Phosphorylation and dephosphorylation of proteins by kinases and phosphatases are central to cellular responses and function. The structural effects of serine and threonine phosphorylation were examined in peptides and in proteins, by circular dichroism, NMR spectroscopy, bioinformatics analysis of the PDB, small-molecule X-ray crystallography, and computational investigations. Phosphorylation of both serine and threonine residues induces substantial conformational restriction in their physiologically more important dianionic forms. Threonine exhibits a particularly strong disorder-to-order transition upon phosphorylation, with dianionic phosphothreonine preferentially adopting a cyclic conformation with restricted φ (φ ~ –60 ̊) stabilized by three noncovalent interactions: a strong intraresidue phosphate-amide hydrogen bond, an n→π* interaction between consecutive carbonyls, and an n→σ* interaction
between the phosphate Oγ lone pair and the antibonding orbital of C–Hβ that restricts the χ2 side chain conformation. Proline is unique among the canonical amino acids for its covalent cyclization on the backbone. Phosphothreonine can mimic proline's backbone cyclization via noncovalent interactions. The preferred torsions of dianionic phosphothreonine are φ,ψ = polyproline II helix > α-helix (φ ~ –60 ̊); χ1 = g–; χ2 ~ +115 ̊ (eclipsed C–H/O–P bonds). This structural signature is observed in diverse proteins, including in the activation loops of protein kinases and in protein-protein interactions. In total, these results suggest a structural basis for the differential use and evolution of threonine versus serine phosphorylation sites in proteins, with serine phosphorylation typically inducing smaller, rheostat-like changes, versus threonine phosphorylation promoting larger, step function-like switches, in proteins.
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Synthesis and conformation o fbackbone N-aminated peptides
The chemical modification of peptides is a promising approach for the design of protein-protein interaction inhibitors and peptide-based drug candidates. Among several peptidomimetic strategies, substitution of the amide backbone maintains side-chain functionality that may be important for engagement of biological targets. Backbone amide substitution has been largely limited to N-alkylation, which can promote cis amide geometry and disrupt important H-bonding interactions. In contrast, N-amination of peptides induces distinct backbone geometries and maintains H-bond donor capacity. In this chapter we discuss the conformational characteristics of designed N-amino peptides and present a detailed protocol for their synthesis on solid support. The described methods allow for backbone N-amino scanning of biologically active parent sequences.
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- Award ID(s):
- 2021265
- NSF-PAR ID:
- 10303791
- Date Published:
- Journal Name:
- Methods in enzymology
- Volume:
- 656
- ISSN:
- 1557-7988
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Peptide backbone amide substitution can dramatically alter the conformational and physiochemical properties of native sequences. Although uncommon relative to N -alkyl substituents, peptides harboring main-chain N -hydroxy groups exhibit unique conformational preferences and biological activities. Here, we describe a versatile method to prepare N -hydroxy peptide on solid support and evaluate the impact of backbone N -hydroxylation on secondary structure stability. Based on previous work demonstrating the β-sheet-stabilizing effect of α-hydrazino acids, we carried out an analogous study with N -hydroxy-α-amino acids using a model β-hairpin fold. In contrast to N -methyl substituents, backbone N -hydroxy groups are accommodated in the β-strand region of the hairpin without energetic penalty. An enhancement in β-hairpin stability was observed for a di- N -hydroxylated variant. Our results facilitate access to this class of peptide derivatives and inform the use of backbone N -hydroxylation as a tool in the design of constrained peptidomimetics.more » « less
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The ability to understand the function of a protein often relies on knowledge about its detailed structure. Sometimes, seemingly insignificant changes in the primary structure of a protein, like an amino acid substitution, can completely disrupt a protein's function. Long-lived proteins (LLPs), which can be found in critical areas of the human body, like the brain and eye, are especially susceptible to primary sequence alterations in the form of isomerization and epimerization. Because long-lived proteins do not have the corrective regeneration capabilities of most other proteins, points of isomerism and epimerization that accumulate within the proteins can severely hamper their functions and can lead to serious diseases like Alzheimer's disease, cancer and cataracts. Whereas tandem mass spectrometry (MS/MS) in the form of collision-induced dissociation (CID) generally excels at peptide characterization, MS/MS often struggles to pinpoint modifications within LLPs, especially when the differences are only isomeric or epimeric in nature. One of the most prevalent and difficult-to-identify modifications is that of aspartic acid between its four isomeric forms: l -Asp, l -isoAsp, d -Asp, and d -isoAsp. In this study, peptides containing isomers of Asp were analyzed by charge transfer dissociation (CTD) mass spectrometry to identify spectral features that could discriminate between the different isomers. For the four isomers of Asp in three model peptides, CTD produced diagnostic ions of the form c n +57 on the N-terminal side of iso-Asp residues, but not on the N-terminal side of Asp residues. Using CTD, the l - and d forms of Asp and isoAsp could also be differentiated based on the relative abundance of y - and z ions on the C-terminal side of Asp residues. Differentiation was accomplished through a chiral discrimination factor, R , which compares an ion ratio in a spectrum of one epimer or isomer to the same ion ratio in the spectrum of a different epimer or isomer. The R values obtained using CTD are as robust and statistically significant as other fragmentation techniques, like radical directed dissociation (RDD). In summary, the extent of backbone and side-chain fragments produced by CTD enabled the differentiation of isomers and epimers of Asp in a variety of peptides.more » « less
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Abstract Here, we report a novel “CyClick” strategy for the macrocyclization of peptides that works in an exclusively intramolecular fashion thereby precluding the formation of dimers and oligomers via intermolecular reactions. The CyClick chemistry is highly chemoselective for the N‐terminus of the peptide with a C‐terminal aldehyde. In this protocol, the peptide conformation internally directs activation of the backbone amide bond and thereby facilitates formation of a stable 4‐imidazolidinone‐fused cyclic peptide with high diastereoselectivity (>99 %). This method is tolerant to a variety of peptide aldehydes and has been applied for the synthesis of 12‐ to 23‐membered rings with varying amino acid compositions in one pot under mild reaction conditions. The reaction generated peptide macrocycles featuring a 4‐imidazolidinone in their scaffolds, which acts as an endocyclic control element that promotes intramolecular hydrogen bonding and leads to macrocycles with conformationally rigid turn structures.
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1016/bs.mie.2021.04.013
