Diana Imhof

The experimental power of FR900359 to study Gq-regulated biological processes

Despite the discovery of heterotrimeric αβγ G proteins ∼25 years ago, their selective perturbation by cell-permeable inhibitors remains a fundamental challenge. Here we report that the plant-derived depsipeptide FR900359 (FR) is ideally suited to this task. Using a multifaceted approach we systematically characterize FR as a selective inhibitor of Gq/11/14 over all other mammalian Gα isoforms and elaborate its molecular mechanism of action. We also use FR to investigate whether inhibition of Gq proteins is an effective post-receptor strategy to target oncogenic signalling, using melanoma as a model system. FR suppresses many of the hallmark features that are central to the malignancy of melanoma cells, thereby providing new opportunities for therapeutic intervention. Just as pertussis toxin is used extensively to probe and inhibit the signalling of Gi/o proteins, we anticipate that FR will at least be its equivalent for investigating the biological relevance of Gq.

Sequence Specificity of SHP-1 and SHP-2 Src Homology 2 Domains CRITICAL ROLES OF RESIDUES BEYOND THE pY+3 POSITION

A combinatorial phosphotyrosyl (pY) peptide library was screened to determine the amino acid preferences at the pY+4 to pY+6 positions for the four SH2 domains of protein-tyrosine phosphatases SHP-1 and SHP-2. Individual binding sequences selected from the library were resynthesized and their binding affinities and specificities to various SH2 domains were further evaluated by SPR studies, stimulation of SHP-1 and SHP-2 phosphatase activity, and in vitro pulldown assays. These studies reveal that binding of a pY peptide to the N-SH2 domain of SHP-2 is greatly enhanced by a large hydrophobic residue (Trp, Tyr, Met, or Phe) at the pY+4 and/or pY+5 positions, whereas binding to SHP-1 N-SH2 domain is enhanced by either hydrophobic or positively charged residues (Arg, Lys, or His) at these positions. Similar residues at the pY+4 to pY+6 positions are also preferred by SHP-1 and SHP-2 C-SH2 domains, although their influence on the overall binding affinities is much smaller compared with the N-SH2 domains. A structural model was generated to qualitatively interpret the contribution of the pY+4 and pY+5 residues to the overall binding affinity. Examination of pY motifs from known SHP-1 and SHP-2-binding proteins shows that many of the pY motifs contain a hydrophobic or positively charged residue(s) at the pY+4 and pY+5 positions.

On the nature of interactions between ionic liquids and small amino-acid-based biomolecules.

During the last decade, ionic liquids (ILs) have revealed promising properties and applications in many research fields, including biotechnology and biological sciences. The focus of this contribution is to give a critical review of the phenomena observed and current knowledge of the interactions occurring on a molecular basis. As opposed to the huge advances made in understanding the properties of proteins in ILs, complementary investigations dealing with interactions between ILs and peptides or oligopeptides are underrepresented and are mostly only of phenomenological nature. However, the field has received more attention in the last few years. This Review features a meta-analysis of the available data and findings and should, therefore, provide a basis for a scientifically profound understanding of the nature and mechanisms of interactions between ILs and structured or nonstructured peptides. Fundamental aspects of the interactions between different peptides/oligopeptides and ILs are complemented by sections on the experimental (spectroscopy, structural biology) and theoretical (computational chemistry) possibilities to explain the phenomena reported so far in the literature. In effect, this should lead to the development of novel applications and support the understanding of IL-solute interactions in general.

Ionic Liquid Applications in Peptide Chemistry: Synthesis, Purification and Analytical Characterization Processes

This review aims to provide a comprehensive overview of the recent advances made in the field of ionic liquids in peptide chemistry and peptide analytics.

New Insight into the Mode of Action of Nickel Superoxide Dismutase by Investigating Metallopeptide Substrate Models

For the first time, the existence of a substrate adduct of a nickel superoxide dismutase (NiSOD) model, based on the first nine residues from the N terminus of the active form of Streptomyces coelicolor NiSOD, has been proven and the adduct has been isolated. This adduct is based on the cyanide anion (CN(-)), as a substrate analogue of the superoxide anion (O(2)(*-)), and the nickel metallopeptide H-HCDLPCGVY-NH(2)-Ni. Spectroscopic studies, including IR, UV/Vis, and liquid- and solid-state NMR spectroscopy, show a single nickel-bound cyanide anion, which is embedded in the metallopeptide structure. This complex sheds new light on the question of whether the mode of action of the NiSOD enzyme is an inner- or outer-sphere mechanism. Whereas discussion was previously biased in favor of an outer-sphere electron-transfer mechanism due to the fact that binding of cyanide or azide moieties to the nickel active site had never been observed, our results are a clear indication in favor of the inner-sphere electron-transfer mechanism for the disproportionation of the O(2)(*-) ion, whereby the substrate is attached to the Ni atom in the active site of the NiSOD.

