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The inhibitors of DNA binding Id1-4 are helix-loop-helix (HLH) proteins that exert their biological function by interacting with members of the basic-HLH (bHLH) transcription-factor family. The HLH domains of the Id and bHLH proteins allow both self- and hetero-association. Due to their abnormal expression in cancer cells, the Id proteins are potential protein targets for cancer treatment. Suitable Id-protein inactivators should promote self-association and/or prevent hetero-association. In this work we evaluated the ability of the Id-protein HLH domain to recognize itself in form of short sequences extracted from the helical and loop regions. We performed a peptide scan of the Id1 HLH domain 64-106 based on three-residue overlapping octapeptides. Interaction of each octapeptide with the natively folded Id1 HLH domain was investigated by CD and fluorescence spectroscopy. The results from both techniques showed that the helix-based but not the loop-based octapeptides interacted with the Id1 HLH domain in the low-micromolar range. In contrast, a nitrotyrosine-containing analog of the Id1 HLH region, which was unable to reproduce the native-like conformation, quenched only the 2-amino-benzoyl-(Abz)-labeled loop-based octapeptides. This opposite self-recognition pattern suggests that the short helix-based and loop-based sequences should be able to distinguish different folding states of the Id1 HLH domain. This feature may be biologically relevant, as the Id proteins are predicted to behave as intrinsically disordered proteins, being in equilibrium between rapidly exchanging monomeric conformations and structurally better-defined homo-/heterodimers displaying the parallel four-helix bundle., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Peptide thioesters are important building blocks in the total synthesis of proteins and protein domains via fragment ligation. However, synthetic access of peptide thioesters still is a bottleneck of this powerful ligation chemistry. The commonly used methods for the Fmoc-based synthesis of peptide thioesters involve nonautomated solution steps that have to be performed after the solid-phase assembly of the peptide. Usually, HPLC purification is required. Herein, a method that enables crude peptides to be used in divergent native chemical ligations reactions is described. We present an Fmoc-based solid-phase synthesis of peptide thioesters with self-purification which facilitates access to these important building blocks, since the often cumbersome HPLC purification can be avoided. Fmoc-protected amino acids are coupled on a safety catch sulfonamide resin. The self-purifying effect is achieved through the combination of (a) N-terminal coupling of a cleavable cyclization linker and subsequent backbone-to-side chain cyclization, (b) activation of the sulfonamide linkage by alkylation, (c) thiolysis for the selective detachment of truncation products, and (d) TFA cleavage for the liberation of the desired peptide thioester in unprotected form. We have previously shown a method wherein cyclization was performed after carboxymethylation of the sulfonamide. However, the automation of this method was difficult and side reactions at methionine residues hampered the general applicability. The new design involves peptide synthesis on a modified carboxy-functionalized sulfonamide linker, a substantially milder activation of the sulfonamide bond and the use of monomethoxytrityl as well as 2-phenyl-isopropyl protecting groups. This approach solved the problems with methionine containing peptides and enabled the complete automation of the self-purifying synthesis of peptide thioesters. The study also addressed problems in the synthesis of difficult peptides. Aggregated truncation products can resist extraction and contaminate full-length thioesters obtained after TFA cleavage. It is shown that significant enhancements of the purity were achieved when mild acidic extractions were included in the wash protocols after thiolysis. The potential of the method was demonstrated in the parallel synthesis of 20-40 amino acid long peptide thioesters, which were obtained in excellent purities. The thioesters and cysteinyl peptides were used without purification in the assembly of immobilized SH3 protein domains of SHO1 in yeast. A cysteine scan by native chemical ligation suggested single amino acid to cysteine substitutions that (a) confer useful ligation yields, (b) support correct folding, and (c) sustain the function of the folded protein domain. The chemical synthesis of the SH3-domain of SHO1 succeeded in highest yields when cysteine placements at positions S23, F24, and E36 were avoided. The synthetic SH3 mutants were examined in a binding assay, which indicated that N27C, L30C, and D34C mutations provide functional SH3-domain.

Synthetic peptides reproducing the helix-loop-helix (HLH) domains of the Id proteins fold into highly stable helix bundles upon self-association. Recently, we have shown that the replacement of the dipeptide Val-Ser at the loop-helix-2 junction with the corresponding O-acyl iso-dipeptide leads to a completely unfolded state that only refolds after intramolecular O --> N acyl migration. Herein, we report on an Id HLH analog based on the substitution of the Pro-Ser motif at the helix-1-loop junction with the corresponding O-acyl iso-dipeptide. This analog has been successfully synthesized by solid-phase Fmoc chemistry upon suppression of DKP formation. No secondary structure could be detected for the O-acyl iso-peptide before its conversion into the native form by O --> N acyl shift. These results show that the loop-helix junctions are determinant for the folded/unfolded state of the Id HLH domain. Further, despite the high risk of DKP formation, peptides containing O-acyl iso-Pro-Ser/Thr units are synthetically accessible by Fmoc chemistry., ((c) 2010 European Peptide Society and John Wiley & Sons, Ltd.)