Position Of Side Chain Research Articles

Subatomic structure of hyper-sweet thaumatin D21N mutant reveals the importance of flexible conformations for enhanced sweetness

One of the sweetest proteins found in tropical fruits (with a threshold of 50 nM), thaumatin, is also used commercially as a sweetener. Our previous study successfully produced the sweetest thaumatin mutant (D21N), designated hyper-sweet thaumatin, which decreases the sweetness threshold to 31 nM. To investigate why the D21N mutant is sweeter than wild-type thaumatin, we compared the structure of the D21N mutant solved at a subatomic resolution of 0.93 Å with that of wild-type thaumatin determined at 0.90 Å. Although the overall structure of the D21N mutant resembles that of wild-type thaumatin, our subatomic resolution analysis successfully assigned and discriminated the detailed atomic positions of side-chains at position 21. The relative B-factor value of the side-chain at position 21 in the D21N mutant was higher than that of wild-type thaumatin, hinting at a greater flexibility of side-chain at 21 in the hyper-sweet D21N mutant. Furthermore, alternative conformations of Lys19, which is hydrogen-bonded to Asp21 in wild-type, were found only in the D21N mutant. Subatomic resolution analysis revealed that flexible conformations at the sites adjacent to positions 19 and 21 play a crucial role in enhancing sweet potency and may serve to enhance the complementarity of electrostatic potentials for interaction with the sweet taste receptor.

Crystal Structure of a ligand-bound LacY–Nanobody Complex

The lactose permease of Escherichia coli (LacY), a dynamic polytopic membrane transport protein, catalyzes galactoside/H+ symport and operates by an alternating access mechanism that exhibits multiple conformations, the distribution of which is altered by sugar-binding. Camelid nanobodies were made against a double-mutant Gly46 → Trp/Gly262 → Trp (LacYWW) that produces an outward-open conformation, as opposed to the cytoplasmic open-state crystal structure of WT LacY. Nanobody 9047 (Nb9047) stabilizes WT LacY in a periplasmic-open conformation. Here, we describe the X-ray crystal structure of a complex between LacYWW, the high-affinity substrate analog 4-nitrophenyl-α-d-galactoside (NPG), and Nb9047 at 3-Å resolution. The present crystal structure demonstrates that Nb9047 binds to the periplasmic face of LacY, primarily to the C-terminal six-helical bundle, while a flexible loop of the Nb forms a bridge between the N- and C-terminal halves of LacY across the periplasmic vestibule. The bound Nb partially covers the vestibule, yet does not affect the on-rates or off-rates for the substrate binding to LacYWW, which implicates dynamic flexibility of the Nb-LacYWW complex. Nb9047-binding neither changes the overall structure of LacYWW with bound NPG, nor the positions of side chains comprising the galactoside-binding site. The current NPG-bound structure exhibits a more occluded periplasmic vestibule than seen in a previous structure of a (different Nb) apo-LacYWW/Nb9039 complex that we argue is caused by sugar-binding, with major differences located at the periplasmic ends of transmembrane helices in the N-terminal half of LacY.

First report of bioactive sterols from the muricid gastropod Chicoreus ramosus

Two unusual △5 sterols with unprecedented skeletons were isolated from the organic extract of muricid gastropod Chicoreus ramosus collected off the Gulf of Mannar Coast. This is the first report of isolation of bioactive sterols endowed with anti-inflammatory potentials from this species. The compounds were characterized as (5Z)-24a-homo-cholesta-5,24a1(24a2) dien-3β-ol (1) and 27(25 → 23)-abeo-(5Z)-3β-hydroxy-24-isopropyl cholesteno-26,23-lactone (2) by the interpretation of a series of spectroscopic techniques involving two-dimensional nuclear magnetic resonance and mass spectral data. The compound 1 is unusual in that it has an ethylene attachment stemming from the additional methylene group at the 24a position of the steroid side chain, whereas compound 2 has a unique side chain bearing a γ-valerolactone ring. The △5 sterol bearing ethylene group (1) displayed comparatively better antidiabetic activity as characterized by inhibitory effects towards α-amylase and α-glucosidase enzymes (IC50 1.97 mM and 1.78 mM, respectively), whereas the cholestenolactone analogue (2) manifested higher anti-inflammatory activity (IC50 1.42 mM) as determined by in vitro 5-lipoxygenase inhibitory potential. Structure-activity correlation study showed that the biological activities of the studied sterols were directly related to their electronic properties. The homosterol (1) exhibiting improved antidiabetic properties showed higher lipophilic character coupled with lesser steric restrictions.