Development of a Functional cis‐Prolyl Bond Biomimetic and Mechanistic Implications for Nickel Superoxide Dismutase

During recent years several peptide-based Ni superoxide dismutase (NiSOD) models have been developed. These NiSOD models show an important structural difference compared to the native NiSOD enzyme, which could cause a completely different mechanism of superoxide dismutation. In the native enzyme the peptide bond between Leu4 and Pro5 is cis-configured, while the NiSOD models exhibit a trans-configured peptide bond between these two residues. To shed light on how the configuration of this single peptide bond influences the activity of the NiSOD model peptides, a new cis-prolyl bond surrogate was developed. As surrogate we chose a leucine/alanine-based disubstituted 1,2,3-triazole, which was incorporated into the NiSOD model peptide replacing residues Leu4 and Pro5. The yielded 1,5-disubstituted triazole nickel peptide exhibited high SOD activity, which was approximately the same activity as its parent trans-configured analogue. Hence, the conformation of the prolyl peptide bond apparently has of minor importance for the catalytic activity of the metallopeptides as postulated in literature. Furthermore, it is shown that the triazole metallopeptide is forming a stable cyanide adduct as a substrate analogue model complex.

Structurally Diverse μ‐Conotoxin PIIIA Isomers Block Sodium Channel NaV1.4

Certain VGSC subtypes (NaV1.3, 1.7, 1.8, and 1.9) are expressed in the peripheral nervous system and mediate the transmission of signals leading to the sensation of different kinds of pain, such as nociception (NaV1.8), acute inflammatory (NaV1.7), and neuropathic (NaV1.3) pain. [2] Therefore, VGSCs are potential targets for novel analgesics, ideally those with strong channel specificity. Among sodium channel antagonists, m- and mO-conotoxins from the venoms of marine cone snails have attracted considerable attention because of their analgesic potency. [2a, 3] m-Conotoxins are 14to 26-mer peptides with six cysteine residues (Supporting Information, Table S1). [4] They inhibit muscle and/or neuronal VGSCs by occluding the ion channel pore. [5] A specific cysteine framework, that is, CCXnCXnCXnCC, confers conformational restriction to their three-dimensional structure upon formation of three disulfide bonds. It is generally accepted that the native fold of the toxins carries the disulfide connectivities Cys1–Cys4, Cys2–Cys5, and Cys3–Cys6 (numbered in the order of occurrence in the amino acid sequence). [3, 5b] However, three-dimensional structures are available only for a limited subset of m-conotoxins, that is, PIIIA, [6]

Development of conformationally restricted analogues of bradykinin and somatostatin using constrained amino acids and different types of cyclization.

The structure-based design of peptide drugs requires the knowledge of the bioactive conformation. Studies on this receptor-bound 3D structure require linear or cyclic analogues with strongly reduced flexibility, but high biological activity, since only analogues with retained potency have preserved the bioactive conformation. Constrained amino acids containing double bonds or bulky substituents at the N(alpha)-, C(alpha)- and C(beta)-atom as well as at the aromatic ring atom were successfully applied to obtain potent and stable analogues of bradykinin and somatostatin, which due to their restricted conformation were suitable objects for conformational studies. Besides the generation of constrained cyclic analogues with improved biological and pharmacological properties, cyclic peptides were used as convenient models for the study of turn formations. Cyclization of the linear peptide bradykinin was performed by linking the N-terminus and the C-terminus, and in both bradykinin and somatostatin by cyclization using the amino acid side chains and by backbone cyclization. The later requires the introduction of N(alpha)-functionalised amino acids for ring closure which can be performed either through incorporation of N(alpha)-functionalised amino acids or dipeptide building units. Conformational analysis of a cyclic bradykinin analogue by means of NMR-studies together with molecular dynamics simulation led to a quasicyclic 3D structure with two turns and together with other 3D structures provided a pharmacophore model of bradykinin antagonists.

Analysis of Fe(III) Heme Binding to Cysteine-Containing Heme-Regulatory Motifs in Proteins

Regulatory heme binds to specific motifs in proteins and controls a variety of biochemical processes. Several of these proteins were recently shown to form complexes with ferric and/or ferrous heme via a cysteine residue as axial ligand. The objective of this study was to examine the heme-binding properties of a series of cysteine-containing peptides with focus on CP motif sequences. The peptides displayed different binding behavior upon Fe(III) heme application with characteristic wavelength shifts of the Soret band to 370 nm or 420-430 nm and in some cases to both wavelengths. Whereas for most of the peptides containing a cysteine only a shift to 420-430 nm was observed, CP-containing peptides exhibited a preference for a shift to 370 nm. Detailed structural investigation using Raman and NMR spectroscopy on selected representatives revealed different binding modes with respect to iron ion coordination, which reflected the results of the UV-vis studies. A predicted short sequence stretch derived from dipeptidyl peptidase 8 was additionally examined with respect to CP motif binding to heme on the peptide as well as on the protein level. The heme association was confirmed with the first solution structure of a CP-peptide-heme complex and, moreover, an inhibitory effect of Fe(III) heme on the enzymes activity. The relevance of both the use of model compounds to elucidate the molecular mechanism underlying regulatory heme binding and its potential for the investigation of regulatory heme control is discussed.

A room temperature ionic liquid as convenient solvent for the oxidative folding of conopeptides

We report the first example of conopeptide oxidation performed in a biocompatible ionic liquid, 1‐ethyl‐3‐methylimidazolium acetate ([C2mim][OAc]), which enables the efficient formation of both hydrophilic and poorly water‐soluble conotoxins compared with conventional methods. Moreover, the method features a high‐concentration approach ultimately leading to higher yields at reduced separation effort. Copyright