Rapid Formation of Intramolecular Disulfide Bridges using Light: An Efficient Method to Control the Conformation and Function of Bioactive Peptides.

Disulfide bonds are widely found in natural peptides and play a pivotal role in stabilizing their secondary structures, which are highly associated with their biological functions. Herein, we introduce a light-mediated strategy to effectively control the formation of disulfides. Our strategy is based on 2-nitroveratryl (oNv), a widely used photolabile motif, which serves both as a photocaging group and an oxidant (after photolysis). We demonstrated that irradiation of oNv-caged thiols with UV light could release free thiols that are rapidly oxidized by locally released byproduct nitrosoarene, leading to a "break-to-bond" fashion. This strategy is highlighted by the in situ restoration of the antimicrobial peptide tachyplesin I (TPI) from its external disulfide-caged analogue TPI-1. TPI-1 exhibits a distorted structure and a diminished function. However, upon irradiation, the ß-hairpin structure and membrane activity of TPI were largely restored via rapid intramolecular disulfide formation. Our study proposes a powerful method to regulate the conformation and function of peptides in a spatiotemporal manner, which has significant potential for the design of disulfide-centered light-responsive systems.

Inviting C5-Trifluoromethylated Pseudoprolines into Collagen Mimetic Peptides.

Numerous collagen mimetic peptides (CMPs) have been engineered using proline derivatives substituted at their C(3) and/or C(4) position in order to stabilize or functionalize collagen triple-helix mimics. However, no example has been reported so far with C(5) substitutions. Here, we introduce a fluorinated CMP incorporating trifluoromethyl groups at the C(5) position of pseudoproline residues. In tripeptide models, our CD, NMR, and molecular dynamics (MD) studies have shown that, when properly arranged, these residues meet the structural requirements for a triple-helix assembly. Two host-guest CMPs were synthesized and analyzed by CD spectroscopy. The NMR analysis in solution of the most stable confirmed the presence of structured homotrimers that we interpret as triple helices. MD calculations showed that the triple-helix model remained stable throughout the simulation with all six trifluoromethyl groups pointing outward from the triple helix. Pseudoprolines substituted at the C(5) positions appeared as valuable tools for the design of new fluorinated collagen mimetic peptides.

Customized Reversible Stapling for Selective Delivery of Bioactive Peptides.

We have developed a new concept for reversible peptide stapling that involves macrocyclization between two amino groups and decyclization promoted via dual 1,4-elimination. Depending on the trigger moiety, this strategy could be employed to selectively deliver peptides to either intracellular or extracellular targets. As a proof of concept, a peptide inhibitor targeting a lysine-specific demethylase 1 (LSD1) was temporarily cyclized to enhance its stability and ability to cross the cell membrane. Once inside the cells, the biologically active linear peptide was released under reducing environment. Moreover, we have developed reversibly stapled peptides using antimicrobial peptides (RStAMPs) whose bioactive helical conformation can be temporarily destabilized by stapling the peptide backbone. The resulting helix-distorted RStAMPs are nontoxic and highly resistant to protease hydrolysis, while at the infection site, RStAMPs can be rapidly activated by the overproduced H2O2 through the dual 1,4-elimination. The latter restored the helical structure of the native peptide and its antimicrobial activity. This work illustrates a highly valuable macrocyclization strategy for the peptide community and should greatly benefit the field of peptide delivery.

Dynamic Amino Acid Side-Chains Grafting on Folded Peptide Backbone.

An efficient strategy for the synthesis of large libraries of conformationally defined peptides is reported, using dynamic combinatorial chemistry as a tool to graft amino acid side chains on a well-ordered 3D (3-dimension) peptide backbone. Combining rationally designed scaffolds with combinatorial side chains selection represents an alternative method to access peptide libraries for structures that are not genetically encodable. This method would allow a breakthrough for the discovery of protein mimetic for unconventional targets for which little is known.

In Vitro Production of Perdeuterated Proteins in H2O for Biomolecular NMR Studies.

