PI by NMR: Probing CH–π Interactions in Protein–Ligand Complexes by NMR Spectroscopy

While CH–π interactions with target proteins are crucial determinants for the affinity of arguably every drug molecule, no method exists to directly measure the strength of individual CH–π interactions in drug–protein complexes. Herein, we present a fast and reliable methodology called PI (π interactions) by NMR, which can differentiate the strength of protein–ligand CH–π interactions in solution. By combining selective amino‐acid side‐chain labeling with ¹H‐¹³C NMR, we are able to identify specific protein protons of side‐chains engaged in CH–π interactions with aromatic ring systems of a ligand, based solely on ¹H chemical‐shift values of the interacting protein aromatic ring protons. The information encoded in the chemical shifts induced by such interactions serves as a proxy for the strength of each individual CH–π interaction. PI by NMR changes the paradigm by which chemists can optimize the potency of drug candidates: direct determination of individual π interactions rather than averaged measures of all interactions.     

Integrative Chemical Biology Approaches for Identification and Characterization of “Erasers” for Fatty‐Acid‐Acylated Lysine Residues within Proteins

Acylation of proteins with fatty acids is important for the regulation of membrane association, trafficking, subcellular localization, and activity of many cellular proteins. While significant progress has been made in our understanding of the two major forms of protein acylation with fatty acids, N‐myristoylation and S‐palmitoylation, studies of the acylation of lysine residues, within proteins, with fatty acids have lagged behind. Demonstrated here is the use of integrative chemical biology approaches to examine human sirtuins as de‐fatty‐acid acylases in vitro and in cells. Photo‐crosslinking chemistry is used to investigate enzymes which recognize fatty‐acid acylated lysine. Human Sirt2 was identified as a robust lysine de‐fatty‐acid acylase in vitro. The results also show that Sirt2 can regulate the acylation of lysine residues, of proteins, with fatty acids within cells.     

CE detection of N‐(3‐dimethylaminopropyl)‐N‐carbodiimide/N‐hydroxysuccinimide‐coupled proteins after homo‐ and hetero‐crosslinking reactions

Protein–protein conjugates formed by carbodiimide crosslinking reactions have been analyzed for the first time using CE. Lysozyme and BSA were chosen as model proteins to study the efficacy of N‐(3‐dimethylaminopropyl)‐N‐ethylcarbodiimide and N‐hydroxysuccinimide as crosslinkers. Detection of the molecular mass increase was checked by SDS‐PAGE. Commercially available, PVA‐coated capillaries showed appropriate selection, while phospho‐deactivated and dynamic PVA‐coated capillaries did not give suitable resolution. CE was found to be an efficient tool to characterize homo‐ (lysozyme–lysozyme) and hetero‐ (lysozyme–BSA) protein coupling by suitable variations of electrophoretic mobilities.     

Understanding chemical reactivity for homo‐ and heterobifunctional protein cross‐linking agents

Chemical cross‐linking, combined with mass spectrometry, has been applied to map three‐dimensional protein structures and protein–protein interactions. Proper choice of the cross‐linking agent, including its reactive groups and spacer arm length, is of great importance. However, studies to understand the details of reactivity of the chemical cross‐linkers with proteins are quite sparse. In this study, we investigated chemical cross‐linking from the aspects of the protein structures and the cross‐linking reagents involved, by using two structurally well‐known proteins, glyceraldehyde 3‐phosohate dehydrogenase and ribonuclease S. Chemical cross‐linking reactivity was compared using a series of homo‐ and hetero‐bifunctional cross‐linkers, including bis(sulfosuccinimidyl) suberate, dissuccinimidyl suberate, bis(succinimidyl) penta (ethylene glycol), bis(succinimidyl) nona (ethylene glycol), m‐maleimidobenzoyl‐N‐hydroxysulfosuccinimide ester, 2‐pyridyldithiol‐tetraoxaoctatriacontane‐N‐hydrosuccinimide and succinimidyl‐[(N‐maleimidopropionamido)‐tetracosaethyleneglycol]ester. The protein structure itself, especially the distances between target amino acid residues, was found to be a determining factor for the cross‐linking efficiency. Moreover, the reactive groups of the chemical cross‐linker also play an important role; a higher cross‐linking reaction efficiency was found for maleimides compared to 2‐pyrimidyldithiols. The reaction between maleimides and sulfhydryl groups is more favorable than that between N‐hydroxysuccinimide esters and amine groups, although cysteine residues are less abundant in proteins compared to lysine residues. Copyright © 2013 John Wiley & Sons, Ltd.     

