15N relaxation NMR studies of prolyl oligopeptidase, an 80 kDa enzyme, reveal a pre-existing equilibrium between different conformational states

Open and closed: The characterization of protein mobility is crucial for the understanding of biological functions. We have applied NMR spectroscopy to study the conformational dynamics of the 80 kDa enzyme prolyl oligopeptidase (POP). Our results revealed that POP is highly dynamic and that inhibition of catalytic activity shifts this conformational equilibrium towards a less dynamic state.     

Investigation into the Feasibility of Thioditaloside as a Novel Scaffold for Galectin‐3‐Specific Inhibitors

Galectin‐3 is extensively involved in metabolic and disease processes, such as cancer metastasis, thus giving impetus for the design of specific inhibitors targeting this β‐galactose‐binding protein. Thiodigalactoside (TDG) presents a scaffold for construction of galectin inhibitors, and its inhibition of galectin‐1 has already demonstrated beneficial effects as an adjuvant with vaccine immunotherapy, thereby improving the survival outcome of tumour‐challenged mice. A novel approach—replacing galactose with its C2 epimer, talose—offers an alternative framework, as extensions at C2 permit exploitation of a galectin‐3‐specific binding groove, thereby facilitating the design of selective inhibitors. We report the synthesis of thioditaloside (TDT) and crystal structures of the galectin‐3 carbohydrate recognition domain in complexes with TDT and TDG. The different abilities of galactose and talose to anchor to the protein correlate with molecular dynamics studies, likely explaining the relative disaccharide binding affinities. The feasibility of a TDT scaffold to enable access to a particular galectin‐3 binding groove and the need for modifications to optimise such a scaffold for use in the design of potent and selective inhibitors are assessed.     

Tuning Sulfur Oxidation States on Thioether‐Bridged Peptide Macrocycles for Modulation of Protein Interactions

Thioethers, sulfoxides, and sulfonium ions, despite diverse physicochemical properties, all engage in noncovalent interactions with proteins. Thioether‐containing macrocycles are also attracting attention as protein–protein interaction (PPI) inhibitors. Here, we used a model PPI between α‐helical mixed lineage leukemia (MLL) protein and kinase‐inducible domain interacting (KIX) domain to evaluate oxidation effects on sulfurcontaining macrocycle structure, stability, and protein affinity. Desolvation effects from various polarity states were evaluated computationally and experimentally at the side chain, amino acid, and peptide level. Sulfur‐containing side chains spanned polarity ranges between all‐hydrocarbon and lactam bridges for modulating solubility, cellular uptake, and affinity. Helical propensity studies showed that, although oxidized sulfur‐containing side chains could be tolerated, conformational effects were sequence‐dependent. In some cases, proteolytic stability, binding capacity with KIX, and increased helicity were obtained as first steps toward developing PPI inhibitors.     

Crystallization Behavior of Molecular Compound in Binary Mixture System of 1,3‐Dioleoyl‐2‐Palmitoyl‐sn‐Glycerol and 1,3‐Dipalmitoyl‐2‐Oleoyl‐sn‐Glycerol

Previous studies showed that the stable β‐form of molecular compound (MC) crystals having a double‐chain‐length structure is formed in a binary mixture system of 1,3‐dioleoyl‐2‐palmitoyl‐sn‐glycerol (OPO) and 1,3‐dipalmitoyl‐2‐oleoyl‐sn‐glycerol (POP) with a 1:1 concentration ratio of OPO and POP. The use of MC crystals made of POP and OPO for edible applications, such as margarine, is advantageous due to no‐trans, low‐saturated, and high‐oleic fats. Industrial manufacturing technology involves rapid cooling processes, and the kinetic properties of crystallization of MC of OPO and POP are required. In this study, we clarified the crystallization of MC of OPO and POP under rapid cooling at rates of 1–150 °C min^?1, using synchrotron radiation time‐resolved X‐ray diffraction and differential scanning calorimetry methods. The main results are as follows: (1) POP and OPO crystallized in separate manners without the formation of MC crystals under rapid cooling (>40 °C min^?1), while MC crystals started to form with decreasing rates of cooling in addition to the POP and OPO crystals (<30 °C min^?1); (2) metastable and stable forms sub‐α, α, β′, and β of POP and OPO were formed, whereas the MC crystals of β were formed during the cooling processes; and (3) the heating processes after crystallization by rapid cooling caused separate melting of the metastable and stable forms of POP and OPO crystals and the formation of MC crystals of β made of POP and OPO, as well as melting of the MC crystals alone.     