The cell-free synthesis is an efficient strategy to produce in large scale protein samples for structural investigations. In vitro synthesis allows for significant reduction of production time, simplification of purification steps and enables production of both soluble and membrane proteins. The cell-free reaction is an open system and can be performed in presence of many additives such as cofactors, inhibitors, redox systems, chaperones, detergents, lipids, nanodisks, and surfactants to allow for the expression of toxic membrane proteins or intrinsically disordered proteins. In this chapter we present protocols to prepare E. coli S30 cellular extracts, T7 RNA polymerase, and their use for in vitro protein expression. Optimizations of the protocol are presented for preparation of protein samples enriched in deuterium, a prerequisite for the study of high-molecular-weight proteins by NMR spectroscopy. An efficient production of perdeuterated proteins is achieved together with a full protonation of all the amide NMR probes, without suffering from residual protonation on aliphatic carbons. Application to the production of the 468 kDa TET2 protein assembly for NMR investigations is presented.

Gramicidin-S-Inspired Cyclopeptidomimetics as Potent Membrane-Active Bactericidal Agents with Therapeutic Potential.

Antimicrobial peptides (AMPs) are promising antibacterial agents often hindered by their undesired hemolytic activity. Inspired by gramicidin S (GS), a well-known cyclodecapeptide, we synthesized a panel of antibacterial cyclopeptidomimetics using ß,γ-diamino acids (ß,γ-DiAAs). We observed that peptidomimetic CP-2 displays a bactericidal activity similar to that of GS while possessing lower side-effects. Moreover, extensive studies revealed that CP-2 likely kills bacteria through membrane disruption. Altogether, CP-2 is a promising membrane-active antibiotic with therapeutic potential.

Development of Therapeutic Gramicidin S Analogues Bearing Plastic ß,γ-Diamino Acids.

Gramicidin S (GS), one of the most widely investigated antimicrobial peptides (AMPs), is known for its robust antimicrobial activity. However, it is restricted to topical application due to undesired hemolytic activity. With the aim of obtaining nontoxic GS analogues, we describe herein a molecular approach in which the native GS ß-turn region is replaced by synthetic ß,γ-diamino acids (ß,γ-DiAAs). Four ß,γ-DiAA diastereomers were employed to mimic the ß-turn structure to afford GS analogues GS3-6, which exhibit diminished hemolytic activity. A comparative structural study demonstrates that the (ßR,γS)-DiAA is the most-stable ß-turn mimic. To further improve the therapeutic index (e. g., high antibacterial activity and low hemolytic activity) and to extend the molecular diversity, GS5 and GS6 were used as structural scaffolds to introduce additional hydrophobic or hydrophilic groups. We show that GS6K, GS6F and GS display comparable antibacterial activity, and GS6K and GS6F have significantly decreased toxicity. Moreover, antibacterial mechanism studies suggest that GS6K kills bacteria mainly through the disruption of the membrane.

Unexpected dimerization of a tripeptide comprising a ß,γ-diamino acid.

We have previously reported the synthesis of enantiopure ß,γ-diamino acids and relevant short α/γ-peptide containing such building blocks. Complete nuclear magnetic resonance (NMR) studies, together with molecular modeling, highlighted the ability of a ß,γ-diamino acid to induce various intramolecular turns. In this paper, we describe for the first time the formation of a dimeric structure constituted by α/γ/α-tripeptide and stabilized by intermolecular interactions. A structural model is proposed based on extensive NMR measurements.

Targeting the Pentose Phosphate Pathway: Characterization of a New 6PGL Inhibitor.

Human African trypanosomiasis, or sleeping sickness, is a lethal disease caused by the protozoan parasite Trypanosoma brucei. However, although many efforts have been made to understand the biochemistry of this parasite, drug development has led to treatments that are of limited efficiency and of great toxicity. To develop new drugs, new targets must be identified, and among the several metabolic processes of trypanosomes that have been proposed as drug targets, carbohydrate metabolism (glycolysis and the pentose phosphate pathway (PPP)) appears as a promising one. As far as the PPP is concerned, a limited number of studies are related to the glucose-6-phosphate dehydrogenase. In this work, we have focused on the activity of the second PPP enzyme (6-phospho-gluconolactonase (6PGL)) that transforms 6-phosphogluconolactone into 6-phosphogluconic acid. A lactam analog of the natural substrate has been synthesized, and binding of the ligand to 6PGL has been investigated by NMR titration. The ability of this ligand to inhibit 6PGL has also been demonstrated using ultraviolet experiments, and protein-inhibitor interactions have been investigated through docking calculations and molecular dynamics simulations. In addition, a marginal inhibition of the third enzyme of the PPP (6-phosphogluconate dehydrogenase) was also demonstrated. Our results thus open new prospects for targeting T. brucei.

