Backbone Substitutions Research Articles

Backbone amides are determinants of Cl− selectivity in CLC ion channels

Chloride homeostasis is regulated in all cellular compartments. CLC-type channels selectively transport Cl− across biological membranes. It is proposed that side-chains of pore-lining residues determine Cl− selectivity in CLC-type channels, but their spatial orientation and contributions to selectivity are not conserved. This suggests a possible role for mainchain amides in selectivity. We use nonsense suppression to insert α-hydroxy acids at pore-lining positions in two CLC-type channels, CLC-0 and bCLC-k, thus exchanging peptide-bond amides with ester-bond oxygens which are incapable of hydrogen-bonding. Backbone substitutions functionally degrade inter-anion discrimination in a site-specific manner. The presence of a pore-occupying glutamate side chain modulates these effects. Molecular dynamics simulations show backbone amides determine ion energetics within the bCLC-k pore and how insertion of an α-hydroxy acid alters selectivity. We propose that backbone-ion interactions are determinants of Cl− specificity in CLC channels in a mechanism reminiscent of that described for K+ channels.

Characterization of hemicelluloses in sugarcane (Saccharum spp. hybrids) culm during xylogenesis

Hemicelluloses are effective renewable biopolymers that can be used in many different industrial processes and preparations. In plants, the content of hemicellulose might change with different developmental stages and/or tissues. Thus, in here chemical and structural differences in hemicellulose isolated from the apical, middle and basal segments of sugarcane stem were characterized using chemical techniques. Further, difference in expression levels of genes related to synthesis of hemicelluloses from these three segments were studied by RNA-seq and qRT-PCR etc. The sugarcane hemicellulose backbone was xylose residues connected via β-1,4 glycosidic linkages which was further substituted with arabinose, acetyl and glucuronic acid side chains. Hemicellulose content was higher in the middle and basal segments with less backbone substitutions compared to apical segments. In terms of gene expression, hemicellulose synthesis and modification genes were intensely expressed in middle and basal segments. Taken together, our research describes differences in hemicellulose content and substitutions in sugarcane during xylogenesis, which will increase our knowledge for finding more refined use of sugarcane bagasse.

Structure of the bacterial ribosome at 2 Å resolution.

Using cryo-electron microscopy (cryo-EM), we determined the structure of the Escherichia coli 70S ribosome with a global resolution of 2.0 Å. The maps reveal unambiguous positioning of protein and RNA residues, their detailed chemical interactions, and chemical modifications. Notable features include the first examples of isopeptide and thioamide backbone substitutions in ribosomal proteins, the former likely conserved in all domains of life. The maps also reveal extensive solvation of the small (30S) ribosomal subunit, and interactions with A-site and P-site tRNAs, mRNA, and the antibiotic paromomycin. The maps and models of the bacterial ribosome presented here now allow a deeper phylogenetic analysis of ribosomal components including structural conservation to the level of solvation. The high quality of the maps should enable future structural analyses of the chemical basis for translation and aid the development of robust tools for cryo-EM structure modeling and refinement.

Chemically Modified Cpf1-CRISPR RNAs Mediate Efficient Genome Editing in Mammalian Cells.

CRISPR-based gene editing is a powerful technology for engineering mammalian genomes. It holds the potential as a therapeutic, although much-needed invivo delivery systems have yet to be established. Here, using the Cpf1-crRNA (CRISPR RNA) crystal structure as a guide, we synthesized a series of systematically truncated and chemically modified crRNAs, and identify positions that are amenable to modification while retaining gene-editing activity. Modified crRNAs were designed with the same modifications that provide protection against nucleases and enable wide distribution invivo. We show crRNAs with chemically modified terminal nucleotides are exonuclease resistant while retaining gene-editing activity. Chemically modified or DNA-substituted nucleotides at select positions and up to 70% of the crRNA DNA specificity region are also well tolerated. In addition, gene-editing activity is maintained with phosphorothioate backbone substitutions in the crRNA DNA specificity region. Finally, we demonstrate that 42-mer synthetic crRNAs from the similar CRISPR-Cas9 system are taken up by cells, an attractive property for invivo delivery. Our study is the first to show that chemically modified crRNAs of the CRISPR-Cpf1 system can functionally replace and mediate comparable gene editing to the natural crRNA, which holds the potential for enhancing both viral- and non-viral-mediated invivo gene editing.