Reactivity of Tyr–Leu and Leu–Tyr dipeptides: identification of oxidation products by liquid chromatography–tandem mass spectrometry

The exposure of peptides and proteins to reactive hydroxyl radicals results in covalent modifications of amino acid side‐chains and protein backbone. In this study we have investigated the oxidation the isomeric peptides tyrosine–leucine (YL) and leucine–tyrosine (LY), by the hydroxyl radical formed under Fenton reaction (Fe²⁺/H₂O₂). Through mass spectrometry (MS), high‐performance liquid chromatography (HPLC‐MS) and electrospray tandem mass spectrometry (HPLC‐MSⁿ) measurements, we have identified and characterized the oxidation products of these two dipeptides. This approach allowed observing and identifying a wide variety of oxidation products, including isomeric forms of the oxidized dipeptides. We detected oxidation products with 1, 2, 3 and 4 oxygen atoms for both peptides; however, oxidation products with 5 oxygen atoms were only present in LY. LY dipeptide oxidation leads to more isomers with 1 and 2 oxygen atoms than YL (3 vs 5 and 4 vs 5, respectively). Formation of the peroxy group occurred preferentially in the C‐terminal residue. We have also detected oxidation products with double bonds or keto groups, dimers (YL–YL and LY–LY) and other products as a result of cross‐linking. Both amino acids in the dipeptides were oxidized although the peptides showed different oxidation products. Also, amino acid residues have shown different oxidation products depending on the relative position on the dipeptide. Results suggest that amino acids in the C‐terminal position are more prone to oxidation. Copyright © 2009 John Wiley & Sons, Ltd.     

Structural and Biochemical Analysis of Protein–Protein Interactions Between the Acyl‐Carrier Protein and Product Template Domain

In fungal non‐reducing polyketide synthases (NR‐PKS) the acyl‐carrier protein (ACP) carries the growing polyketide intermediate through iterative rounds of elongation, cyclization and product release. This process occurs through a controlled, yet enigmatic coordination of the ACP with its partner enzymes. The transient nature of ACP interactions with these catalytic domains imposes a major obstacle for investigation of the influence of protein–protein interactions on polyketide product outcome. To further our understanding about how the ACP interacts with the product template (PT) domain that catalyzes polyketide cyclization, we developed the first mechanism‐based crosslinkers for NR‐PKSs. Through in vitro assays, in silico docking and bioinformatics, ACP residues involved in ACP–PT recognition were identified. We used this information to improve ACP compatibility with non‐cognate PT domains, which resulted in the first gain‐of‐function ACP with improved interactions with its partner enzymes. This advance will aid in future combinatorial biosynthesis of new polyketides.     

A Photochemical Switch for Controlling Protein–Protein Interactions

Incorporation of an unnatural amino acid containing a photolabile group in the side chain allows specific interactions between two proteins to be prevented. The photocaged ras protein in which Asp 38 has been substituted by its β-nitrobenzyl ester (Nb) is unable to interact with its effector protein p120–GAP (see drawing below) although it has the same intrinsic GTPase activity. After photocleavage of the Nb group, 50% of the p120–GAP-dependent GTPase activity relative to the wild-type protein is restored.     

