Lanmodulin peptides - unravelling the binding of the EF-Hand loop sequences stripped from the structural corset.

Lanmodulin (LanM), a naturally lanthanide (Ln)-binding protein with a remarkable selectivity for Lns over Ca(ii) and affinities in the picomolar range, is an attractive target to address challenges in Ln separation. Why LanM has such a high selectivity is currently not entirely understood; both specific amino acid sequences of the EF-Hand loops and cooperativity effects have been suggested. Here, we removed the effect of cooperativity and synthesised all four 12-amino acid EF-Hand loop peptides, and investigated their affinity for two Lns (Eu(iii) and Tb(iii)), the actinide Cm(iii) and Ca(ii). Using isothermal titration calorimetry and time-resolved laser fluorescence spectroscopy (TRLFS) combined with parallel factor analysis, we show that the four short peptides behave very similarly, having affinities in the micromolar range for Eu(iii) and Tb(iii). Ca(ii) was shown not to bind to the peptides, which was verified with circular dichroism spectroscopy. This technique also revealed an increase in structural organisation upon Eu(iii) addition, which was supported by molecular dynamics simulations. Lastly, we put Eu(iii) and Cm(iii) in direct competition using TRLFS. Remarkably, a slightly higher affinity for Cm(iii) was found. Our results demonstrate that the picomolar affinities in LanM are largely an effect of pre-structuring and therefore a reduction of flexibility in combination with cooperative effects, and that all EF-Hand loops possess similar affinities when detached from the protein backbone, albeit still retaining the high selectivity for lanthanides and actinides over calcium.

Transforming Chemical Proteomics Enrichment into a High-Throughput Method Using an SP2E Workflow.

Protein post-translational modifications (PTMs) play a critical role in the regulation of protein catalytic activity, localization, and protein-protein interactions. Attachment of PTMs onto proteins significantly diversifies their structure and function, resulting in proteoforms. However, the sole identification of post-translationally modified proteins, which are often cell type and disease-specific, is still a highly challenging task. Substoichiometric amounts and modifications of low abundant proteins necessitate the purification or enrichment of the modified proteins. Although the introduction of mass spectrometry-based chemical proteomic strategies has enabled the screening of protein PTMs with increased throughput, sample preparation remains highly time-consuming and tedious. Here, we report an optimized workflow for the enrichment of PTM proteins in a 96-well plate format, which could be extended to robotic automation. This platform allows us to significantly lower the input of total protein, which opens up the opportunity to screen specialized and difficult-to-culture cell lines in a high-throughput manner. The presented SP2E protocol is robust and time- and cost-effective, as well as suitable for large-scale screening of proteoforms. The application of the SP2E protocol will thus enable the characterization of proteoforms in various processes such as neurodevelopment, neurodegeneration, and cancer. This may contribute to an overall acceleration of the recently launched Human Proteoform Project.

A set of rhamnosylation-specific antibodies enables detection of novel protein glycosylations in bacteria.

Despite its potential importance for bacterial virulence, protein rhamnosylation has not yet been sufficiently studied. Specific anti-SerRha, anti-ThrRha and anti-AsnRha antibodies allowed the identification of previously unknown monorhamnosylated proteins in cytosol and membrane fractions of bacterial cell lysates. Mapping of the complete rhamnoproteome in pathogens should facilitate development of targeted therapies against bacterial infections.

Folding and Unfolding of the Short Light-Triggered ß-Hairpin Peptide AzoChignolin Occurs within 100 ns.

To map the underlying molecular mechanisms of folding dynamics in proteins, light-operated peptides have emerged as promising tools. In this study, we reveal the complete sequence of light-induced structural changes of AzoChignolin, a short ß-hairpin peptide containing an azobenzene photoswitch in its loop region. Light-triggered structural changes were monitored by time-resolved IR spectroscopy. Formation and destruction of the hairpin structure is very fast and occurs within 100 ns for AzoChignolin in methanol. Atomistic molecular dynamics simulations using two explicit solvents, methanol and water, revealed the underlying molecular processes and allowed us to gain further insight into the reaction mechanism. Despite its rapid reaction time, hairpin formation in these solvents is not force-driven by the molecular switch but proceeded via formation of interstrand hydrogen bonds and contacts between aromatic residues. Moreover, the combined experimental and theoretical study demonstrates that the solvent (methanol vs water) does not dictate the velocity of ß-hairpin formation in the AzoChignolin peptide comprising only a few hydrophobic residues in the strands.