The study of palm‐oil‐based bio‐polyol on the morphological,acoustic and mechanical properties of flexible polyurethane foams

The focus of this paper was to explore the acoustic properties of flexible polyurethane (FPU) foam modified by palm‐oil‐based polyol (POP). The presence of POP showed a marked influence on the microstructure and mechanical properties of FPU foam. A smaller mean pore diameter can be observed at lower POP content. Indeed, the introduction of POP caused a higher closed pore ratio and an increased air‐flow resistivity, which consequently improved the sound absorption coefficient and transmission loss. In particular, the acoustic performance of the all bio‐based FPU foam was enhanced at low frequency, and the density was lower than that of the reference foam. Additionally, the addition of POP also improved the compressive strength. Conversely, the tensile strength of FPU foam declined with increasing POP content. From this study, the outstanding acoustic ability of bio‐based FPU foam has been proved, with additional advantages of lower density and higher compressive strength. © 2019 Society of Chemical Industry     

The Predicted Ensemble of Low‐Energy Conformations of Human Somatostatin Receptor Subtype 5 and the Binding of Antagonists

Human somatostatin receptor subtype 5 (hSSTR5) regulates cell proliferation and hormone secretion. However, the identification of effective therapeutic small‐molecule ligands is impeded because experimental structures are not available for any SSTR subtypes. Here, we predict the ensemble of low‐energy 3D structures of hSSTR5 using a modified GPCR Ensemble of Structures in Membrane BiLayer Environment (GEnSeMBLE) complete sampling computational method. We find that this conformational ensemble displays most interhelical interactions conserved in class A G protein‐coupled receptors (GPCRs) plus seven additional interactions (e.g., Y2.43–D3.49, T3.38–S4.53, K5.64–Y3.51) likely conserved among SSTRs. We then predicted the binding sites for a series of five known antagonists, leading to predicted binding energies consistent with experimental results reported in the literature. Molecular dynamics (MD) simulation of 50 ns in explicit water and lipid retained the predicted ligand‐bound structure and formed new interaction patterns (e.g. R3.50–T6.34) consistent with the inactive μ‐opioid receptor X‐ray structure. We suggest more than six mutations for experimental validation of our prediction. The final predicted receptor conformations and antagonist binding sites provide valuable insights for designing new small‐molecule drugs targeting SSTRs.     

Molecular Insights into the Thermal Stability of mAbs with Variable‐Temperature Ion‐Mobility Mass Spectrometry

The aggregation of protein‐based therapeutics such as monoclonal antibodies (mAbs) can affect the efficacy of the treatment and can even induce effects that are adverse to the patient. Protein engineering is used to shift the mAb away from an aggregation‐prone state by increasing the thermodynamic stability of the native fold, which might in turn alter conformational flexibility. We have probed the thermal stability of three types of intact IgG molecules and two Fc‐hinge fragments by using variable‐temperature ion‐mobility mass spectrometry (VT‐IM‐MS). We observed changes in the conformations of isolated proteins as a function of temperature (300–550 K). The observed differences in thermal stability between IgG subclasses can be rationalized in terms of changes to higher‐order structural organization mitigated by the hinge region. VT‐IM‐MS provides insights into mAbs structural thermodynamics and is presented as a promising tool for thermal‐stability studies for proteins of therapeutic interest.     