Rates of Chemical Reactions Embedded in a Metabolic Network by Dissolution Dynamic Nuclear Polarisation NMR.

The isomerisation of 6-phosphogluconolactones and their hydrolyses into 6-phosphogluconic acid form a non enzymatic side cycle of the pentose-phosphate pathway (PPP) in cells. Dissolution dynamic nuclear polarisation can be used for determining the kinetic rates of the involved transformations in real time. It is found that the hydrolysis of both lactones is significantly slower than the isomerisation process, thereby shedding new light onto this subtle chemical process.

Homochiral versus Heterochiral Trifluoromethylated Pseudoproline Containing Dipeptides: A Powerful Tool to Switch the Prolyl-Amide Bond Conformation.

The design of constrained peptides is of prime importance in the development of bioactive compounds and for applications in supramolecular chemistry. Due to its nature, the peptide bond undergoes a spontaneous cis-trans isomerism, and the cis isomers are much more difficult to stabilize than the trans forms. By using oxazolidine-based pseudoprolines (ΨPro) substituted by a trifluoromethyl group, we show that the cis peptide bond can be readily switched from 0% to 100% in Xaa-ΨPro dipeptides. Our results prove that changing the configuration of the Cα in Xaa or in ΨPro is sufficient to invert the cis:trans populations while changing the nature of the Xaa side chain finely tuned the conformers ratio. Moreover, a strong correlation is found between the puckering of the oxazolidine ring and the peptide bond conformation. This finding highlights the role of the trifluoromethyl group in the stabilization of the peptide bond geometry. We anticipate that such templates will be very useful to constrain the backbone geometry of longer peptides.

Application of Circular Dichroism Spectroscopy to the Analysis of the Interaction Between the Estrogen Receptor Alpha and Coactivators: The Case of Calmodulin.

The estrogen receptor α ligand-binding domain (ERα-LBD) binds the natural hormone 17ß-estradiol (E2) to induce transcription and cell proliferation. This process occurs with the contribution of protein and peptide partners (also called coactivators) that can modulate the structure of ERα, and therefore its specificity of action. As with most transcription factors, ERα exhibits a high content of α helix, making it difficult to routinely run spectroscopic studies capable of deciphering the secondary structure of the different partners under binding conditions. Ca(2+)-calmodulin, a protein also highly structured in α-helix, is a key coactivator for ERα activity. Here, we show how circular dichroism can be used to study the interaction of ERα with Ca(2+)-calmodulin. Our approach allows the determination not only of the conformational changes induced upon complex formation but also the dissociation constant (K d) of this interaction.

Β,γ-diamino acid: an original building block for hybrid α/γ-peptide synthesis with extra hydrogen bond donating group.

Using a ß,γ-diamino acid, several small hybrid α/γ peptides have been synthesized and their conformations investigated through extensive NMR studies and molecular dynamics. A tripeptide and a tetrapeptide have thus shown several hydrogen bonds in solution, including a 13-membered ring involving the ß-nitrogen.

Toward Quantitative Measurements of Enzyme Kinetics by Dissolution Dynamic Nuclear Polarization.

Dissolution dynamic nuclear polarization (D-DNP) experiments enabled us to study the kinetics of the enzymatic phosphorylation reaction of glucose to form glucose-6-phosphate (G6P) by hexokinase (HK), with or without the presence of an excess of G6P, which is known to be an inhibitor of the enzyme. Against all expectations, our observations demonstrate that the phosphorylation of both α and ß glucose anomers occurs with comparable kinetics. The catalytic constant of the reaction was estimated based on a simple kinetic model tailored for hyperpolarized systems.

Incorporation of CF3-pseudoprolines into peptides: a methodological study.