Affinity Polymers Tailored for the Protein A Binding Site of Immunoglobulin G Proteins

Rational design in combination with a screening process was used to develop affinity polymers for a specific binding site on the surface of immunoglobulin G (IgG) proteins. The concept starts with the identification of critical amino acid residues on the protein interface and their topological arrangement. Appropriate binding monomers were subsequently synthesized. Together with a sugar monomer (2–5 equiv) for water solubility and a dansyl monomer (0.5 equiv) as a fluorescent label, they were subjected in aqueous solution to linear radical copolymerization in various compositions (e.g., azobisisobutyronitrile (AIBN), homogeneous water/DMF mixtures). After ultrafiltration and lyophilization, colorless dry water‐soluble powders were obtained. NMR spectroscopic and gel permeation chromatography (GPC) characterization indicated molecular weights between 30 and 500 kD and confirmed retention of monomer composition as well as the absence of monomers. In a competitive enzyme‐linked immunosorbent assay (ELISA) screen of the polymer libraries (20–50 members), few copolymers qualified as strong and selective binders for the protein A binding site on the Fc fragment of the antibody. Their monomer composition precisely reflected the critical amino acids found at the interface. The simple combination of a charged and a nonpolar binding monomer sufficed for selective submicromolar IgG recognition by the synthetic polymer. Affinities were confirmed by fluorescence titrations; they increased with decreasing salt load but remained largely unaltered at lowered pH. Other proteins, including those of similar size and isoelectric point (pI), were bound 10–1000 times less tightly. This example indicates that interaction domains in other proteins may also be targeted by synthetic polymers if their comonomer composition reflects the nature and arrangement of amino acid residues on the protein surface.     

A Non‐Invasive NMR Method Based on Histidine Imidazoles to Analyze the pH‐Modulation of Protein–Nucleic Acid Interfaces

A useful ²J(N?H) coupling‐based NMR spectroscopic approach is proposed to unveil, at the molecular level, the contribution of the imidazole groups of histidines from RNA/DNA‐binding proteins on the modulation of binding to nucleic acids by pH. Such protonation/deprotonation events have been monitored on the single His96 located at the second RNA/DNA recognition motif (RRM2) of T‐cell intracellular antigen‐1 (TIA‐1) protein. The pK_a values of the His96 ionizable groups were substantially higher in the complexes with short U‐rich RNA and T‐rich DNA oligonucleotides than those of the isolated TIA‐1 RRM2. Herein, the methodology applied to determine changes in pK_a of histidine side chains upon DNA/RNA binding, gives valuable information to understand the pH effect on multidomain DNA/RNA‐binding proteins that shuttle among different cellular compartments.     

Serine‐Selective Aerobic Cleavage of Peptides and a Protein Using a Water‐Soluble Copper–Organoradical Conjugate

The site‐specific cleavage of peptide bonds is an important chemical modification of biologically relevant macromolecules. The reaction is not only used for routine structural determination of peptides, but is also a potential artificial modulator of protein function. Realizing the substrate scope beyond the conventional chemical or enzymatic cleavage of peptide bonds is, however, a formidable challenge. Here we report a serine‐selective peptide‐cleavage protocol that proceeds at room temperature and near neutral pH value, through mild aerobic oxidation promoted by a water‐soluble copper–organoradical conjugate. The method is applicable to the site‐selective cleavage of polypeptides that possess various functional groups. Peptides comprising D ‐amino acids or sensitive disulfide pairs are competent substrates. The system is extendable to the site‐selective cleavage of a native protein, ubiquitin, which comprises more than 70 amino acid residues.     

Multicomponent Peptide Stapling as a Diversity‐Driven Tool for the Development of Inhibitors of Protein–Protein Interactions

Stapled peptides are chemical entities in‐between biologics and small molecules, which have proven to be the solution to high affinity protein–protein interaction antagonism, while keeping control over pharmacological performance such as stability and membrane penetration. We demonstrate that the multicomponent reaction‐based stapling is an effective strategy for the development of α‐helical peptides with highly potent dual antagonistic action of MDM2 and MDMX binding p53. Such a potent inhibitory activity of p53‐MDM2/X interactions was assessed by fluorescence polarization, microscale thermophoresis, and 2D NMR, while several cocrystal structures with MDM2 were obtained. This MCR stapling protocol proved efficient and versatile in terms of diversity generation at the staple, as evidenced by the incorporation of both exo‐ and endo‐cyclic hydrophobic moieties at the side chain cross‐linkers. The interaction of the Ugi‐staple fragments with the target protein was demonstrated by crystallography.     