Mechanism to Trigger Unfolding in O6‐Alkylguanine‐DNA Alkyltransferase

O⁶‐alkylguanine‐DNA alkyltransferase (AGT) adopts a non‐enzymatic suicide mechanism for the repair of methylated guanine bases by transferring the methyl adduct to itself, thereby initiating unfolding and fast degradation. Classical molecular dynamics simulations provide quantitative evidence that two conserved glycine residues at the centre of an α‐helix make the structure susceptible to structural perturbations. The stability of this helix, designated the “recognition helix”, is an important factor during the early onset of unfolding of human AGT (hAGT). By combining theory and experiment, we found that helical stability is controlled by key factors in the surrounding protein structure. By using a “double‐clip” mechanism, nearby residues hydrogen bond to both the base and centre of the helix. This double clip stabilises this site in the protein in the absence of substrate, but the helix is destabilised upon alkylation. The present investigation aimed to establish why alkylation of hAGT leads to conformational changes and how the protein environment functions as a switch, thus turning the stability of the protein “on” or “off” to tune degradability.     

Pathogenic Mutations Shift the Equilibria of α‐Synuclein Single Molecules towards Structured Conformers

α‐synuclein (α‐Syn) is an abundant brain protein whose mutations have been linked to early‐onset Parkinson's disease (PD). We recently demonstrated, by means of a single‐molecule force spectroscopy (SMFS) methodology, that the conformational equilibrium of monomeric wild‐type (WT) α‐Syn shifts toward β‐containing structures in several unrelated conditions linked to PD pathogenicity. Herein, we follow the same methodology previously employed for WT α‐Syn to characterize the conformational heterogeneity of pathological α‐Syn mutants A30P, A53T, and E46K. Contrary to the bulk ensemble‐averaged spectroscopies so far employed to this end by different authors, our single‐molecule methodology monitored marked differences in the conformational behaviors of the mutants with respect to the WT sequence. We found that all the mutants have a much higher propensity than the WT to adopt a monomeric compact conformation that is compatible with the acquiring of β structure. Mutants A30P and A53T show a similar conformational equilibrium that is significantly different from that of E46K. Another class of conformations, stabilized by mechanically weak interactions (MWI), shows a higher variety in the mutants than in the WT protein. In the A30P mutant these interactions are relatively stronger, and therefore the corresponding conformations are possibly more structured. The more structured and globular conformations of the mutants can explain their higher propensity to aggregate with respect to the WT.     

Substrate Binding Drives Active‐Site Closing of Human Blood Group B Galactosyltransferase as Revealed by Hot‐Spot Labeling and NMR Spectroscopy Experiments

Crystallography has shown that human blood group A (GTA) and B (GTB) glycosyltransferases undergo transitions between “open”, “semiclosed”, and “closed” conformations upon substrate binding. However, the timescales of the corresponding conformational reorientations are unknown. Crystal structures show that the Trp and Met residues are located at “conformational hot spots” of the enzymes. Therefore, we utilized ¹⁵N side‐chain labeling of Trp residues and ¹³C‐methyl labeling of Met residues to study substrate‐induced conformational transitions of GTB. Chemical‐shift perturbations (CSPs) of Met and Trp residues in direct contact with substrate ligands reflect binding kinetics, whereas the CSPs of Met and Trp residues at remote sites reflect conformational changes of the enzyme upon substrate binding. Acceptor binding is fast on the chemical‐shift timescale with rather small CSPs in the range of less than approximately 20 Hz. Donor binding matches the intermediate exchange regime to yield an estimate for exchange rate constants of approximately 200–300 Hz. Donor or acceptor binding to GTB saturated with acceptor or donor substrate, respectively, is slow (<10 Hz), as are coupled protein motions, reflecting mutual allosteric control of donor and acceptor binding. Remote CSPs suggest that substrate binding drives the enzyme into the closed state required for catalysis. These findings should contribute to better understanding of the mechanism of glycosyl transfer of GTA and GTB.     