Backbone Amide Nitrogen Atoms are Key Determinants of Inter-Anion Discrimination in CLCs

Most known Cl- channels are more permeable to other anions, such as I- or NO3-, than to their namesake Cl- ion. CLC channels, in contrast, are more permeable to Cl- and have a unique selectivity sequence of Cl->Br->NO3->I-, suggesting that the mechanism of ion selectivity in the CLCs is different from that of other Cl- channels. It has been proposed that CLC selectivity is primarily determined by interactions of pore-lining side chains with the permeating ions. Specifically, a serine residue (Sercen) which participates in the central Cl- binding site was shown to control the Cl- vs NO3- selectivity of CLC channels and transporters. Surprisingly, in the structure of the ClC-K channel, which follows the signature CLC selectivity sequence, Sercen points away from the pore. Here, we show that mutations at Sercen do not alter the selectivity sequence of the CLC-K channel, suggesting that this residue is not a general determinant of CLC selectivity. Since all CLCs share a common selectivity sequence, we hypothesize that the backbone amides, which coordinate the ions in the pore, might be the conserved determinants of selectivity. To test this possibility directly, we used atomic-scale mutagenesis with non-canonical amino acids to replace pore-lining amide nitrogen atoms with oxygen atoms, by introducing α-hydroxy acids in ClC-0 and ClC-K. This maneuver eliminates the hydrogen bonding capability of the backbone NH groups while leaving the side-chain composition unperturbed. We found that CIC-0 and CIC-K channels with individual backbone substitutions displayed macroscopic currents in Xenopus laevis oocytes with a strongly degraded inter-anionic selectivity. The data suggests that the pore-lining backbone amides are the general determinants of inter-anion selectivity in CLCs and that side chains contribute to selectivity in only a subset of CLCs.

Chemical substitutions in the selectivity filter of potassium channels do not rule out constricted-like conformations for C-type inactivation

In many K+ channels, prolonged activating stimuli lead to a time-dependent reduction in ion conduction, a phenomenon known as C-type inactivation. X-ray structures of the KcsA channel suggest that this inactivated state corresponds to a "constricted" conformation of the selectivity filter. However, the functional significance of the constricted conformation has become a matter of debate. Functional and structural studies based on chemically modified semisynthetic KcsA channels along the selectivity filter led to the conclusion that the constricted conformation does not correspond to the C-type inactivated state. The main results supporting this view include the observation that C-type inactivation is not suppressed by a substitution of D-alanine at Gly77, even though this modification is believed to lock the selectivity filter into its conductive conformation, whereas it is suppressed following amide-to-ester backbone substitutions at Gly77 and Tyr78, even though these structure-conserving modifications are not believed to prevent the selectivity filter from adopting the constricted conformation. However, several untested assumptions about the structural and functional impact of these chemical modifications underlie these arguments. To make progress, molecular dynamics simulations based on atomic models of the KcsA channel were performed. The computational results support the notion that the constricted conformation of the selectivity filter corresponds to the functional C-type inactivated state of the KcsA. Importantly, MD simulations reveal that the semisynthetic KcsAD-ala77 channel can adopt an asymmetrical constricted-like nonconductive conformation and that the amide-to-ester backbone substitutions at Gly77 and Tyr78 perturb the hydrogen bonding involving the buried water molecules stabilizing the constricted conformation.