Stimuli‐Responsive Biomolecule‐Based Hydrogels and Their Applications

This Review presents polysaccharides, oligosaccharides, nucleic acids, peptides, and proteins as functional stimuli‐responsive polymer scaffolds that yield hydrogels with controlled stiffness. Different physical or chemical triggers can be used to structurally reconfigure the crosslinking units and control the stiffness of the hydrogels. The integration of stimuli‐responsive supramolecular complexes and stimuli‐responsive biomolecular units as crosslinkers leads to hybrid hydrogels undergoing reversible triggered transitions across different stiffness states. Different applications of stimuli‐responsive biomolecule‐based hydrogels are discussed. The assembly of stimuli‐responsive biomolecule‐based hydrogel films on surfaces and their applications are discussed. The coating of drug‐loaded nanoparticles with stimuli‐responsive hydrogels for controlled drug release is also presented.     

A Convergent Strategy for the Synthesis of Type‐1 Elongated Mucin Cores 1–3 and the Corresponding Glycopeptides

Mucins are a class of highly O‐glycosylated proteins found on the surface of cells in epithelial tissues. O‐Glycosylation is crucial for the functionality of mucins and changes therein can have severe consequences for an organism. With that in mind, the elucidation of interactions of carbohydrate binding proteins with mucins, whether in morbidly altered or unaltered conditions, continue to shed light on mechanisms involved in diseases like chronic inflammations and cancer. Despite the known importance of type‐1 and type‐2 elongated mucin cores 1–4 in glycobiology, the corresponding type‐1 structures are much less well studied. Here, the first chemical synthesis of extended mucin type‐1 O‐glycan core 1–3 amino acid structures based on a convergent approach is presented. By utilizing differentiation in acceptor reactivity, shared early stage Tn‐ and T‐acceptor intermediates were elongated with a common type‐1 [β‐D ‐Gal‐1,3‐β‐D ‐GlcNAc] disaccharide, which allows for straightforward preparation of diverse glycosylated amino acids carrying the type‐1 mucin core 1–3 saccharides. The obtained glycosylated 9‐fluorenylmethoxycarbonyl (Fmoc)‐protected amino acid building blocks were employed in synthesis of type‐1 mucin glycopeptides, which are useful in biological applications.     

Fluorine Pseudocontact Shifts Used for Characterizing the Protein–Ligand Interaction Mode in the Limit of NMR Intermediate Exchange

The characterization of protein–ligand interaction modes becomes recalcitrant in the NMR intermediate exchange regime as the interface resonances are broadened beyond detection. Here, we determined the ¹⁹F low‐populated bound‐state pseudocontact shifts (PCSs) of mono‐ and di‐fluorinated inhibitors of the BRM bromodomain using a highly skewed protein/ligand ratio. The bound‐state ¹⁹F PCSs were retrieved from ¹⁹F chemical exchange saturation transfer (CEST) in the presence of the lanthanide‐labeled protein, which was termed the ¹⁹F PCS‐CEST approach. These PCSs enriched in spatial information enabled the identification of best‐fitting poses, which agree well with the crystal structure of a more soluble analog in complex with the BRM bromodomain. This approach fills the gap of the NMR structural characterization of lead‐like inhibitors with moderate affinities to target proteins, which are essential for structure‐guided hit‐to‐lead evolution.     

Utilizing thiol–ene chemistry for crosslinked nickel cation‐based anion exchange membranes

Metal cation‐based anion exchange membranes (AEMs) are a unique class of materials that have shown potential to be highly stable AEMs with competitive conductivities. Here, we expand upon previous work to report the synthesis of crosslinked nickel cation‐based AEMs formed using the thiol–ene reaction. These thiol–ene‐based samples were first characterized for their morphology, both with and without nickel cations, where the nickel‐containing membranes demonstrated a disordered scattering peak characteristic of ionic clusters. The samples were then characterized for their water uptake, chemical and mechanical stability, and conductivity. They showed a combination of high water content and extreme brittleness, which also resulted in fairly low conductivity. The brittleness resulted from large water swelling as well as the need for each nickel cation to act as a crosslinker, necessary with the current nickel‐coordination chemistry. Therefore, increasing the ion exchange capacity (IEC) for these types of AEMs, important for enhancing conductivity, also increased the crosslink density. The low conductivity and brittleness seen in this work demonstrated the need to develop non‐crosslinking metal‐complexes. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2018 , 56, 328–339     