H/D Isotope Effects on Nuclear Shielding As a Tool for the Study of Interionic Hydrogen Bonds in Ionic Liquids

H/D isotope effects on nuclear shielding (IENS) have found applications in the study of reaction kinetics, chemical and conformational equilibria, and non-bonded interactions. In particular, they are especially well-suited to investigate hydrogen bonded (H-bonded) systems. In this presentation, we demonstrate that H/D IENS are indeed a powerful tool to scrutinize interionic H-bonds in ionic liquids (ILs), using 1-n-alkyl-3-methylimidazolium salts as examples. Results obtained with protonated and deuterated isotopologues of 1-n-butyl-3-methylimidazolium chloride ([C4mim]Cl), tetrafluroborate ([C4mim]BF4), and hexafluorofosphate ([C4mim]PF6) indicate that IENS on the 35/37Cl and 19F resonances of the anions, or D35/37Cl(H,D) and D19F(H,D), allow to identify and differentiate H-bonds between the anion and the different aromatic protons on the cation. Furthermore, IENS measured on sequentially deuterated [C4mim]BF4 and [C4mim]PF6 isotopologues reveal the formation of aliphatic C-H⋅⋅⋅X hydrogen bonds between the fluorinated anions and hydrogens along the N-alkyl sidechains of the cations. Our findings also indicate that the interactions between the fluorinated anions and hydrogens on the C-1’ and C-1” position of the N-alkyl sidechains are comparable to, and in some cases stronger than, those involving protons on the aromatic ring, underscoring the role that these weak interionic forces have on the local ordering of imidazolium salts in the liquid state. The methodology also shows that the length of the N-alkyl sidechain anchored on the N-1 position has no sizable effect on the strength of interionic H-bonds in this type of ILs. Current studies involving variations on the sidechain structure, was well as applications of the approach to the study of other systems bearing NMR-active nuclei, are discussed. Figure 1

Effects of solvent vapor annealing on organic photovoltaics with a new type of solution-processable oligothiophene-based electronic donor material

We synthesized a pair of A–D–A–D–A (A = acceptor, D = donor unit) oligomers containing a benzothiadiazole (BT) unit, namely, DRCN5BT-HH and DRCN5BT-TT, which differs in the positions of alkyl side chains. Solution-processed bulk heterojunction solar cells consisting of PC61BM and DRCN5BT-HH gave a high Voc of 1.10 V and markedly improved power conversion efficiencies up to 3.26% after solvent vapor annealing (SVA). The effects of SVA on the morphology and microstructure of the active layer were investigated by atomic force microscopy (AFM) and grazing-incidence wide angle X-ray scattering (GIWAXS). For DRCN5BT-HH, crystallization, orientation, and molecular ordering were improved, and a fibrillar structure was formed with acicular aggregates of the donor. For DRCN5BT-TT, no marked structural changes were induced by SVA. We found that these morphological and microstructural changes induced by SVA correlate with the differences in the photovoltaic properties of both of the isomers.