Synthesis and characterization of conjugated polyphenols derived from azomethine formation containing terephtaldehyde via oxidative polycondensation

ABSTRACTPreparation of conjugated aromatic polyimines, containing phenols in the backbone and side group substitutions, has been successfully achieved by a chemical oxidative polycondensation of a series of Schiff bases using NaOCl as the oxidant at optimum reaction temperature of 90°C. The molecular structures of synthesized compounds were verified by the FT-IR, UV-Vis, 1H-NMR and 13C-NMR techniques. Using thermogravimetric analysis-differential thermal analysis (TG-DTA) and size exclusion chromatography (SEC), the molecular weight distribution parameters and the characterization of all compounds could be specified. The initial degradation temperatures of the polymers were found in the range of 170–271°C. DSC analyses of the polyazomethines (PAMs) were carried out to assign the glass transition temperatures (Tg). UV-Vis measurements gave information about the optical (Eg) band gaps. Fluorescence measurements were carried out to obtain the maximum PL intensities. The spectral analysis outcome signified a blue and a green light emission behavior when irradiated at different wavelengths. Cyclic Voltammetry (CV) was an effective method to explore the HOMO and LUMO energy levels and electrochemical () band gaps of the polymers. The HOMO energy levels of the synthesized polyimines were lower than those of the monomers indicating the more conjugated structures of the polymers. Four-point collinear probe was used to determine the electrical conductivities of both doped and undoped states of the synthesized polymers with respect to doping time with iodine. The morphological properties of the polymers were illustrated using Scanning Electron Microscopy (SEM) images at different amplifications.

Effects of backbone substitutions on the conformational behavior of Shigella flexneri O-antigens: implications for vaccine strategy

The O-antigen (O-Ag), the polysaccharide part of the lipopolysaccharide, is the major target of the serotype-specific protective humoral response elicited upon host infection by Shigella flexneri, the main causal agent of the endemic form of bacillary dysentery. The O-Ag repeat units (RUs) of 12 S. flexneri serotypes share the tetrasaccharide backbone →2)-α-l-Rhap-(1→2)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-β-d-GlcpNAc-(1→, with site-selective glucosylation(s) and/or O-acetylation defining the serotypes. To investigate the conformational basis of serotype specificity, we sampled conformational behaviors during 60ns of molecular dynamic simulations for oligosaccharides representing three RUs of each one of the O-Ags corresponding to serotypes 1a, 1b, 2a, 2b, 3a, 3b, 4a, 4b, 5a, 5b, X and Y, respectively. The calculated trajectories were checked by nuclear magnetic resonance (NMR) for 1a, 2a, 3a and 5a O-Ags. The simulations predict that in all O-Ags, but 1a and 1b, serotype-specific substitutions of the backbone do not induce any new backbone conformations compared with the linear type O-Ag Y, although they restrain locally the accessible conformational space. Moreover, the influence of any given substituent on the backbone is independent of the eventual presence of other substituents. Finally, only slight differences in conformational behavior between terminal and inner RUs were observed. These results suggest that the reported serotype-specificity of the protective immune response is not due to recognition of distinct backbone conformations, but to binding of the serotype-defining substituents in the O-Ag context. The gained knowledge is discussed in terms of impact on the development of a broad-serotype coverage vaccine.

Abstract 1031: Sphingolipid metabolism as a therapeutic target in chemotherapy resistant and hormone therapy resistant breast cancer

Abstract Endocrine- and chemo-resistance are major causes for failure of treatment in recurrent breast cancer. Normal apoptotic pathways targeted by chemotherapeutic agents become altered, diminishing their efficacy and increasing resistance mechanisms. The proapoptotic sphingolipid compound N-Octanoyl-D-erythro-sphingosine (C8-ceramide) mimics endogenous long chain ceramide and is a potent drug in endocrine-/chemo-resistant breast cancer cells. In this study, we examined basal expression levels of modifier enzymes for endogenous ceramide regulation across drug sensitive, chemoresistant and hormone therapy- resistant breast cancer cells lines. Differences were found among UDP-galactose ceramide galactosyltransferase, sphingosine kinase-1, and acid sphingomyelinase gene expression in: MCF-10A, MCF-7, MDA-MB-231 and MCF-7TN-R cell lines. Using C8-ceramide as the lead compound, 8 novel ceramide analogs were synthesized with either enol or amide backbone substitutions and tested for biological activity. The enol substitution was found to be the most potent, with (2S,3R,E)-2-((E)benzylideneamino)octadec-4-ene1,3-diol (Analog C) displaying the greatest reduction in proliferation. Analog B appeared to be the most potent cytotoxic drug in MCF-7 and MDA-MB-231 cell lines with IC50 values of 6.2uM and 3.0 uM respectively. In the cytotoxicity assay MCF-7TN-R cell line, Analog A appeared to be the most potent with an IC50 of 2.4uM. The clonogenic proliferation assay provided very different, yet promising results. Analog D had the lowest IC50 of 460nM in MDA-MB-231, Analog C had an IC50 of 100nM in MCF-7TN-R, and Analog A had an IC50 of 1.48uM in the MCF-7 breast cancer cell line. All of the IC50s for proliferation assays were at least an order of magnitude more potent than the control drug C8 (IC50 of 5uM). Due to the potency of these drugs, these results exemplify the potential of novel ceramide-targeted therapies in chemo-resistant and endocrine-resistant breast cancer cell lines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1031.

