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The elucidation of internal dynamics in proteins is essential for the understanding of their stability and functionality. Breaking the symmetry of the degenerate rotation of the phenyl side chain provides additional structural information and allows a detailed description of the dynamics. Based on this concept, we propose a combination of synthetic and computational methods, to study the rotational mobility of the Phe ring in a sensitive zinc finger motif. The systematic methyl hopping around the phenylalanine ring yields o -, m -, p -tolyl and xylyl side chains that provide a vast array of additional NOE contacts, allowing the precise determination of the orientation of the aromatic ring. MD simulations and metadynamics complement these findings and facilitate the generation of free energy profiles for each derivative. Previous studies used a wide temperature window in combination with NMR spectroscopy to elucidate the side chain mobility of stable proteins. The zinc finger moiety exhibits a limited thermodynamic stability in a temperature range of only 40 K, making this approach impractical for this compound class. Therefore, we have developed a method that can be applied even to thermolabile systems and facilitates the detailed investigation of protein dynamics., Competing Interests: We declare no conflict of interest., (This journal is © The Royal Society of Chemistry.)

A generalized synthetic strategy is proposed here for the synthesis of asymmetric β-indoylated amino acids by 8-aminoquinoline (8AQ)-directed C(sp 3 )-H functionalization of suitably protected precursors. Peptides containing one of the four stereoisomers of (indol-3-yl)-3-phenylalanine at position 2 of the parent peptide KwFwLL-NH 2 (w=d-Trp) cover a wide range of activities as ghrelin receptor inverse agonists, among them the most active described until now. This application exemplarily shows how β-indoylated amino acids can be used for the systematic variation of the position of an indole group in a bioactive peptide., (© 2020 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

A designed disulfide-rich β-hairpin peptide that dimerizes spontaneously served as a hinge-type connection between proteins. Here, we analyze the range of dynamics of this hinge dimer with the aim of proposing new applications for the DNA-encodable peptide and establishing guidelines for the computational analysis of other disulfide hinges. A recent structural analysis based on nuclear magnetic resonance spectroscopy and ion mobility spectrometry revealed an averaged conformation in the hinge region which motivated us to investigate the dynamic behavior using a combination of molecular dynamics simulation, metadynamics and free energy surface analysis to characterize the conformational space available to the hinge. Principal component analysis uncovered two slow modes of the peptide, namely, the opening and closing motion and twisting of the two β-hairpins assembling the hinge. Applying a collective variable (CV) that mimics the first dominating mode, led to a major expansion of the conformational space. The description of the dynamics could be achieved by analysis of the opening angle and the twisting of the β-hairpins and, thus, offers a methodology that can also be transferred to other derivatives. It has been demonstrated that the hinge peptide's lowest energy conformation consists of a large opening angle and strong twist but is separated by small energy barriers and can, thus, adopt a closed and untwisted structure. With the aim of proposing further applications for the hinge peptide, we simulated its behavior in the sterically congested environment of a four-helix bundle. Preliminary investigations show that one helix is pushed out and a three-helix bundle forms. The insights gained into the dynamics of the tetra-disulfide peptide and analytical guidelines developed in this study may contribute to the understanding of the structure and function of more complex hinge-type proteins, such as the IgG antibody family., Competing Interests: The authors declare that no competing interests exist.

A pair of intermolecular disulfide bonds connecting two protein domains restricts their relative mobility in a systematic way. The bi-disulfide hinge cannot rotate like a single intermolecular disulfide bond yet is less restrained than three or more intermolecular disulfides which restrict the relative motion to a minimum. The intermediate mobility of bi-disulfide linked domains is characterized by their dominating opening and closing modes comparable to the mechanics of a door hinge on the macroscopic scale. Here we compare the central hinge region of Immunoglobulin G1 (IgG1) which is highly conserved among different species, with a recently designed hinge-type motif CHWECRGCRLVC from our lab, that was successfully used for the dimerization of the IgG1/κ-ab CL4 monocolonal antibody (mab). The minimal length of these synthetic hinges comprises only 12 amino acids, rendering them ideal models for computational studies. Well-tempered metadynamics was performed to adequately describe the available conformational space defined by the different hinges. In spite of the differences in amino acid composition and ring sizes, there are characteristic similarities of designed and natural hinges like the dependent mobility of the individual strands of each hinge domain., (Copyright © 2020 Horx and Geyer.)

The 8-aminoquinoline (8AQ) directed C(sp 3 )-H functionalization was applied in the synthesis of β-arylated tryptophan derivatives. The laborious protecting group reorganization towards α-amino acids compatible for solid phase peptide synthesis (SPPS) was cut short by the transformation of the directing group into an activated amide, which was either used directly in peptide coupling or in the gram scale synthesis of storable Fmoc-protected amino acids for SPPS. In this work, directed C-H activation and nonplanar amide chemistry complement each other for the synthesis of hybrids between phenylalanine and tryptophan with restricted side chain mobility., (This journal is © The Royal Society of Chemistry 2019.)

Maleimide-thiol coupling is a popular bioconjugation strategy, but little is known about the stereoselectivity and the stereodynamics of the succinimide thioether formed in a biopolymer environment. We used thiol 1,4-addition for the macrocyclisation of 5 designed pentapeptides with the ringsize of hexapeptides because they incorporate the succinimide thioether (4-8). Both succinimide diastereomers are observed in the constrained macrocyclic rings in each case. In spite of the low diastereoselectivity of the macrocyclisation reaction, there is a significant influence of the amino acid environment on the epimerization rate of the succinimide. Its half life can be as short as several hours at room temperature when Gly is the amino acid following the succinimide (peptide 8). On the contrary, no epimerization is detectable even after several weeks in the case of d-Phe C-terminal to the succinimide in peptide 4. Already the small selection of examples shows how big the differences in epimerization rates can be and that the local environment has a significant influence. The variation of amino acids in the vicinity of the ligation site points the way towards the synthesis of bioconjugates which are obtained as stable and separable diastereomers., (Copyright © 2018 European Peptide Society and John Wiley & Sons, Ltd.)