High-Throughput Synthesis and Analysis of Intact Glycoproteins Using SAMDI-MS.

High-throughput quantification of the post-translational modification of many individual protein samples is challenging with current label-based methods. This paper demonstrates an efficient method that addresses this gap by combining Escherichia coli-based cell-free protein synthesis (CFPS) and self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI-MS) to analyze intact proteins. This high-throughput approach begins with polyhistidine-tagged protein substrates expressed from linear DNA templates by CFPS. Here, we synthesized an 87-member library of the E. coli Immunity Protein 7 (Im7) containing an acceptor sequence optimized for glycosylation by the Actinobacillus pleuropneumoniae N-glycosyltransferase (NGT) at every possible position along the protein backbone. These protein substrates were individually treated with NGT and then selectively immobilized to self-assembled monolayers presenting nickel-nitrilotriacetic acid (Ni-NTA) complexes before final analysis by SAMDI-MS to quantify the conversion of substrate to glycoprotein. This method offers new opportunities for rapid synthesis and quantitative evaluation of intact glycoproteins.

A Conditionally Fluorescent Peptide Reporter of Secondary Structure Modulation.

Proteins containing intrinsic disorder often form secondary structure upon interaction with a binding partner. Modulating such structures presents an approach for manipulating the resultant functional outcomes. Translational repressor protein 4E-BP1 is an example of an intrinsically disordered protein that forms an α-helix upon binding to its protein ligand, eIF4E. Current biophysical methods for analyzing binding-induced structural changes are low-throughput, require large amounts of sample, or are extremely sensitive to signal interference by the ligand itself. Herein, we describe the discovery and development of a conditionally fluorescent 4E-BP1 peptide that reports structural changes of its helix in high-throughput format. This reporter peptide is based on conditional quenching of fluorescein by thioamides. In this case, fluorescence signal increases as the peptide becomes more ordered. Conversely, destabilization of the α-helix results in decreased fluorescence signal. The low concentration and low volume of peptide required make this approach amenable for high-throughput screening to discover ligands that alter peptide secondary structure.

Preparation of a monoPEGylated derivative of cyanovirin-N and its virucidal effect on acyclovir-resistant strains of herpes simplex virus type 1.

The long-term administration of acyclovir (ACV) for therapy against herpes simplex virus type 1 (HSV-1) infections can result in the emergence of ACV-resistant HSV strains. It is therefore urgent to develop new anti-herpetic compounds with mechanisms that differ from that of ACV. Cyanovirin-N (CV-N) is an antiviral agent that has an inhibitory effect on HSV-1 infections, and PEGylation of CV-N is potentially useful for pharmaceutical applications. Here, a (Gly4Ser)3 linker molecule was attached to the N-terminus of CV-N, and the resulting compound, linker-CV-N (LCV-N), was produced on a pilot scale with purity up to 95%. Then, PEG10k-LCV-N was synthesized by modifying at the α-amine group of the N-terminus of LCV-N with 10-kDa polyethylene glycol propionaldehyde (mPEG-ALD). CV-N, LCV-N and PEG10k-LCV-N were all found to have potent inhibitory activity against ACV-resistant HSV strains with IC50 values in the nM range. LCV-N was the most potent of these three compounds against both normal and ACV-resistant HSV strains. Although PEG10k-LCV-N showed less antiviral activity than CV-N and LCV-N, it still exhibited significant and universal virucidal activity against drug-resistant viruses. The toxicity and immunogenicity of PEG10k-LCV-N were dramatically lower than those of CV-N and LCV-N. In conclusion, we suggest that LCV-N and PEG10k-LCV-N are promising and safe microbicides for the control and/or treatment of ACV-resistant HSV infection.

Development of a strategy for the total chemical synthesis of an allergenic protein: the peach LTP Pru p 3.