Polyketanils: Preparation of π-Conjugated Polymer Bases from p-dibenzoylbenzene with Various Diamines. Protonation with DL-Camphor-10-sulfonic Acid

New luminescent materials - aromatic polyketanils (PKs) containing p-phenylene and biphenylene linkages in the backbone and electron-donating side-group substitutions have been synthesized via melt polycondensation (180°C, 24 h). Their structures were confirmed by elemental and spectral Fourier transform infrared, 13Cand 1H NMR, and ultraviolet-visible (UV-vis) analysis. All the PKs were solution-processable and thermally stable, making them potential candidate materials for applications in microelectronics and aerospace. These ketanil polymers showed high thermal stability and well-defined glass transition temperatures ( Tg). The temperature at which the polymers lose 10% of their weight ( T10) ranged from 420 to 520°C. Depending on the polymer structure, PKs showed Tg values in the range of 133 to 244°C. Moreover, the polymers emit either blue radiation or green radiation depending on the mutual presence of conjugated and saturated chain segments. The HOMO/LUMO energy gap of the polymers was in the range of 2.74-2.19 eV. The photoluminescence properties of the PKs after protonation with DL-camphor-10-sulfonic acid (CSA) were tested. The structure formation of (PKs)1(CSA)2 complexes are discussed on the basis of 1HNMRspectroscopy.

Simple machine-assisted protocol for solid-phase synthesis of depsipeptides

A straightforward machine-assisted protocol for the synthesis of linear depsipeptides is reported. The synthesis was performed on a 433A Peptide Synthesizer (Applied Biosystems) using preprogrammed and optimized modules for Boc chemistry and without any need for hardware modification. The robustness of the protocol was demonstrated with 12 examples of 26-membered depsipeptides with single and multiple (up to 6) ester backbone substitutions.

Combinatorial Selection, Inhibition, and Antiviral Activity of DNA Thioaptamers Targeting the RNase H Domain of HIV-1 Reverse Transcriptase

Despite the key role played by the RNase H of human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT) in viral proliferation, only a few inhibitors of RNase H have been reported. Using in vitro combinatorial selection methods and the RNase H domain of the HIV RT, we have selected double-stranded DNA thioaptamers (aptamers with selected thiophosphate backbone substitutions) that inhibit RNase H activity and viral replication. The selected thioaptamer sequences had a very high proportion of G residues. The consensus sequence for the selected thioaptamers showed G clusters separated by single residues at the 5'-end of the sequence. Gel electrophoresis mobility shift assays and nuclear magnetic resonance spectroscopy showed that the selected thioaptamer binds to the isolated RNase H domain, but did not bind to a structurally similar RNase H from Escherichia coli. The lead thioaptamer, R12-2, showed specific binding to HIV-1 RT with a binding constant (K(d)) of 70 nM. The thioaptamer inhibited the RNase H activity of intact HIV-1 RT. In cell culture, transfection of thioaptamer R12-2 (0.5 microg/mL) markedly inhibited viral production and exhibited a dose response of inhibition with R12-2 concentrations ranging from 0.03 to 2.0 microg/mL (IC(50) < 100 nM). Inhibition was also seen across a wide range of virus inoculum, ranging from a multiplicity of infection (moi) of 0.0005 to 0.05, with a reduction of the level of virus production by more than 50% at high moi. Suppression of virus was comparable to that seen with AZT when moi <or= 0.005.