Effect of substitution positions of alkyl side chains in phenanthrodithiophene–isoindigo copolymers: The enhancement of crystallinity and control of molecular orders

ABSTRACTThe synthesis, characterization, and solar cell application of newly developed two semiconducting polymers containing phenanthro[1,2‐b:8,7‐b′]dithiophene (PDT) and an isoindigo (IID) unit are described. In addition, a relationship between substitution position of side chains and molecular weights of the polymers and their solar cell performance are also discussed. Because of the installation of alkyl side chains onto sterically less hindered positions, PDT‐IID copolymers 12OD‐2 and 8OD‐2 have stronger intermolecular interaction than that of the previously reported copolymer 12OD. In low‐Mn polymers 12OD‐2 and 8OD‐2 formed high‐crystalline thin film with higher face‐on ratio than that of 12OD, but their unsuitable large‐scale phase separation suppressed their efficient photocurrent generation, leading to poor PCE of 2–3%. However, the surface morphology of 12OD‐2 and 8OD‐2 blended films are drastically improved by increasing Mn, which leads to the enhancement of Jsc and higher PCE of up to 4.3%. However, high‐Mn polymers 12OD‐2 and 8OD‐2 formed high‐crystalline film with about 10–15% lower face‐on ratio than that of high‐Mn polymer 12OD, leading to poor hole transporting ability, and thus lower Jsc and PCE. From this result, too much strong intermolecular interaction promotes the formation of unsuitable edge‐on orientation in blended films. © 2018 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2018, 56, 1757–1767

Structurally Well-Defined Anion Conductive Aromatic Copolymers: Effect of the Side-Chain Length

For improving the alkaline stability and other properties of aromatic semiblock copolymer [QPE-bl-11a(C1)] membranes containing benzyltrimethylammonium groups, several novel hydrophilic monomers with different side-chain lengths and substitution positions were designed and synthesized for the polymerization. The pendant-type preaminated copolymers PE-bl-11s were quaternized using iodomethane to obtain the target QPE-bl-11s with well-defined chemical structure. In TEM analyses, QPE-bl-11a(C3) and QPE-bl-11a(C5) membranes with propyl and pentyl side-chains, respectively, showed more developed phase-separated morphology with greater hydrophilic domains (ca. 10–20 nm in width) than that of the C1 equivalent. The phase separation was more distinct and larger for the QPE-bl-11a membranes linked with p-phenylene groups in the hydrophilic part than for the QPE-bl-11b membranes with m-phenylene groups. In particular, QPE-bl-11b(C5) membrane exhibited considerably smaller hydrophilic/hydrophobic domains compared to...

Mortalin Caught in Action: Structures of the Nucleotide Binding Domain in the Apo, ADP‐bound and AMP+PPi‐bound States

The heat shock protein 70, Hsp70, chaperone family plays key roles in cellular homeostasis and stress response. Hsp70s not only regulate the folding of nascent proteins, but also prevent aggregation, promote disaggregation, and refold misfolded proteins. The mitochondrial Hsp70, Mortalin, functions in a variety of processes such as mitochondrial protein import and quality control, Fe‐S cluster biogenesis, mitochondrial homeostasis, and regulation of p53, an important tumor suppressor. Mortalin has been implicated in the regulation of apoptosis, cell stress response, neurodegenerative diseases, and has an anti‐apoptotic role in cancer cells. We have elucidated crystal structures of the nucleotide binding domain of human Mortalin in the apo, ADP‐, AMP and AMP states at 2.8 Å, 2.78 Å, and 1.75 Å resolutions, respectively. The crystal structure of Mortalin‐NBD binding to the modified ADP homolog N6propargyl‐ADP has also been determined at 1.49 Å. The overall structures and active site organizations are similar, with a few key differences in side chain positions. Specific residues in the nucleotide binding pocket, which are not conserved in other Hsp70‐family members, lead to lower nucleotide affinity and slower turnover relative to cytosolic Hsp70. ITC and biolayer interferometry experiments performed on the R126W and Y128C disease variants of Mortalin to determine ADP binding and interactions with an inter‐domain linker peptide elucidate how the mutations change Mortalin's functions and how these interactions differ between Mortalin, Hsp70, and Hsc70. ITC experiments have also been performed to test how the MortalinNBD can accommodate modified ADP nucleotides. This structural and binding data may also contribute to the understanding of disease‐associated Mortalin mutations and to improved Mortalin‐targeting antitumor compounds.Support or Funding InformationNSF 15‐52113This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Breaking the Bimolecular Crystal: The Effect of Side-Chain Length on Oligothiophene/Fullerene Intercalation