Rigidified Clicked Dimeric Ligands for Studying the Dynamics of the PDZ1‐2 Supramodule of PSD‐95

PSD‐95 is a scaffolding protein of the MAGUK protein family, and engages in several vital protein–protein interactions in the brain with its PDZ domains. It has been suggested that PSD‐95 is composed of two supramodules, one of which is the PDZ1‐2 tandem domain. Here we have developed rigidified high‐affinity dimeric ligands that target the PDZ1‐2 supramodule, and established the biophysical parameters of the dynamic PDZ1‐2/ligand interactions. By employing ITC, protein NMR, and stopped‐flow kinetics this study provides a detailed insight into the overall conformational energetics of the interaction between dimeric ligands and tandem PDZ domains. Our findings expand our understanding of the dynamics of PSD‐95 with potential relevance to its biological role in interacting with multivalent receptor complexes and development of novel drugs.     

Protein‐Specific Imaging of O‐GlcNAcylation in Single Cells

Thousands of intracellular proteins are post‐translationally modified with O‐GlcNAc, and O‐GlcNAcylation impacts the function of modified proteins and mediates diverse biological processes. However, the ubiquity of this important glycosylation makes it highly challenging to probe the O‐GlcNAcylation state of a specific protein at the cellular level. Herein, we report the development of a FLIM–FRET‐based strategy, which exploits the spatial proximity of the O‐GlcNAc moiety and the attaching protein, for protein‐specific imaging of O‐GlcNAcylation in single cells. We demonstrated this strategy by imaging the O‐GlcNAcylation state of tau and β‐catenin inside the cells. Furthermore, the changes in tau O‐GlcNAcylation were monitored when the overall cellular O‐GlcNAc was pharmacologically altered by using the OGT and OGA inhibitors. We envision that the FLIM–FRET strategy will be broadly applicable to probe the O‐GlcNAcylation state of various proteins in the cells.     

Multilevel Changes in Protein Dynamics upon Complex Formation of the Calcium‐Loaded S100A4 with a Nonmuscle Myosin IIA Tail Fragment

Dysregulation of Ca²⁺‐binding S100 proteins plays important role in various diseases. The asymmetric complex of Ca²⁺‐bound S100A4 with nonmuscle myosin IIA has high stability and highly increased Ca²⁺ affinity. Here we investigated the possible causes of this allosteric effect by NMR spectroscopy. Chemical shift‐based secondary‐structure analysis did not show substantial changes for the complex. Backbone dynamics revealed slow‐timescale local motions in the H1 helices of homodimeric S100A4; these were less pronounced in the complex form and might be accompanied by an increase in dimer stability. Different mobilities in the Ca²⁺‐coordinating EF‐hand sites indicate that they communicate by an allosteric mechanism operating through changes in protein dynamics; this must be responsible for the elevated Ca²⁺ affinity. These multilevel changes in protein dynamics as conformational adaptation allow S100A4 fine‐tuning of its protein–protein interactions inside the cell during Ca²⁺ signaling.     

Tailoring Encodable Lanthanide‐Binding Tags as MRI Contrast Agents

Lanthanide‐binding tags (LBTs), peptide‐based coexpression tags with high affinity for lanthanide ions, have previously been applied as luminescent probes to provide phasing for structure determination in X‐ray crystallography and to provide restraints for structural refinement and distance information in NMR. The native affinity of LBTs for Gd³⁺ indicates their potential as the basis for engineering of peptide‐based MRI agents. However, the lanthanide coordination state that enhances luminescence and affords tightest binding would not be ideal for applications of LBTs as contrast agents, due to the exclusion of water from the inner coordination sphere. Herein, we use structurally defined LBTs as the starting point for re‐engineering the first coordination shell of the lanthanide ion to provide for high contrast through direct coordination of water to Gd³⁺ (resulting in the single LBT peptide, m‐sLBT). The effectiveness of LBTs as MRI contrast agents was examined in vitro through measurement of binding affinity and proton relaxivity. For imaging applications that require targeted observation, fusion to specific protein partners is desirable. However, a fusion protein comprising a concatenated double LBT (dLBT) as an N‐terminal tag for the model protein ubiquitin had reduced relaxivity compared with the free dLBT peptide. This limitation was overcome by the use of a construct based on the m‐sLBT sequence (q‐dLBT–ubiquitin). The structural basis for the enhanced contrast was examined by comparison of the X‐ray crystal structure of xq‐dLBT–ubiquitin (wherein two tryptophan residues are replaced with serine), to that of dLBT‐ubiquitin. The structure shows that the backbone conformational dynamics of the MRI variant may allow enhanced water exchange. This engineered LBT represents a first step in expanding the current base of specificity‐targeted agents available.     