Progress in Thioaptamer Development

Thioaptamers are thiophosphate ester modified nucleic acids that are isolated via in vitro or bead-based thioaptamer selection against a target molecule such as a protein. Thioaptamers offer advantages over traditional aptamers in their enhanced affinity and specificity and higher stability, largely due to the properties of the sulfur backbone-modifications. An in vitro thioaptamer selection procedure that simultaneously selects for sequence and optimized hybrid phosphoromonothioate or phosphate backbone substitutions is outlined. A novel bead-based thioaptamer selection protocol that can produce mixed phosphorodithioate, phosphoromonothioate or phosphate hybrid backbones is also described. Several examples of thioaptamers targeting specific protein are provided. Such thioaptamers are shown to modulate protein activity in vivo.

Probing backbone hydrogen bonds in the hydrophobic core of GCN4.

Backbone amide hydrogen bonds play a central role in protein secondary and tertiary structure. Previous studies have shown that substitution of a backbone ester (-COO-) in place of a backbone amide (-CONH-) can selectively destabilize backbone hydrogen bonds in a protein while maintaining a similar conformation to the native backbone structure. The majority of these studies have focused on backbone substitutions that were accessible to solvent. The GCN4 coiled coil domain is an example of a stable alpha-helical dimer that possesses a well-packed hydrophobic core. Amino acids in the a and d positions of the GCN4 helix, which pack the hydrophobic core, were replaced with the corresponding alpha-hydroxy acids in the context of a chemoselectively ligated heterodimer. While the overall structure and oligomerization state of the heterodimer were maintained, the overall destabilization of the ester analogues was greater (average DeltaDeltaG of 3+ kcal mol(-1)) and more variable than previous studies. Since burial of the more hydrophobic ester should stabilize the backbone and reduce the DeltaDeltaG, the increased destabilization must come from another source. However, the observed destabilization is correlated with the protection factors for individual amide hydrogens from previous hydrogen exchange experiments. Therefore, our results suggest that backbone engineering through ester substitution is a useful approach for probing the relative strength of backbone hydrogen bonds.

Aptamers containing thymidine 3′-O-phosphorodithioates: Synthesis and binding to nuclear factor-κB

Aptamers targeting NF-κB containing thymidine 3′-O-phosphorodithioates in selected positions of an oligonucleotide duplex were synthesized. Binding affinities to NF-κB varied with the number and positions of the dithioate backbone substitutions. One of the aptamers showed specific binding to a single NF-gkB dimer in cell culture extracts.

Potent pseudopeptide bombesin‐like agonists and antagonists

Bombesin-like pseudopeptides have been synthesized, and certain physicochemical properties and biological activities have been examined. Bombesin and the related peptide litorin were modified at positions 13-14 and 8-9, respectively, with psi[CH2S] and psi[CH2N(CH3)]. [Phe13 psi[CH2S]Leu14]bombesin and [Phe8 psi[CH2S]-Leu9]litorin bound to the murine pancreatic bombesin/gastrin releasing peptide receptor with similar dissociation constants (Kd = 3.9 and 3.4 nM, respectively). Increased potency was achieved by oxidation of the thiomethylene ether to two diastereomeric sulfoxides (isomer I, Kd = 1.6 nM and isomer II, Kd = 0.89 nM. Further oxidation to the sulfone decreased potency ([Phe8 psi[CH2SO2]Leu9]litorin, Kd = 9.9 nM). All five analogs were receptor antagonists as determined by phosphatidylinositol turnover in murine pancreas. In contrast to these peptide backbone substitutions, a psi[CH2N(CH3)] at the 8-9 amide bond position resulted in an agonist. The analogs were compared with those of litorin (Kd = 0.1 nM) and [Leu9]litorin (Kd = 0.17 nM) by CD and fluorescence spectroscopy. The CD spectra demonstrated ordered conformation for all the peptides in TFE. Different conformations corresponding to agonist and antagonist peptides were suggested by CD. Based on the pH-dependence of the fluorescence spectra of the peptides in a zwitterionic detergent, two titratable groups were identified (pKa = 6.3 and 8.5). The lower pKa is found in the agonist analogs but not in the psi [CH2S]-containing antagonist.