Polymer/fullerene bimolecular crystal formation has been investigated using a variety of conjugated polymers and fullerenes to understand the design rules that influence donor–acceptor interaction. Modifications of the polymer by varying the substitution side-chain position, density, and branching have demonstrated the importance of the “pocket” dimensions (free volume between side chains where the fullerene resides) for controlling intercalation. Yet the effect of pocket height has not been systematically explored because of the solubility limitations in polymers. In this report, we present an experimental investigation into the effect of the pocket height by synthesizing poly[2,5-bis(3-alkylthiophen-2-yl)thieno[3,2-b]thiophene] dimers with varied side chain lengths and track the morphological changes of the dimer/fullerene blends using grazing-incidence X-ray scattering, thermal measurements, and photoluminescence quenching. We identify two regimes: (1) oligomers with side chains greater than or equal t...

A predictive model of interfacial interactions between functionalised carbon fibre surfaces cross-linked with epoxy resin

Attachment of amine-bearing molecules, denoted surface grafted molecules (SGMs), onto the surface of carbon fibre has been previously shown to enhance interfacial interactions at the carbon fibre/epoxy interface. However, the design principles inherent to optimising this enhancement are not yet established. Here, we investigate the influence of SGM design criteria on the interfacial mechanical response for three types of SGM via predictions based on molecular dynamics simulations, and by experimental measurements. The SGMs are covalently grafted to the graphitic fibre surface via in situ generated and decomposed phenyl diazo salts. All three SGMs possess a phenyl ring as the surface attachment point, and differ by the number and/or position (meta or para) of the amine-terminated side-chain(s) attached to the ring. The single-chain meta-substituted SGM produces the least interfacial enhancement, while the double-chain meta-substituted SGM enhances the interfacial shear strength by 29% relative to the control. In contrast, the single-chain para-SGM performs almost comparably to the double-chain meta-SGM. Our modelling predictions recover this trend and offer molecular-scale explanations for these findings, providing guidance in the design of effective surface-tailoring strategies to realise enhancements in the shear response of carbon fibre–epoxy interfaces.

The impact of molecular symmetry and shape on the stability of discotic liquid crystals

Three series of dibenzo[a,c]phenazines were prepared in an order to assess the impact of side chain position on the phase stability of columnar liquid crystals. Each series was composed of four isomeric compounds differing only in the disposition of hexyloxy and decyloxy chains around the central aromatic core, giving rise to electronically similar compounds with varying shapes and symmetries. The substitution pattern was found to have a moderate effect on the clearing transition of the liquid crystal but a larger impact on the melting temperatures. These observations suggest a viable strategy for controlling the phase range of liquid crystals via judicious choice of peripheral chain structure and location.

Highly potent visnagin derivatives inhibit Cyp1 and prevent doxorubicin cardiotoxicity.

Anthracyclines such as doxorubicin are highly effective chemotherapy agents used to treat many common malignancies. However, their use is limited by cardiotoxicity. We previously identified visnagin as protecting against doxorubicin toxicity in cardiac but not tumor cells. In this study, we sought to develop more potent visnagin analogs in order to use these analogs as tools to clarify the mechanisms of visnagin-mediated cardioprotection. Structure-activity relationship studies were performed in a zebrafish model of doxorubicin cardiomyopathy. Movement of the 5-carbonyl to the 7 position and addition of short ester side chains led to development of visnagin analogs with 1,000-fold increased potency in zebrafish and 250-fold increased potency in mice. Using proteomics, we discovered that doxorubicin caused robust induction of Cytochrome P450 family 1 (CYP1) that was mitigated by visnagin and its potent analog 23. Treatment with structurally divergent CYP1 inhibitors, as well as knockdown of CYP1A, prevented doxorubicin cardiomyopathy in zebrafish. The identification of potent cardioprotective agents may facilitate the development of new therapeutic strategies for patients receiving cardiotoxic chemotherapy. Moreover, these studies support the idea that CYP1 is an important contributor to doxorubicin cardiotoxicity and suggest that modulation of this pathway could be beneficial in the clinical setting.