Protein Synthesis in Sub‐Micrometer Water‐in‐Oil Droplets

Water‐in‐oil (w/o) emulsions are used as a cellular model because of their unique cell‐like architecture. Previous works showed the capability of eukaryotic‐cell‐sized w/o droplets (5–50 μm) to support protein synthesis efficiently; however data about smaller w/o compartments (<1 μm) are lacking. This work focuses on the biosynthesis of the enhanced green fluorescent protein (EGFP) inside sub‐micrometric lecithin‐based w/o droplets (0.8–1 μm) and on its dependence on the compartments’ dynamic properties in terms of solute exchange mechanisms. We demonstrated that protein synthesis is strongly affected by the nature of the lipid interface. These findings could be of value and interest for both basic and applied research.     

Tampering with Cell Division by Using Small‐Molecule Inhibitors of CDK–CKS Protein Interactions

Cyclin‐dependent kinases (CDKs) control many cellular processes and are considered important therapeutic targets. Large collections of inhibitors targeting CDK active sites have been discovered, but their use in chemical biology or drug development has been often hampered by their general lack of specificity. An alternative approach to develop more specific inhibitors is targeting protein interactions involving CDKs. CKS proteins interact with some CDKs and play important roles in cell division. We discovered two small‐molecule inhibitors of CDK–CKS interactions. They bind to CDK2, do not inhibit its enzymatic activity, inhibit the proliferation of tumor cell lines, induce an increase in G1 and/or S‐phase cell populations, and cause a decrease in CDK2, cyclin A, and p27^Kip1 levels. These molecules should help decipher the complex contributions of CDK–CKS complexes in the regulation of cell division, and they might present an interesting therapeutic potential.     

Effects of FlAsH/Tetracysteine (TC) Tag on PrP Proteolysis and PrPres Formation by TC‐Scanning

Protein–protein interactions associated with proteolytic processing and aggregation are integral to normal and pathological aspects of prion protein (PrP) biology. Characterization of these interactions requires the identification of amino acid residues involved. The FlAsH/tetracysteine (FlAsH/TC) tag is a small fluorescent tag amenable to insertion at internal sites in proteins. In this study, we used serial FlAsH/TC insertions (TC‐scanning) as a probe to characterize sites of protein–protein interaction between PrP and other molecules. To explore this application in the context of substrate–protease interactions, we analyzed the effect of FlAsH/TC insertions on proteolysis of cellular prion protein (PrPsen) in in vitro reactions and generation of the C1 metabolic fragment of PrPsen in live neuroblastoma cells. The influence of FlAsH/TC insertion was evaluated by TC‐scanning across the cleavage sites of each protease. The results showed that FlAsH/TC inhibited protease cleavage only within limited ranges of the cleavage sites, which varied from about one to six residues in width, depending on the protease, providing an estimate of the PrP residues interacting with each protease. TC‐scanning was also used to probe a different type of protein–protein interaction: the conformational conversion of FlAsH‐PrPsen to the prion disease‐associated isoform, PrPres. PrP constructs with FlAsH/TC insertions at residues 90–96 but not 97–101 were converted to FlAsH‐PrPres, identifying a boundary separating loosely versus compactly folded regions of PrPres. Our observations demonstrate that TC‐scanning with the FlAsH/TC tag can be a versatile method for probing protein–protein interactions and folding processes.     