Optical Control of Cytokine Production Using Photoswitchable Galactosylceramides.

α-Galactosylceramides are glycosphingolipids that show promise in cancer immunotherapy. After presentation by CD1d, they activate natural killer T cells (NKT), which results in the production of a variety of pro-inflammatory and immunomodulatory cytokines. Herein, we report the synthesis and biological evaluation of photochromic derivatives of KRN-7000, the activity of which can be modulated with light. Based on established structure-activity relationships, we designed photoswitchable analogues of this glycolipid that control the production of pro-inflammatory cytokines, such as IFN-γ. The azobenzene derivative α-GalACer-4 proved to be more potent than KRN-7000 itself when activated with 370 nm light. Photolipids of this type could improve our mechanistic understanding of cytokine production and could open new directions in photoimmunotherapy.

Conditional and Reversible Activation of Class A and B G Protein-Coupled Receptors Using Tethered Pharmacology.

Understanding the activation and internalization of G protein-coupled receptors (GPCRs) using conditional approaches is paramount to developing new therapeutic strategies. Here, we describe the design, synthesis, and testing of ExONatide, a benzylguanine-linked peptide agonist of the glucagon-like peptide-1 receptor (GLP-1R), a class B GPCR required for maintenance of glucose levels in humans. ExONatide covalently binds to SNAP-tagged GLP-1R-expressing cells, leading to prolonged cAMP generation, Ca2+ rises, and intracellular retention of the receptor. These effects were readily switched OFF following cleavage of the introduced disulfide bridge using the cell-permeable reducing agent beta-mercaptoethanol (BME). A similar approach could be extended to a class A GPCR using GhrelON, a benzylguanine-linked peptide agonist of the growth hormone secretagogue receptor 1a (GHS-R1a), which is involved in food intake and growth. Thus, ExONatide and GhrelON allow SNAP-tag-directed activation of class A and B GPCRs involved in gut hormone signaling in a reversible manner. This tactic, termed reductively cleavable agONist (RECON), may be useful for understanding GLP-1R and GHS-R1a function both in vitro and in vivo, with applicability across GPCRs.

Structural Basis for EarP-Mediated Arginine Glycosylation of Translation Elongation Factor EF-P.

Glycosylation is a universal strategy to posttranslationally modify proteins. The recently discovered arginine rhamnosylation activates the polyproline-specific bacterial translation elongation factor EF-P. EF-P is rhamnosylated on arginine 32 by the glycosyltransferase EarP. However, the enzymatic mechanism remains elusive. In the present study, we solved the crystal structure of EarP from Pseudomonas putida The enzyme is composed of two opposing domains with Rossmann folds, thus constituting a B pattern-type glycosyltransferase (GT-B). While dTDP-ß-l-rhamnose is located within a highly conserved pocket of the C-domain, EarP recognizes the KOW-like N-domain of EF-P. Based on our data, we propose a structural model for arginine glycosylation by EarP. As EarP is essential for pathogenicity in P. aeruginosa, our study provides the basis for targeted inhibitor design.IMPORTANCE The structural and biochemical characterization of the EF-P-specific rhamnosyltransferase EarP not only provides the first molecular insights into arginine glycosylation but also lays the basis for targeted-inhibitor design against Pseudomonas aeruginosa infection.

Optical control of a receptor-linked guanylyl cyclase using a photoswitchable peptidic hormone.

The optical control over biological function with small photoswitchable molecules has gathered significant attention in the last decade. Herein, we describe the design and synthesis of a small library of photoswitchable peptidomimetics based upon human atrial natriuretic peptide (ANP), in which the photochromic amino acid [3-(3-aminomethyl)phenylazo]phenylacetic acid (AMPP) is incorporated into the peptide backbone. The endogeneous hormone ANP signals via the natriuretic peptide receptor A (NPR-A) through raising intracellular cGMP concentrations, and is involved in blood pressure regulation and sodium homeostasis, as well as lipid metabolism and pancreatic function. The cis- and trans-isomers of one of our peptidomimetics, termed TOP271, exhibit a four-fold difference in NPR-A mediated cGMP synthesis in vitro. Despite this seemingly small difference, TOP271 enables large, optically-induced conformational changes ex vivo and transforms the NPR-A into an endogenous photoswitch. Thus, application of TOP271 allows the reversible generation of cGMP using light and remote control can be afforded over vasoactivity in explanted murine aortic rings, as well as pancreatic beta cell function in islets of Langerhans. This study demonstrates the broad applicability of TOP271 to enzyme-dependent signalling processes, extends the toolbox of photoswitchable molecules to all classes of transmembrane receptors and utilizes photopharmacology to deduce receptor activation on a molecular level.