α,β-Unsubstitutedmeso-positioning thienyl BODIPY: a promising electron deficient building block for the development of near infrared (NIR) p-type donor–acceptor (D–A) conjugated polymers

Development of new ultra low bandgap (Eoptg < 1 eV) π-conjugated D–A polymers consisting of α,β-unsubstitutedmeso-positioning thienyl BODIPY.

Structure–activity relationship investigation of benzamide and picolinamide derivatives containing dimethylamine side chain as acetylcholinesterase inhibitors

A series of benzamide and picolinamide derivatives containing dimethylamine side chain (4a–4c and 7a–7i) were synthesised and evaluated for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity in vitro. Structure–activity relationship investigation revealed that the substituted position of dimethylamine side chain markedly influenced the inhibitory activity and selectivity against AChE and BChE. In addition, it seemed that the bioactivity of picolinamide amide derivatives was stronger than that of benzamide derivatives. Among them, compound 7a revealed the most potent AChE inhibitory activity (IC50: 2.49 ± 0.19 μM) and the highest selectivity against AChE over BChE (Ratio: 99.40). Enzyme kinetic study indicated that compound 7a show a mixed-type inhibition against AChE. The molecular docking study revealed that this compound can bind with both the catalytic site and the peripheral site of AChE.

Photoresponsive Nano-Coumarin with Indole Auxin Hormone

In this work, we designed and synthesized two photoresponsive materials with 3-indoleacetic acid (IAA) plant hormone which can be monitored from the photoreposive properties of coumarin. The varied position of the long alkoxy side chain (-OC16H33) was purposely introduced to adhesive on the plant leaves. Two coumarin-caged nanomaterials showed average particle diameter about 400 nm and gave the maximum emission wavelength at 425 and 450 nm. The formulated nanoemulsion showed good wettability (θ=48o) withCassia fistulaleave surface. Interestingly, CM2 gave the short photoresponse of photolysis within one day.

Mimicking Intermolecular Interactions of Tight Protein-Protein Complexes for Small-Molecule Antagonists.

Tight protein-protein interactions (Kd <100 nm) that occur over a large binding interface (>1000 Å2 ) are highly challenging to disrupt with small molecules. Historically, the design of small molecules to inhibit protein-protein interactions has focused on mimicking the position of interface protein ligand side chains. Here, we explore mimicry of the pairwise intermolecular interactions of the native protein ligand with residues of the protein receptor to enrich commercial libraries for small-molecule inhibitors of tight protein-protein interactions. We use the high-affinity interaction (Kd =1 nm) between the urokinase receptor (uPAR) and its ligand urokinase (uPA) to test our methods. We introduce three methods for rank-ordering small molecules docked to uPAR: 1) a new fingerprint approach that represents uPA's pairwise interaction energies with uPAR residues; 2) a pharmacophore approach to identify small molecules that mimic the position of uPA interface residues; and 3) a combined fingerprint and pharmacophore approach. Our work led to small molecules with novel chemotypes that inhibited a tight uPAR⋅uPA protein-protein interaction with single-digit micromolar IC50 values. We also report the extensive work that identified several of the hits as either lacking stability, thiol reactive, or redox active. This work suggests that mimicking the binding profile of the native ligand and the position of interface residues can be an effective strategy to enrich commercial libraries for small-molecule inhibitors of tight protein-protein interactions.

A novel SH2 recognition mechanism recruits Spt6 to the doubly phosphorylated RNA polymerase II linker at sites of transcription.

We determined that the tandem SH2 domain of S. cerevisiae Spt6 binds the linker region of the RNA polymerase II subunit Rpb1 rather than the expected sites in its heptad repeat domain. The 4 nM binding affinity requires phosphorylation at Rpb1 S1493 and either T1471 or Y1473. Crystal structures showed that pT1471 binds the canonical SH2 pY site while pS1493 binds an unanticipated pocket 70 Å distant. Remarkably, the pT1471 phosphate occupies the phosphate-binding site of a canonical pY complex, while Y1473 occupies the position of a canonical pY side chain, with the combination of pT and Y mimicking a pY moiety. Biochemical data and modeling indicate that pY1473 can form an equivalent interaction, and we find that pT1471/pS1493 and pY1473/pS1493 combinations occur in vivo. ChIP-seq and genetic analyses demonstrate the importance of these interactions for recruitment of Spt6 to sites of transcription and for the maintenance of repressive chromatin.