The possibility to obtain allergenic proteins by means of total chemical synthesis would be a big step forward in the development of cures to food allergy and in the study of the mechanism of allergic reactions, because this would allow to achieve control at the molecular level over the structure of the product and to study its relationship with the allergenic activity in fine details. This is instead not possible by using allergens produced by extraction from natural sources or by recombinant DNA techniques. In this work, we aimed to test for the first time the feasibility of the total chemical synthesis of an allergenic protein. Pru p 3, the most studied member of the family of lipid transfer proteins, relevant plant food pan-allergens, was used as model target. Strategies for the convergent assembly of the target protein, starting from five peptide fragments to be bound by means of either native chemical ligation or peptide hydrazide ligation, followed by desulfurization, to achieve ligations at alanine, were developed and tested. All the reaction conditions were set up and optimized. Two large peptides covering the two halves of the protein sequence were synthesized and structurally characterized by means of circular dichroism, and their immunogenicity was proved by means of immunoblot, using antibodies against Pru p 3, and immunoCAP inhibition tests. Finally, the five peptides were bound together to produce the whole protein stretch. The obtained results demonstrate the feasibility of total chemical synthesis as a new way to obtain pure allergens. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Chemically synthesized peptide libraries as a new source of BBB shuttles. Use of mass spectrometry for peptide identification.

The blood-brain barrier (BBB) is a biological barrier that protects the brain from neurotoxic agents and regulates the influx and efflux of molecules required for its correct function. This stringent regulation hampers the passage of brain parenchyma-targeting drugs across the BBB. BBB shuttles have been proposed as a way to overcome this hurdle because these peptides can not only cross the BBB but also carry molecules which would otherwise be unable to cross the barrier unaided. Here we developed a new high-throughput screening methodology to identify new peptide BBB shuttles in a broadly unexplored chemical space. By introducing d-amino acids, this approach screens only protease-resistant peptides. This methodology combines combinatorial chemistry for peptide library synthesis, in vitro models mimicking the BBB for library evaluation and state-of-the-art mass spectrometry techniques to identify those peptides able to cross the in vitro assays. BBB shuttle synthesis was performed by the mix-and-split technique to generate a library based on the following: Ac-d-Arg-XXXXX-NH2 , where X were: d-Ala (a), d-Arg (r), d-Ile (i), d-Glu (e), d-Ser (s), d-Trp (w) or d-Pro (p). The assays used comprised the in vitro cell-based BBB assay (mimicking both active and passive transport) and the PAMPA (mimicking only passive diffusion). The identification of candidates was determined using a two-step mass spectrometry approach combining LTQ-Orbitrap and Q-trap mass spectrometers. Identified sequences were postulated to cross the BBB models. We hypothesized that some sequences cross the BBB through passive diffusion mechanisms and others through other mechanisms, including paracellular flux and active transport. These results provide a new set of BBB shuttle peptide families. Furthermore, the methodology described is proposed as a consistent approach to search for protease-resistant therapeutic peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Design and engineering of an O(2) transport protein.

The principles of natural protein engineering are obscured by overlapping functions and complexity accumulated through natural selection and evolution. Completely artificial proteins offer a clean slate on which to define and test these protein engineering principles, while recreating and extending natural functions. Here we introduce this method with the design of an oxygen transport protein, akin to human neuroglobin. Beginning with a simple and unnatural helix-forming sequence with just three different amino acids, we assembled a four-helix bundle, positioned histidines to bis-histidine ligate haems, and exploited helical rotation and glutamate burial on haem binding to introduce distal histidine strain and facilitate O(2) binding. For stable oxygen binding without haem oxidation, water is excluded by simple packing of the protein interior and loops that reduce helical-interface mobility. O(2) affinities and exchange timescales match natural globins with distal histidines, with the remarkable exception that O(2) binds tighter than CO.

X-ray crystal structure of teicoplanin A2-2 bound to a catalytic peptide sequence via the carrier protein strategy.

