Highly Chemo-, Regio- and Stereoselective Cysteine Modification of Peptides and Proteins with Ynamides

Chemoselective modification of peptides and proteins has wide applications in chemical biology and pharmaceutical development. An efficient cysteine (Cys) precise modification protocol via rationally designed β-addition of ynamides is reported. The strong electron-withdrawing triflyl group on the nitrogen atom of ynamides plays a crucial role for controlling the chemo-, stereo- and regioselectivities of this protocol. Another substituent of the terminal ynamides offers a handle for functionality diversification. This Cys modification with ynamides proceeds efficiently in a slightly basic aqueous media (pH 8) to provide a series of Z-isomer of the corresponding conjugated products with excellent stereoselectivity (> 99%) and superior stability. All the reactive peptide side chain functional groups such as amino, carboxyl, primary amide, and hydroxyl groups, as well as the unprotected imidazole and indole rings are compatible. This method displays a broad substrates scope including linear and cyclic peptides and proteins. The potential application of this method in peptide and protein chemical biology was exemplified by Cys-bioconjugation with ynamides containing different functional molecules, including drug, fluorescent and affinity tags. In addition, this strategy is also compatible with click chemistry (performed in one pot), which remarkably extends the toolbox for further applications. The chemoselective biotinylation of ubiquitin(G47C) variant with a biotinylated ynamide, as well as the regioselective modification of Cys14 and Cys38 in bovine pancreatic trypsin inhibitor (BPTI) were accomplished under the optimized conditions and in high yield, without perturbation of disulfide bonds.

Free Energy Landscape and Rate Estimation of the Aromatic Ring Flips in Basic Pancreatic Trypsin Inhibitor Using Metadynamics

Aromatic side-chains (phenylalanine and tyrosine) of a protein flip by 180° around the Cβ-Cγ axis (χ2 dihedral of side-chain) producing two symmetry-equivalent states. The ring-flip dynamics act as an NMR probe to understand local conformational fluctuations. Ring-flips are categorized as slow (ms onwards) or fast (ns to near ms) based on timescales accessible to NMR experiments. In this study, we investigated the ability of the infrequent metadynamics approach to discriminate between slow and fast ring-flips for eight individual aromatic side-chains (F4, Y10, Y21, F22, Y23, F33, Y35, F45) of basic pancreatic trypsin inhibitor (BPTI). Well-tempered metadynamics simulations were performed to observe ring-flipping free energy surfaces for all eight aromatic residues. The results indicate that χ2 as a standalone collective variable (CV) is not sufficient to classify fast and slow ring-flips. Most of the residues needed χ1 (N−Cχα) as a complementary CV, indicating the importance of librational motions in ring-flips. Multiple pathways and mechanisms were observed for residues F4, Y10, and F22. Recrossing events are observed for residues F22 and F33, indicating a possible role of friction effects in the ring-flipping. The results demonstrate the successful application of the metadynamics based approach to estimate ring-flip rates of aromatic residues in BPTI and identify certain limitations of the approach.

Resistance to the antifungal activity of Aprotinin occurs through mutations in genes that function in cation homeostasis

An increase in the prevalence of fungal infections is coinciding with an increase of resistance to current clinical antifungals, placing pressure on the discovery of new antifungal candidates. One option is to investigate drugs that have been approved for use for other medical conditions that have secondary antifungal activity. Aprotinin, also known as Bovine Pancreatic Trypsin inhibitor (BPTI), is an antifibrinolytic that has been approved for systemic use in patients in some countries. Bleackley and coworkers (2014) revealed that BPTI also has antifungal activity against S. cerevisiae and C. albicans and does this by targeting the magnesium transporter ALR1. Here we have further investigated the potential for aprotinin to be used as an antifungal by assessing the development of resistance. We used an in vitro model to assess the evolution of BPTI resistance/tolerance whereby BPTI was serial passaged with the model organism S. cerevisiae. Resistance to BPTI developed more quickly than resistance to the plant defensin NaD1 and the clinical antifungal, caspofungin. Full genome sequencing of resistant lines revealed that resistance to BPTI developed as the result of a deleterious mutation in either the ptk2 or sky1 genes. This revealed that cation homeostasis and transport functions were particularly affected in S. cerevisiae after exposure to BPTI. Therefore, the mutations in these genes probably decreases release of magnesium and other cations from the cell, protecting the yeast from the limiting intracellular magnesium levels that arise when BPTI blocks the magnesium transporter Alr1p.