One-Pot Synthesis of Substituted Trifluoromethylated 2,3-Dihydro-1H-imidazoles.

An operationally simple one-pot reaction for the preparation of a novel class of racemic trifluoromethylated 2,3-dihydro-1H-imidazoles derived from electron-poor N,O-acetals and aryl Grignard reagents is described. In addition, access to highly functionalized 2-trifluoromethyl-2,3-dihydro-1H-imidazoles was accomplished by reaction of N-aryl hemiaminal ethers and N-aryl trifluoroethylamines in the presence of an excess of n-butyllithium.

Optical Control of Insulin Secretion Using an Incretin Switch.

Incretin mimetics are set to become a mainstay of type 2 diabetes treatment. By acting on the pancreas and brain, they potentiate insulin secretion and induce weight loss to preserve normoglycemia. Despite this, incretin therapy has been associated with off-target effects, including nausea and gastrointestinal disturbance. A novel photoswitchable incretin mimetic based upon the specific glucagon-like peptide-1 receptor (GLP-1R) agonist liraglutide was designed, synthesized, and tested. This peptidic compound, termed LirAzo, possesses an azobenzene photoresponsive element, affording isomer-biased GLP-1R signaling as a result of differential activation of second messenger pathways in response to light. While the trans isomer primarily engages calcium influx, the cis isomer favors cAMP generation. LirAzo thus allows optical control of insulin secretion and cell survival.

Synthesis and biological evaluation of a novel MUC1 glycopeptide conjugate vaccine candidate comprising a 4'-deoxy-4'-fluoro-Thomsen-Friedenreich epitope.

The development of selective anticancer vaccines that provide enhanced protection against tumor recurrence and metastasis has been the subject of intense research in the scientific community. The tumor-associated glycoprotein MUC1 represents a well-established target for cancer immunotherapy and has been used for the construction of various synthetic vaccine candidates. However, many of these vaccine prototypes suffer from an inherent low immunogenicity and are susceptible to rapid in vivo degradation. To overcome these drawbacks, novel fluorinated MUC1 glycopeptide-BSA/TTox conjugate vaccines have been prepared. Immunization of mice with the 4'F-TF-MUC1-TTox conjugate resulted in strong immune responses overriding the natural tolerance against MUC1 and producing selective IgG antibodies that are cross-reactive with native MUC1 epitopes on MCF-7 human cancer cells.

Antibody recognition of fluorinated haptens and antigens.

Regarding its many roles for lead optimization and drug development, fluorine will definitely continue to be of major importance in medicinal chemistry. With safe and selective fluorinating agents at hands, the use of fluorinated compounds has become routine in pharmaceutical and material sciences and many of the well-appreciated organofluorine inductive effects are now understood. In contrast, our knowledge of how fluorine affects binding affinity and selectivity of proteins and antibodies at the molecular level is by far less advanced. Thus, applications of fluorinated haptens and antigens in the mapping of binding epitopes for various antibodies of diagnostic and therapeutic relevance, as well as for the generation of immune modulating agents and ligands with enhanced T cell affinity are reviewed. Moreover, recent examples of vaccines against cocaine and cancer based on selectively fluorinated antigens with improved antigenic properties are presented.

Designed peptides for biomineral polymorph recognition: a case study for calcium carbonate.

With their unique ability for substrate recognition and their sequence-specific self-assembly properties, peptides play an important role in controlling the mineralization of inorganic materials in natural systems and in controlling the assembly of soft materials into complex structures required for biological functions. Here we report the use of an engineered heptapeptide that can differentiate between the crystalline anhydrous polymorphs of calcium carbonate. This peptide contains the positively charged amino acid arginine as well as proline rather than the prototypical negatively charged aspartate or glutamate units. Its affinity to vaterite compared to aragonite was demonstrated by fluorescence microscopy using biotinylated peptides. Crystallization experiments in the presence of the vaterite-affine peptide afforded only vaterite, whereas a mutant peptide, where a proline residue was replaced by glycine, exclusively leads to the formation of calcite.

Antibody recognition of fluorinated MUC1 glycopeptide antigens.

The syntheses of various fluorinated MUC1 glycopeptide antigens and their specific binding to serum antibodies from mice immunized with natural and fluorinated TF(6)-MUC1-TTox conjugate vaccines are presented.

