural motifs for these toxins is the inhibitor cystine knot (ICK) scaffold, referred to as a “knottin” fold These peptides contain six Cys residues connected together by disulfide bonds in a Cys(I)-Cys(IV), Cys(II)-Cys(V), Cys(III)-Cys(VI) connectivity. Analogs of ProTx-II (1, Figure 1) (Park et al, 2014; Henriques et al, 2016; Flinspach et al, 2017), GpTx-1 (Murray et al, 2015; Chen et al, 2018; Lawrence et al, 2019), JzTx-V (Moyer et al, 2018; Wu et al, 2018), and HwTx-IV (Revell et al, 2013; Agwa et al, 2017) have been identified as potent and selective antagonists of the Nav1.7 receptor
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