We report the X-ray crystal structure of a site-selective peptide catalyst moiety and teicoplanin A2-2 complex. The expressed protein ligation technique was used to couple T4 lysozyme (T4L) and a synthetic peptide catalyst responsible for the selective phosphorylation of the N-acetylglucosamine sugar in a teicoplanin A2-2 derivative. The T4L-Pmh-dPro-Aib-dAla-dAla construct was crystallized in the presence of teicoplanin A2-2. The resulting 2.3 Å resolution protein-peptide-teicoplanin complex crystal structure revealed that the nucleophilic nitrogen of N-methylimidazole in the Pmh residue is in closer proximity (7.6 Å) to the N-acetylglucosamine than the two other sugar rings present in teicoplanin (9.3 and 20.3 Å, respectively). This molecular arrangement is consistent with the observed selectivity afforded by the peptide-based catalyst when it is applied to a site-selective phosphorylation reaction involving a teicoplanin A2-2 derivative.

Stabilization of branched oligosaccharides: Lewis(x) benefits from a nonconventional C-H···O hydrogen bond.

Although animal lectins usually show a high degree of specificity for glycan structures, their single-site binding affinities are typically weak, a drawback which is often compensated in biological systems by an oligovalent presentation of carbohydrate epitopes. For the design of monovalent glycomimetics, structural information regarding solution and bound conformation of the carbohydrate lead represents a valuable starting point. In this paper, we focus on the conformation of the trisaccharide Le(x) (Gal[Fucα(1-3)]ß(1-4)GlcNAc). Mainly because of the unfavorable tumbling regime, the elucidation of the solution conformation of Le(x) by NMR has only been partially successful so far. Le(x) was therefore attached to a (13)C,(15)N-labeled protein. (13)C,(15)N-filtered NOESY NMR techniques at ultrahigh field allowed increasing the maximal NOE enhancement, resulting in a high number of distance restraints per glycosidic bond and, consequently, a well-defined structure. In addition to the known contributors to the conformational restriction of the Le(x) structure (exoanomeric effect, steric compression induced by the NHAc group adjacent to the linking position of L-fucose, and the hydrophobic interaction of L-fucose with the ß-face of D-galactose), a nonconventional C-H···O hydrogen bond between H-C(5) of L-fucose and O(5) of D-galactose was identified. According to quantum mechanical calculations, this C-H···O hydrogen bond is the most prominent factor in stabilization, contributing 40% of the total stabilization energy. We therefore propose that the nonconventional hydrogen bond contributing to a reduction of the conformational flexibility of the Le(x) core represents a novel element of the glycocode. Its relevance to the stabilization of related branched oligosaccharides is currently being studied.

Generation of the first structure-based pharmacophore model containing a selective "zinc binding group" feature to identify potential glyoxalase-1 inhibitors.

Within this study, a unique 3D structure-based pharmacophore model of the enzyme glyoxalase-1 (Glo-1) has been revealed. Glo-1 is considered a zinc metalloenzyme in which the inhibitor binding with zinc atom at the active site is crucial. To our knowledge, this is the first pharmacophore model that has a selective feature for a "zinc binding group" which has been customized within the structure-based pharmacophore model of Glo-1 to extract ligands that possess functional groups able to bind zinc atom solely from database screening. In addition, an extensive 2D similarity search using three diverse similarity techniques (Tanimoto, Dice, Cosine) has been performed over the commercially available "Zinc Clean Drug-Like Database" that contains around 10 million compounds to help find suitable inhibitors for this enzyme based on known inhibitors from the literature. The resultant hits were mapped over the structure based pharmacophore and the successful hits were further docked using three docking programs with different pose fitting and scoring techniques (GOLD, LibDock, CDOCKER). Nine candidates were suggested to be novel Glo-1 inhibitors containing the "zinc binding group" with the highest consensus scoring from docking.

Fmoc synthesis of peptide thioesters without post-chain-assembly manipulation.

An operationally simple method for the synthesis of peptide thioesters is developed using standard Fmoc solid-phase peptide synthesis procedures. The method relies on the use of a premade enamide-containing amino acid which, in the final TFA cleavage step, renders the desired thioester functionality through an irreversible intramolecular N-to-S acyl transfer.

Potent lentiviral restriction by a synthetic feline TRIM5 cyclophilin A fusion.