Synthesis of a MUC1-glycopeptide-BSA conjugate vaccine bearing the 3'-deoxy-3'-fluoro-Thomsen-Friedenreich antigen.

A novel MUC1-glycopeptide-BSA conjugate vaccine with a specifically fluorinated Thomsen-Friedenreich antigen side chain at Thr6 was prepared. Preliminary immunological experiments reveal specific binding of the tumor-associated glycopeptide antigen analog by anti-MUC1-mouse antibodies.

Synthesis of fluorinated Thomsen-Friedenreich antigens: direct deoxyfluorination of αGalNAc-threonine tert-butyl esters.

Selectively 6-fluorinated analogs of the tumor-associated T(N) antigen Fmoc-Thr(α-O-GalNAc)-OtBu can be efficiently prepared using DAST-mediated de(hydr)oxyfluorination reactions of preformed and orthogonally protected glycosyl amino esters without affecting the labile protecting groups and O-glycosidic linkages. The resulting mono- and difluorinated T(N) analogs are interesting building blocks for non-hydrolyzable mucin-type antigen mimetics, as illustrated by the unprecedented synthesis of two different multiply fluorinated Thomsen-Friedenreich derivatives. The reported deoxyfluoro antigen analogs represent important functional probes for carbohydrate-binding proteins and glycosyl-processing enzymes.

Perfluoroalkylated amphiphilic MUC1 glycopeptide antigens as tools for cancer immunotherapy.

The synthesis of perfluoroalkylated glycopeptide antigens and their specific binding to anti-MUC1 mouse antibodies is reported.

Langmuir-Blodgett films of fluorinated glycolipids and polymerizable lipids and their phase separating behavior.

This paper describes the phase separating behavior of Langmuir monolayers from mixtures of different lipids that (i) either carry already a glycopeptide recognition site or can be easily modified to carry one and (ii) polymerizable lipids. To ensure demixing during compression, we used fluorinated lipids for the biological headgroups and hydrocarbon based lipids as polymerizable lipids. As a representative for a lipid monomer, which can be polymerized in the hydrophilic headgroup, a methacrylic monomer was used. As a monomer, which can be polymerized in the hydrophobic tail, a lipid with a diacetylene unit was used (pentacosadiynoic acid, PDA). The fluorinated lipids were on the one hand a perfluorinated lipid with three chains and on the other hand a partially fluorinated lipid with a T(N)-antigen headgroup. The macroscopic phase separation was observed by Brewster angle microscopy, whereas the phase separation on the nanoscale level was observed by atomic force microscopy. It turned out that all lipid mixtures showed (at least) a partial miscibility of the hydrocarbon compounds in the fluorinated compounds. This is positive for pattern formation, as it allows the formation of small demixed 2D patterned structures during crystallization from the homogeneous phase. For miscibility especially a liquid analogue phase proved to be advantageous. As lipid 3 with three fluorinated lipid chains (very stable monolayer) is miscible with the polymerizable lipids 1 and 2, it was mostly used for further investigations. For all three lipid mixtures, a phase separation on both the micrometer and the nanometer level was observed. The size of the crystalline domains could be controlled not only by varying the surface pressure but also by varying the molar composition of the mixtures. Furthermore, we showed that the binary mixture can be stabilized via UV polymerization. After polymerization and subsequent expansion of the barriers, the locked-in polymerized structures are stable even at low surface pressures (10 mN/m), where the unpolymerized mixture did not show any segregation.

Synthesis of glycosylated ß³-homo-threonine conjugates for mucin-like glycopeptide antigen analogues.

Glycopeptides from the mucin family decorated with tumour-associated carbohydrate antigens (TACA) have proven to be important target structures for the development of molecularly defined anti-cancer vaccines. The strategic incorporation of ß-amino acid building blocks into such mucin-type sequences offers the potential to create pseudo-glycopeptide antigens with improved bioavailability for tumour immunotherapy. Towards this end, T(N) and TF antigen conjugates O-glycosidically linked to Fmoc-ß³-homo-threonine were prepared in good yield via Arndt-Eistert homologation of the corresponding glycosyl α-amino acid derivative. By incorporation of T(N)-Fmoc-ß³hThr conjugate into the 20 amino acid tandem repeat sequence of MUC1 using sequential solid-phase glycopeptide synthesis, a first example of a mixed α/ß-hybrid glycopeptide building block was obtained. The latter is of interest for the development of novel glycoconjugate mimics and model structures for anti-cancer vaccines with increased biological half-life.