The peptide coupling reactions allowing the incorporation of trifluoromethyl substituted oxazolidine-type pseudoprolines (CF3-ΨPro) into peptide chains have been studied. While standard protocols can be used for the peptide coupling reaction at the C-terminal position of the CF3-ΨPro, acid chloride activation has to be used for the peptide coupling reaction at the N-terminal position to overcome the decrease of nucleophilicity of the CF3-ΨPro. We demonstrate that the N-amidification of a diastereomeric mixture of CF3-ΨPro using Fmoc-protected amino acid chloride without base gave the corresponding dipeptides as a single diastereomer (6 examples). The ratio of the cis and trans amide bond conformers was determined by NMR study, highlighting the role of the Xaa side chains in the control of the peptide backbone conformation. Finally a tripeptide bearing a central CF3-ΨPro has been successfully synthesized.

Original ß,γ-diamino acid as an inducer of a γ-turn mimic in short peptides.

Original αγα tripeptides containing one ß,γ-diamino acid have been synthesized and their conformation determined by extensive NMR and molecular dynamic studies. These studies revealed the presence of a C(9) hydrogen bonded turn around the ß,γ-diamino acid which was stabilized by bulky side chains of the preceding residue. This turn can be considered as a mimic of the well-known γ-turn.

Local control of the cis-trans isomerization and backbone dihedral angles in peptides using trifluoromethylated pseudoprolines.

NMR studies and theoretical calculations have been performed on model peptides Ac-Ser(ΨPro)-NHMe, (S,S)Ac-Ser(Ψ(H,CF3)Pro)-NHMe, and (R,S)Ac-Ser(Ψ(CF3,H)Pro)-NHMe. Their thermodynamic and kinetic features have been analyzed in chloroform, DMSO, and water, allowing a precise description of their conformational properties. We found that trifluoromethyl C(δ)-substitutions of oxazolidine-based pseudoprolines can strongly influence the cis-trans rotational barriers with only moderate effects on the cis/trans population ratio. In CHCl(3), the configuration of the CF(3)-C(δ) entirely controls the ψ-dihedral angle, allowing the stabilization of γ-turn-like or PPI/PPII-like backbone conformations. Moreover, in water and DMSO, this C(δ)-configuration can be used to efficiently constrain the ring puckering without affecting the cis/trans population ratio. Theoretical calculations have ascertained the electronic and geometric properties induced by the trifluoromethyl substituent and provided a rational understanding of the NMR observations.

Biophysical studies of the interaction between calmodulin and the R²87-T³¹¹ region of human estrogen receptor α reveals an atypical binding process.

The transcriptional activity of human estrogen receptor ERα is modulated by a number of coregulatory proteins among which calmodulin (CaM). Segment 295-311 in the hinge region of ERα has previously been proposed to be the CaM binding site. In this work, we investigate the molecular mechanism of the interaction of CaM with peptides derived from the hinge region of ERα, using a biophysical approach combining isothermal titration calorimetry, fluorescence, CD and NMR. The ERα17p peptide, corresponding to the previously identified 295-311 region of ERα, recruits mainly the C-terminal domain of Ca(4)CaM, as shown by NMR spectroscopy. In contrast, a longer peptide, ERα25p, extended on the N-terminal side (residues 287-311) interacts with both N- and C-terminal domains of Ca(4)CaM. These results lead to a new delineation of the CaM binding site, encompassing residues 287-294. In particular, fluorescence spectroscopy reveals that the conserved W(292) residue is engaged within hydrophobic pockets on Ca(4)CaM. ITC results show that ERα25p binds Ca(4)CaM with an atypical 2:1 stoichiometry and a dissociation constant in the micromolar range. Based on the NMR titration of Ca(4)CaM by ERα25p showing a biphasic behavior for several residues, we suggest that concerted conformational changes of CaM domains may be required to accommodate the binding of a second peptide. CD spectra indicate that ERα25p partially folds into an α-helix upon binding to Ca(4)CaM. Hence, ERα25p is a new CaM-binding ligand that could be appropriate for the synthesis of derivatives able to control ER-dependent transcription, particularly in the context of hormone-dependent breast tumors.

Constrained α/γ-peptides: a new stable extended structure in solution without any hydrogen bond and characterized by a four-fold symmetry.

Small α/γ-peptides alternating α-aminoisobutyric acid and cyclic γ-amino acid residues are described. NMR studies together with restrained simulated annealing revealed that an extended backbone conformation largely dominates in solution for as short as 4-residues long oligomers. This new fold type is devoid of any hydrogen bond and characterized by a four-fold symmetry.