X‐ray Structural Analysis of Tau‐Tubulin Kinase 1 and Its Interactions with Small Molecular Inhibitors

Tau‐tubulin kinase 1 (TTBK1) is a serine/threonine/tyrosine kinase that putatively phosphorylates residues including S422 in tau protein. Hyperphosphorylation of tau protein is the primary cause of tau pathology and neuronal death associated with Alzheimer’s disease. A library of 12 truncation variants comprising the TTBK1 kinase domain was screened for expression in Escherichia coli and insect cells. One variant (residues 14–313) could be purified, but mass spectrometric analysis revealed extensive phosphorylation of the protein. Co‐expression with lambda phosphatase in E. coli resulted in production of homogeneous, nonphosphorylated TTBK1. Binding of ATP and several compounds to TTBK1 was characterized by surface plasmon resonance. Crystal structures of TTBK1 in the unliganded form and in complex with ATP, and two high‐affinity ATP‐competitive inhibitors, 3‐[(6,7‐dimethoxyquinazolin‐4‐yl)amino]phenol ( 1 ) and methyl 2‐bromo‐5‐(7H‐pyrrolo[2,3‐d]pyrimidin‐4‐ylamino)benzoate ( 2 ), were elucidated. The structure revealed two clear basic patches near the ATP pocket providing an explanation of TTBK1 for phosphorylation‐primed substrates. Interestingly, compound 2 displayed slow binding kinetics to TTBK1, the structure of TTBK1 in complex with this compound revealed a reorganization of the L199–D200 peptide backbone conformation together with altered hydrogen bonding with compound 2 . These conformational changes necessary for the binding of compound 2 are likely the basis of the slow kinetics. This first TTBK1 structure can assist the discovery of novel inhibitors for the treatment of Alzheimer’s disease.     

Dynamic real‐time optimization with closed‐loop prediction

Process plants are operating in an increasingly global and dynamic environment, motivating the development of dynamic real‐time optimization (DRTO) systems to account for transient behavior in the determination of economically optimal operating policies. This article considers optimization of closed‐loop response dynamics at the DRTO level in a two‐layer architecture, with constrained model predictive control (MPC) applied at the regulatory control level. A simultaneous solution approach is applied to the multilevel DRTO optimization problem, in which the convex MPC optimization subproblems are replaced by their necessary and sufficient Karush–Kuhn–Tucker optimality conditions, resulting in a single‐level mathematical program with complementarity constraints. The performance of the closed‐loop DRTO strategy is compared to that of the open‐loop prediction counterpart through a multi‐part case study that considers linear dynamic systems with different characteristics. The performance of the proposed strategy is further demonstrated through application to a nonlinear polymerization reactor grade transition problem. © 2017 American Institute of Chemical Engineers AIChE J, 63: 3896–3911, 2017     

Rotational molding of polypropylene/ultra‐low‐density ethylene‐α‐olefin copolymer blends

This work investigates the performance of blends in rotational molding, by using an industrially relevant system consisting of polypropylene (PP) and an ultra‐low‐density ethylene‐α‐olefin copolymer (or polyolefin plastomer, POP). Specifically, the effect of POP content and PP type on the sintering and densification behavior, as well as the rotomolded part properties and morphology, was examined. The sinter‐melting curves of these blends exhibited bimodality, due to the wide melting point difference between the two polymers. Increasing POP content resulted in higher sintering and densification rates, as well as improved impact properties and elongation at break, counteracted by lower stiffness. Selection of a polypropylene component with lower viscosity led to better sintering and densification characteristics, due to enhanced flow properties. Better overall performance in terms of mechanical properties was obtained when polypropylene/polyethylene impact copolymers, as opposed to a PP homopolymer, were used. Polym. Eng. Sci. 44:1662–1669, 2004. © 2004 Society of Plastics Engineers.