![]() The potency and portability of disulfide-rich peptides have attracted much attention from the growing biotechnology sector as a potential source of leads for development of therapeutics or agricultural agents ( Gongora-Benitez et al., 2014). This property is perhaps most dramatically demonstrated in venom peptides, where disulfide-rich peptide toxins are not only excreted but further injected into a foreign host where they exert their function, often with devastating consequences ( Undheim et al., 2015). In these molecules, the disulfide bonds serve to stabilize the protein fold in the extracellular environment. The significance of disulfide bonds in proteins can be appreciated by their prevalence accounting for ~18% of all known protein structures (9,709 of the 55,032 proteins deposited in the PDB contain at least one disulfide bond – excluding structures with >90% identity, PDB accessed ).ĭisulfide-rich peptides and proteins are commonly secreted, and include biopharmaceutical targets such as hormones and antibodies ( Lewis and Garcia, 2003 Mamathambika and Bardwell, 2008 Gongora-Benitez et al., 2014). The observed improvements in resolution when using RDCs is remarkable considering the small size of these peptides.ĭisulfide bridges are naturally occurring cross-links formed between the side chains of two cysteine residues and are one of the most important post-translational modifications in proteins. At this resolution the sidechain of ordered amino acids can be defined accurately, allowing the geometry of the cysteine bridges to be better defined, and allowing for disulfide-bond connectivities to be determined with high confidence. We find that structures based primarily on NOEs, yield ensembles that are equivalent to a crystallographic resolution of 2-3 Å in resolution, and that incorporation of RDCs reduces this to ~1-1.5 Å resolution. Here, we use an extensive set of long-range residual dipolar couplings (RDCs) to assess the resolution of the NMR structure of a disulfide-rich peptide. Given the central role of disulfide bonds in the structure of these molecules, it is unclear what the inherent resolution of such NMR structures is when using traditional NMR methods. However, in NMR the sulfur atoms that are involved in three of the five dihedral angles in a disulfide bond cannot be readily measured. NMR is the most commonly used method for studying such molecules, where the relatively small size of these molecules results in highly precise structural ensembles defined by a large number of distance and dihedral angle restraints per amino acid. Structural characterization of small disulfide rich peptides (DRPs) presents unique challenges when using commonly applied biophysical methods. These structural scaffolds have, therefore, proven to be very attractive in bioengineering efforts to develop novel biologics with applications in health and agriculture. The arrangement of the multiple disulfide bonds directs the peptide fold into distinct structural motifs that have evolved for resistance against biochemical and physical insults. These cross-links play a critical role in stabilizing the 3D-structure of small disulfide rich polypeptides such as hormones and venom toxins. 2Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD, United Statesĭisulfide bridges in proteins are formed by the oxidation of pairs of cysteine residues.1Centre for Advanced Imaging, The University of Queensland, St Lucia, QLD, Australia.Venkatraman Ramanujam 1,2 Yang Shen 2 Jinfa Ying 2 Mehdi Mobli 1 *
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