A systematic mutational analysis identifies a 5-residue proline tag that enhances the in vivo immunogenicity of a non-immunogenic model protein 240 folds

Small proteins are generally non-immunogenic, which can be a major hurdle in developing protein and peptide vaccines or producing antibodies for biopharmaceutical usage. For improving a protein’s immunogenicity, we previously proposed to use short Solubility Controlling Peptide (SCP) tags that oligomerize proteins into soluble aggregates. Here, we systematically analyzed the effect of SCP-tags that do not induce oligomerization on the immunogenicity of a small, non-immunogenic, model protein, Bovine Pancreatic Trypsin Inhibitor (BPTI-19A; 6 kDa). We assessed the effect of the following ten SCP-tags: Six tags made of five consecutive Arg, Lys, His, Asp, Asn, Pro; one made of seven Pro; two tags made of consecutive Arg-lle and Asn-Ile, all attached at the C-terminus of BPTI-19A; and a 5-proline tag attached at the N-terminus. Circular dichroism, fluorescence, dynamic light scattering measurements, and analytical ultra-centrifugation indicated that the addition of the SCP-tags did not change the secondary structure content nor the tertiary structures of the protein nor its monomeric state. On the other hand, the C-terminus 5-proline (C5P) tag unexpectedly increased the immunogenicity (IgG level) of BPTI-19A by up to 240 fold as assessed by ELISA. Additionally, the 5-arginine tag (C5R) increased the titer by up to 73 fold. The titer increase lasted for several weeks, and the effect was cumulative to that of the Freund’s adjuvant, which is commonly used to boost a protein’s immunogenicity. Altogether, SCP-tags that do not oligomerize proteins substantially increased the immunogenicity of a non-immunogenic protein, suggesting that the 5-proline and the 5-arginine SCP-tags may provide a novel tool for facilitating the production of antibodies or improving the effectiveness of protein-based vaccines.

Crystal structures of full length DENV4 NS2B-NS3 reveal the dynamic interaction between NS2B and NS3

Flavivirus is a genus of emerging and re-emerging arboviruses which include many significant human pathogens. Non-structural protein 3 (NS3), a multifunctional protein with N-terminal protease and C-terminal helicase, is essential in viral replication. The NS3 protease together with NS2B cofactor is an attractive antiviral target. A construct with an artificial glycine linker connecting the NS2B cofactor and NS3 protease has been used for structural, biochemical and drug-screening studies. The effect of this linker on dynamics and enzymatic activity of the protease was studied by several biochemical and NMR methods but the findings remained inconclusive. Here, we designed constructs of NS2B cofactor joined to full length DENV4 NS3 in three different manners, namely bNS2B47NS3 (bivalent), eNS2B47NS3(enzymatically cleavable) and gNS2B47NS3 (glycine-rich G4SG4 linker). We report the first crystal structures of linked and unlinked full-length NS2B-NS3 enzyme in its free state and also in complex with Bovine Pancreatic Trypsin Inhibitor (BPTI). These structures demonstrate that the NS2B-NS3 protease mainly adopts a closed conformation. BPTI binding is not essential to but favors the closed conformation by interacting with both NS2B and NS3. The artificial linker between NS2B and NS3 tends to induce the open conformation and interfere with the protease activity. This negative impact on the enzyme structure and function is restricted to the protease domain as the ATPase activities of these constructs are not affected.

Generation of “nanometer-size aggregates” using Solubility Controlling Peptide tags and their ability to increase a protein’s immunogenicity in vivo

Sub-visible aggregates of proteins are suspected to cause adverse immune response, and a recent FDA guideline has recommended the monitoring of micrometer-size aggregates (2-10 μm) though recognizing that the underlying mechanism behind aggregation and immunogenicity remains unclear. Here, we report a correlation between the immunogenicity and the size of nanometer-scale aggregates of a small 6.5 kDa model protein, Bovine Pancreatic Trypsin Inhibitor (BPTI) variant. BPTI-19A, a monomeric and non-immunogenic protein, was oligomerized into sub-visible aggregates with hydrodynamic radii (Rh) of 3~4 nm by attaching hydrophobic solubility controlling peptide (SCP) tags to its C-terminus. The results showed that the association of non-immunogenic BPTI into nanometer-size aggregates made it highly immunogenic, as assessed by the IgG antibody titers of the mice’s sera. Overall, the study emphasizes that sub-visible aggregates, as small as a few nanometers, which are presently ignored, are worth monitoring for deciphering the origin of undesired immunogenicity of therapeutic proteins.