A synthetic feline TRIM5-cyclophilin A fusion protein (feTRIMCyp) was generated and transduced into feline cells. feTRIMCyp was highly efficient at preventing infection with human (HIV) and feline (FIV) immunodeficiency virus pseudotypes, and feTRIMCyp-expressing cells resisted productive infection with either FIV-Fca or FIV-Pco. The restriction of FIV infection by feTRIMCyp was reversed by the cyclosporine (Cs) derivatives NIM811 and Debio-025 but less so by Cs itself. FeTRIMCyp and FIV infections of the cat offer a unique opportunity to evaluate TRIMCyp-based approaches to genetic therapy for HIV infection and the treatment of AIDS.

Isolation and identification of diadenosine 5',5'''-P1,P4-tetraphosphate binding proteins using magnetic bio-panning.

We report the development of a synthetic, biotin-conjugated diadenosine tetraphosphate (Ap(4)A)-'molecular hook' attached to magnetic beads enabling the isolation of Ap(4)A-binding proteins from bacterial cells or mammalian tissue lysates. Characterisation and identification of isolated binding proteins is performed sequentially by mass spectrometry. The observation of positive controls suggests that these newly observed proteins are putative Ap(4)A-binding partners, and we have expectations that others can be found with further technical improvements in our methods.

New multi-determinant strategy for a group A streptococcal vaccine designed for the Australian Aboriginal population.

Infection with group A streptococci can result in acute and post-infectious pathology, including rheumatic fever and rheumatic heart disease. These diseases are associated with poverty and are increasing in incidence, particularly in developing countries and amongst indigenous populations, such as Australia's Aboriginal population, who suffer the highest incidence worldwide. Immunity to group A streptococci is mediated by antibodies against the M protein, a coiled-coil alpha helical surface protein of the bacterium. Vaccine development faces two substantial obstacles. Although opsonic antibodies directed against the N terminus of the protein are mostly responsible for serotypic immunity, more than 100 serotypes exist. Furthermore, whereas the pathogenesis of rheumatic fever is not well understood, increasing evidence indicates an autoimmune process. To develop a suitable vaccine candidate, we first identified a minimum, helical, non-host-cross-reactive peptide from the conserved C-terminal half of the protein and displayed this within a non-M-protein peptide sequence designed to maintain helical folding and antigenicity, J14 (refs. 8,9). As this region of the M protein is identical in only 70% of group A streptococci isolates, the optimal candidate might consist of the conserved determinant with common N-terminal sequences found in communities with endemic group A streptococci. We linked seven serotypic peptides with J14 using a new chemistry technique that enables the immunogen to display all the individual peptides pendant from an alkane backbone. This construct demonstrated excellent immunogenicity and protection in mice.

Recent Advances in the Scaffold Engineering of Protein Binders.

In recent years, extensive attention has been given to the generation of new classes of ligand- specific binding proteins to supplement monoclonal antibodies. A combination of protein engineering and display technologies has been used to manipulate non-human antibodies for humanization and stabilization purposes or even the generation of new binding proteins. Engineered protein scaffolds can now be directed against therapeutic targets to treat cancer and immunological disorders. Although very few of these scaffolds have successfully passed clinical trials, their remarkable properties such as robust folding, high solubility, and small size motivate their employment as a tool for biology and applied science studies. Here, we have focused on the generation of new non-Ig binding proteins and single domain antibody manipulation, with a glimpse of their applications.

Molecular Details of a Coupled Binding and Folding Reaction between the Amyloid Precursor Protein and a Folded Domain.