Synthesis and relative potencies of new constrained CRF antagonists

Two series of CRF antagonists with N alpha- and C alpha-methylated alanine and leucines were evaluated for their biological activities in vitro and in vivo in several systems. The poly-N-methylated analogue of alpha-helical-CRF9-41, [N alpha MeLeu10,15,27,37,N alpha MeAla22,32,41]-alpha-Hel-CRF9-41, was found to be considerably less potent than the parent non-N-methylated analogue. This result was expected on the basis that alpha-helicity was thought to be required for biological activity and the prediction that backbone substitutions on the nitrogen have a tendency to break alpha-helices (a hypothesis that was confirmed by circular dichroism). Next, a series of constrained analogues of the potent CRF antagonist, [DPhe12,Nle21,38]h/rCRF12-41, was synthesized that contained C alpha-methylleucine and/or C alpha-methylalanine (Aib) residues at selected positions. Because C alpha-methylation is recognized to increase alpha-helicity, and because there is now strong NMR data suggesting that residues 6-36 assume a well-defined alpha-helix, it was expected that these analogues would be more potent. Although usual solid-phase peptide synthesis procedures were followed, success in coupling the C alpha-methyl amino acids was obtained only with a 1:1 mixture of BOP/HOBt. In vitro potencies of the synthesized compounds were measured in a collagenase-dispersed anterior pituitary cell culture bioassay. Monosubstituted analogues were shown to be twice to one fourth as potent as the parent compound; while the pluri-substituted peptides were slightly less potent. This decrease in potency might be correlated to an unexpected lower helical content of the pluri-substituted compounds (as determined by CD spectroscopy), as it was suggested that the bioactive conformation of the CRF was predominantly alpha-helical. Interestingly, one analogue, [DPhe12,Nle21,38,C alpha-MeLeu37]h/rCRF12-41, was found to be more potent and longer acting than the parent compound in two in vivo assays measuring ACTH release after intravenous administration to adrenalectomized rats and reversal of stress-induced delay in gastric emptying in the rat after intracisternal administration. The molecular basis for this increased duration of action and potency is being investigated.

Structure‐permeability relationships in silicone polymers

AbstractPermeability coefficients P for He, O2, N2, CO2 CH4, C2H4, C2H6, and C3H8 in 12 different silicone polymer membranes were determined at 35.0°C and pressures up to 9 atm. Values of P for CO2, CH4, and C3H8 were also determined at 10.0 and 55.0°C. In addition, mean diffusion coefficients D and solubility coefficients S were obtained for CO2, CH4, and C3H8 in 6 silicone polymers at 10.0, 35.0, and 55.0°C. Substitution of increasingly bulkier functional groups in the side and backbone chains of silicone polymers results in a significant decrease in P for a given penetrant gas. This is due mainly to a decrease in D, whereas S decreases to a much lesser extent. Backbone substitutions appear to have a somewhat lesser effect in depressing P than equivalent side‐chain substitutions. The selectivity of a silicone membrane for a gas A relative to a gas B, i.e., the permeability ratio P(A)/P(B), may increase or decrease as a result of such substitutions, but only if the substituted groups are sufficiently bulky. The selectivity of the more highly permeable silicone membranes is controlled by the ratio S(A)/S(B), whereas the selectivity of the less permeable membranes depends on both the ratios D(A)/D(B) and S(A)/S(B). The permeability as well as the selectivity of one silicone membrane toward CO2 were significantly enhanced by the substitution of a fluorine‐containing side group that increased the solubility of CO2 in that polymer.