Double‐crosslinked reversible redox‐responsive hydrogels based on disulfide–thiol interchange

We present novel redox‐responsive hydrogels based on poly(N‐isopropylacrylamide) or poly(acrylamide), consisting of a reversible disulfide crosslinking agent N,N′‐bis(acryloyl)cystamine and a permanent crosslinking agent N,N′‐methylenebisacrylamide for microfluidic applications. The mechanism of swelling/deswelling behavior starts with the cleavage and reformation of disulfide bonds, leading to a change of crosslinking density and crosslinking points. Raman and ultraviolet‐visible spectroscopy confirm that conversion efficiency of thiol–disulfide interchange up to 99%. Rheological analysis reveals that the E modulus of hydrogel is dependent on the crosslinking density and can be repeatedly manipulated between high‐ and low‐stiffness states over at least 5 cycles without significant decrease. Kinetic studies showed that the mechanical strength of the gels changes as the redox reaction proceeds. This process is much faster than the autonomous diffusion in the hydrogel. Moreover, cooperative diffusion coefficient (D_coop) indicates that the swelling process of the hydrogel is affected by the reduction reaction. Finally, this reversibly switchable redox behavior of bulky hydrogel could be proven in microstructured hydrogel dots through short‐term photopatterning process. These hydrogel dots on glass substrates also showed the desired short response time on cyclic swelling and shrinking processes known from downsized hydrogel shapes. Such stimuli‐responsive hydrogels with redox‐sensitive crosslinkers open a new pathway in exchanging analytes for sensing and separating in microfluidics applications. © 2019 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2019 , 57, 2590–2601     

A Unified Strategy for the Synthesis of Mucin Cores 1–4 Saccharides and the Assembled Multivalent Glycopeptides

By displaying different O‐glycans in a multivalent mode, mucin and mucin‐like glycoproteins are involved in a plethora of protein binding events. The understanding of the roles of the glycans and the identification of potential glycan binding proteins are major challenges. To enable future binding studies of mucin glycan and glycopeptide probes, a method that gives flexible and efficient access to all common mucin core‐glycosylated amino acids was developed. Based on a convergent synthesis strategy starting from a shared early stage intermediate by differentiation in the glycoside acceptor reactivity, a common disaccharide building block allows for the creation of extended glycosylated amino acids carrying the mucin type‐2 cores 1–4 saccharides. Formation of a phenyl‐sulfenyl‐N‐Troc (Troc=trichloroethoxycarbonyl) byproduct during N‐iodosuccinimide‐promoted thioglycoside couplings was further characterized and a new methodology for the removal of the Troc group is described. The obtained glycosylated 9‐fluorenylmethoxycarbonyl (Fmoc)‐protected amino acid building blocks are incorporated into peptides for multivalent glycan display.     

Stabilizer‐Guided Inhibition of Protein–Protein Interactions

The discovery of novel protein–protein interaction (PPI) modulators represents one of the great molecular challenges of the modern era. PPIs can be modulated by either inhibitor or stabilizer compounds, which target different though proximal regions of the protein interface. In principle, protein–stabilizer complexes can guide the design of PPI inhibitors (and vice versa). In the present work, we combine X‐ray crystallographic data from both stabilizer and inhibitor co‐crystal complexes of the adapter protein 14‐3‐3 to characterize, down to the atomic scale, inhibitors of the 14‐3‐3/Tau PPI, a potential drug target to treat Alzheimer’s disease. The most potent compound notably inhibited the binding of phosphorylated full‐length Tau to 14‐3‐3 according to NMR spectroscopy studies. Our work sets a precedent for the rational design of PPI inhibitors guided by PPI stabilizer–protein complexes while potentially enabling access to new synthetically tractable stabilizers of 14‐3‐3 and other PPIs.