Intrinsically disordered regions in proteins often function as binding motifs in protein-protein interactions. The mechanistic aspects and molecular details of such coupled binding and folding reactions, which involve formation of multiple noncovalent bonds, have been broadly studied theoretically, but experimental data are scarce. Here, using a combination of protein semisynthesis to incorporate phosphorylated amino acids, backbone amide-to-ester modifications, side chain substitutions, and binding kinetics, we examined the interaction between the intrinsically disordered motif of amyloid precursor protein (APP) and the phosphotyrosine binding (PTB) domain of Mint2. We show that the interaction is regulated by a self-inhibitory segment of the PTB domain previously termed ARM. The helical ARM linker decreases the association rate constant 30-fold through a fast pre-equilibrium between an open and a closed state. Extensive side chain substitutions combined with kinetic experiments demonstrate that the rate-limiting transition state for the binding reaction is governed by native and non-native hydrophobic interactions and hydrogen bonds. Hydrophobic interactions were found to be particularly important during crossing of the transition state barrier. Furthermore, linear free energy relationships show that the overall coupled binding and folding reaction involves cooperative formation of interactions with roughly 30% native contacts formed at the transition state. Our data support an emerging picture of coupled binding and folding reactions following overall chemical principles similar to those of folding of globular protein domains but with greater malleability of ground and transition states.

Determination of penetratin secondary structure in live cells with Raman microscopy.

Cell penetrating peptides (CPPs) have attracted recent interest as drug delivery tools, although the mechanisms by which CPPs are internalized by cells are not well-defined. Here, we report a new experimental approach for the detection and secondary structure determination of CPPs in live cells using Raman microscopy with heavy isotope labeling of the peptide. As a first demonstration of principle, penetratin, a 16-residue CPP derived from the Antennapedia homeodomain protein of Drosophila, was measured in single, living melanoma cells. Carbon-13 labeling of the Phe residue of penetratin was used to shift the intense aromatic ring-breathing vibrational mode from 1003 to 967 cm(-1), thereby enabling the peptide to be traced in cells. Difference spectroscopy and principal components analysis (PCA) were used independently to resolve the Raman spectrum of the peptide from the background cellular Raman signals. On the basis of the position of the amide I vibrational band in the Raman spectra, the secondary structure of the peptide was found to be mainly random coil and beta-strand in the cytoplasm, and possibly assembling as beta-sheets in the nucleus. The rapid entry and almost uniform cellular distribution of the peptide, as well as the lack of correlation between peptide and lipid Raman signatures, indicated that the mechanism of internalization under the conditions of study was probably nonendocytotic. This experimental approach can be used to study a wide variety of CPPs as well as other classes of peptides in living cells.

RXP-E: a connexin43-binding peptide that prevents action potential propagation block.

Gap junctions provide a low-resistance pathway for cardiac electric propagation. The role of GJ regulation in arrhythmia is unclear, partly because of limited availability of pharmacological tools. Recently, we showed that a peptide called "RXP-E" binds to the carboxyl terminal of connexin43 and prevents chemically induced uncoupling in connexin43-expressing N2a cells. Here, pull-down experiments show RXP-E binding to adult cardiac connexin43. Patch-clamp studies revealed that RXP-E prevented heptanol-induced and acidification-induced uncoupling in pairs of neonatal rat ventricular myocytes. Separately, RXP-E was concatenated to a cytoplasmic transduction peptide (CTP) for cytoplasmic translocation (CTP-RXP-E). The effect of RXP-E on action potential propagation was assessed by high-resolution optical mapping in monolayers of neonatal rat ventricular myocytes, containing approximately 20% of randomly distributed myofibroblasts. In contrast to control experiments, when heptanol (2 mmol/L) was added to the superfusate of monolayers loaded with CTP-RXP-E, action potential propagation was maintained, albeit at a slower velocity. Similarly, intracellular acidification (pH(i) 6.2) caused a loss of action potential propagation in control monolayers; however, propagation was maintained in CTP-RXP-E-treated cells, although at a slower rate. Patch-clamp experiments revealed that RXP-E did not prevent heptanol-induced block of sodium currents, nor did it alter voltage dependence or amplitude of Kir2.1/Kir2.3 currents. RXP-E is the first synthetic molecule known to: (1) bind cardiac connexin43; (2) prevent heptanol and acidification-induced uncoupling of cardiac gap junctions; and (3) preserve action potential propagation among cardiac myocytes. RXP-E can be used to characterize the role of gap junctions in the function of multicellular systems, including the heart.