Determining the relative strengths of aromatic and aliphatic C-H⋅⋅⋅X hydrogen bonds in imidazolium ionic liquids through measurement of H/D isotope effects on 19 F nuclear shielding.

The relative strengths of aromatic and aliphatic C-H⋅⋅⋅X hydrogen bonds in imidazolium ionic liquids were investigated through measurement of H/D isotope effects on the 19 F nuclear shielding of deuterated isotopologues of 1-n-butyl-3-methylimidazolium hexafluorophosphate and tetrafluoroborate ([C4 mim]PF6 and [C4 mim]BF4 ). Δ19 F(H,D) values ranging from 9.7 to 49.7ppb were observed for [C4 mim]PF6 isotopologues, while for the [C4 mim]BF4 series these went from 26.2 to 83.8ppb. Our findings indicate that the interactions between the fluorinated anions and protons on the C-1' and C-1″ position of the N-alkyl sidechains are comparable to, and in some cases stronger than, those involving protons on the aromatic ring, underscoring the role that these weak interionic forces have on the local ordering of imidazolium salts in the liquid state. Copyright © 2017 John Wiley & Sons, Ltd.

Highly Selective Stable Isotope Labeling of Histidine Residues by Using a Novel Precursor in E. coli‐Based Overexpression Systems

The importance of NMR spectroscopy in unraveling the structural and dynamic properties of proteins is ever-expanding owing to progress in experimental techniques, hardware development, and novel labeling approaches. Multiple sophisticated methods of aliphatic residue labeling can be found in the literature, whereas the selective incorporation of NMR active isotopes into other amino acids still holds the potential for improvement. In order to close this methodological gap, we present a novel metabolic precursor for cell-based protein overexpression to assemble 13 C/2 H isotope patterns in the peptide backbone, as well as in side chain positions of a mechanistically distinguished histidine residue.

Prediction of Bond Vector Autocorrelation Functions from Larmor Frequency-Selective Order Parameter Analysis of NMR Relaxation Data.

Protein molecular dynamics interpretation of the standard R1, R2, and heteronuclear NOE relaxation measurements has typically been limited to a single S2 order parameter which is often insufficient to characterize the rich content of these NMR experiments. In the absence of exchange linebroadening, an optimized reduced spectral density analysis of these measurements can yield spectral density values at three distinct frequencies. Surprisingly, these three discrete spectral density values have proven to be sufficient for a Larmor frequency-selective order parameter analysis of the 223 methine and methylene H-C bonds of the B3 domain of Protein G (GB3) to accurately back-calculate the entire curve of the corresponding bond vector autocorrelation functions upon which the NMR relaxation behavior depends. The 13C relaxation values calculated from 2 μs of CHARMM36 simulation trajectories yielded the corresponding autocorrelation functions to an average rmsd of 0.44% with only three bond vectors having rmsd errors slightly greater than 1.0%. Similar quality predictions were obtained using the CHARMM22/CMAP, AMBER ff99SB, and AMBER ff99SB-ILDN force fields. Analogous predictions for the backbone 15N relaxation values were 3-fold more accurate. Excluding seven residues for which either experimental data is lacking or previous MD studies have indicated markedly divergent dynamics predictions, the CHARMM36-derived and experimentally derived 15N relaxation values for the remaining 48 amides of GB3 agree to an average of 0.016, 0.010, and 0.020 for the fast limit (Sf2) and Larmor frequency-selective (SH2 and SN2) order parameters, respectively. In contrast, for a substantial fraction of side chain positions, the statistical uncertainties obtained in the relaxation value predictions from each force field were appreciably less than the much larger differences predicted among these force fields, indicating a significant opportunity for experimental NMR relaxation measurements to provide structurally interpretable guidance for further optimizing the prediction of protein dynamics.