Scaled-up Synthesis and Characterization of Oxytocin Trisulfide

Hammer, Robert P.; Butrie, Melissa A.; Davidson, Karen; Goldblatt, Phillip T.; Schrader, Alex M.; Dalluge, Joseph J.; Becker, Allyn; Barany, George

doi:10.1007/s10989-023-10580-9

Cited by 1 publication

(1 citation statement)

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“…Disul de bonds play a critical role in the structures and functions of both natural and designed proteins and peptides. Disul de bonds covalently link distal regions within a wide range of peptides, including oxytocin (Gimpl & Fahrenholz, 2001;Hammer et al, 2023;Muttenthaler et al, 2010), cyclotides (de Veer et al, 2019; North eld et al, 2014), disul de-rich peptides (Singh et al, 2010), insulin (Karas et al, 2021), and proteins (Dombkowski et al, 2014;Mitchinson & Wells, 1989;Pace et al, 1988;Zavodszky et al, 2001), including antibodies (Goto & Hamaguchi, 1979;McAuley et al, 2008;Thies et al, 2002). Threedimensional folding imposed by disul de bonds is critical for the structure and function of these molecules, many of which have signi cant clinical value.…”

Section: Introductionmentioning

confidence: 99%

Structural Characterization of Disulfide-Linked p53-Derived Peptide Dimers

DiGiorno,

Vithanage,

Victorio

et al. 2024

Preprint

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Disulfide bonds provide a convenient method for chemoselective alteration of peptide and protein structure and function. We previously reported that mild oxidation of a p53-derived bisthiol peptide (CTFANLWRLLAQNC) under dilute non-denaturing conditions led to unexpected disulfide-linked dimers as the exclusive product. The dimers were antiparallel, significantly α-helical, resistant to protease degradation, and easily reduced back to the original bisthiol peptide. Here we examine the intrinsic factors influencing peptide dimerization using a combination of amino acid substitution, circular dichroism (CD) spectroscopy, and X-ray crystallography. CD analysis of peptide variants suggests critical roles for Leu6 and Leu10 in the formation of stable disulfide-linked dimers. The 1.0 Å resolution crystal structure of the peptide dimer supports these data, revealing a leucine-rich LxxLL dimer interface with canonical knobs-into-holes packing. Two levels of higher-order oligomerization are also observed in the crystal: an antiparallel “dimer of dimers” mediated by Phe3 and Trp7 residues in the asymmetric unit and a tetramer of dimers mediated by Trp7 and Leu10. In CD spectra of Trp-containing peptide variants, minima at 227 nm provide evidence for the dimer of dimers in dilute aqueous solution. Importantly, and in contrast to the original dimer model, the canonical leucine-rich core and robust dimerization of most peptide variants suggests a tunable molecular architecture to target various proteins and evaluate how folding and oligomerization impact various properties, such as cell permeability.

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