Sequential Linkage of Carbohydrate Antigens to Mimic Capsular Polysaccharides: Toward Semisynthetic Glycoconjugate Vaccine Candidates against Streptococcus pneumoniae Serotype 14.

Vaccines based on isolated polysaccharides successfully protect humans from bacterial pathogens such as Streptococcus pneumoniae. Because polysaccharide production and isolation can be technically challenging, glycoconjugates containing synthetic antigens are an attractive alternative. Typically, the shortest possible oligosaccharide antigen is preferable as syntheses of longer structures are more difficult and time-consuming. Combining several protective epitopes or polysaccharide repeating units as blocks by bonds other than glycosidic linkages would greatly reduce the synthetic effort if the immunological response to the polysaccharide could be retained. To explore this concept, we bridged the well-understood and immunologically potent RU of S. pneumoniae serotype 14 (ST14) with an aliphatic spacer and conjugated it to the carrier protein CRM197. Mice immunized with the spacer-bridged glycan conjugates produced high levels of specific antibodies after just one or two vaccine doses, while the tetrasaccharide repeating unit alone required three doses. The antibodies recognized specifically ST14 CPS, while no significant antibody levels were raised against the spacer or unrelated CPS. Synthetic vaccines generated antibodies with opsonic activity. Mimicking polysaccharides by coupling repeating unit antigens via an aliphatic spacer may prove useful also for the development of other glycoconjugate vaccine candidates, thereby reducing the synthetic complexity while enhancing a faster immune response.

Design and Synthesis of Lipopolysaccharide-Binding Antimicrobial Peptides Based on Truncated Rabbit and Human CAP18 Peptides and Evaluation of Their Action Mechanism.

Lipopolysaccharide (LPS) is a toxic and immunogenic agent for human. Additionally, LPS is a good target for some antimicrobial compounds, including antimicrobial peptides (AMPs). LPS-binding peptides (LBPs) can recognize and neutralize LPS. Rabbit and human cathelicidins are AMPs with LPS-binding activity. In this study, we designed and synthesized two new truncated LBPs from rabbit and human CAP18 peptides by in silico methods. After synthesis of peptides, the antimicrobial properties and LPS-binding activity of these peptides were evaluated. The parental rabbit and human CAP18 peptides were selected as positive controls. Next, the changes in the secondary structure of these peptides before and after treatment with LPS were measured by circular dichroism (CD). Human cytotoxicity of the peptides was evaluated by MTT and red blood cells (RBCs) hemolysis assays. Finally, field emission scanning electron microscopy (FE-SEM), confocal microscopy, and flow cytometry were performed to study the action mechanism of these peptides. Results indicated that the hCap18 and rCap18 had antibacterial activity (at a MIC of 4-128 µg/mL). The results of the quantitative LAL test demonstrated that LPS-binding activity of hCap18 peptide was better than rCap18, while rCap18 peptide had better antimicrobial properties. Furthermore, rCap18 had less cytotoxicity than hCap18. However, both peptides were nontoxic for normal human skin fibroblast cell in MIC range. In conclusion, rCap18 has good antibacterial properties, while hCap18 can be tested as a diagnostic molecule in our future studies.

Synthesis and studies on cell-penetrating peptides.

The ability of different synthetic cell penetrating peptides, as Antennapedia (wild and Phe(6) mutated penetratins), flock house virus, and integrin peptides to form complexes with a 25mer antisense oligonucleotide was compared and their conformation was determined by circular dichroism spectroscopy. The efficiency for oligonucleotide delivery into cells was measured using peptides labeled with a coumarin derivative showing blue fluorescence and the fluorescein-labeled antisense oligonucleotide showing green fluorescence. Fluorescence due to the excitation energy transfer confirmed the interaction of the antisense oligonucleotide and cell-penetrating peptides. The most efficient oligonucleotide delivery was found for penetratins. Comparison of the two types of penetratins shows that the wild-type penetratin proved to be more efficient than mutated penetratin. The paper also emphasizes that the attachment of a fluorescent label may have an effect on the conformation and flexibility of cell-penetrating peptides that must be taken into consideration when evaluating biological experiments.