Binding of nucleobases with graphene and carbon nanotube: a review of computational studies

Functionalized carbon nanotubes (CNTs) constitute a new class of nanostructured materials that have vast applications in CNT purification and separation, biosensing, drug delivery, etc. Hybrids formed from the functionalization of CNT with biological molecules have shown interesting properties and have attracted great attention in recent years. Of particular interest is the hybridization of single- or double-stranded nucleic acid (NA) with CNT. Nucleobases, as the building blocks of NA, interact with CNT and contribute strongly to the stability of the NA–CNT hybrids and their properties. In this work, we present a thorough review of previous studies on the binding of nucleobases with graphene and CNT, with a focus on the simulation works that attempted to evaluate the structure and strength of binding. Discrepancies among these works are identified, and factors that might contribute to such discrepancies are discussed.     

Spectroscopic and DFT investigation of interactions between cyclophosphamide and aspirin with lysozyme as binary and ternary systems

Multi-spectroscopic and density functional theory (DFT) calculations was used to study the interaction between cyclophosphamide (CYP) and aspirin (ASA) with lysozyme (LYS). The experimental results showed that fluorescence quenching of LYS by drug was a result of the formation of drug–LYS complex; static quenching was confirmed to result in fluorescence quenching. Modified Stern–Volmer plots of interaction between CYP and ASA with protein in the binary and ternary systems were used to determine the binding parameters. Molecular distances between the donor (LYS) and acceptor (CYP and ASA) for all systems were estimated according to Forster’s theory. The quantitative analysis obtained by CD spectra suggested that the presence of ASA and CYP decreased the α-helical content of LYS and induced the destabilizing of it. Theoretical studies on the interaction between LYS with ASA and CYP have been carried out using DFT at the B3LYP/6-31G level in the solvent phase. Binding energy of the mentioned complexes was calculated. It showed that tryptophan (Trp) 62 had the most affinity toward ASA and CYP. Analyzing the calculated results revealed that the five member ring of Trp has a key role in interaction of LYS with ASA and CYP.     

The Modular Organisation and Stability of a Thermostable Family 10 Xylanase

The thermophilic marine bacterium Rhodothermus marinus produces a modular family 10 xylanase (Xyn10A). It consists of two N-terminal family 4 carbohydrate binding modules (CBMs) followed by a domain of unknown function (D3), and a catalytic module (CM) flanked by a small fifth domain (D5) at its C-terminus. Several truncated mutants of the enzyme have been produced and characterised with respect to biochemical properties and stability. Multiple calcium binding sites are shown to be present in the two N-terminal CBMs and recent evidence suggests that the third domain of the enzyme also has the ability to bind the same metal ligand. The specific binding of Ca²⁺ was demonstrated to have a pronounced effect on thermostability as shown by differential scanning calorimetry and thermal inactivation studies. Furthermore, deletion mutants of the enzyme were less stable than the full-length enzyme suggesting that module interactions contributed to the stability of the enzyme. Finally, recent evidence indicates that the fifth domain of Xyn10A is a novel type of module mediating cell-attachment.     

Modelling drug elution from stents: effects of reversible binding in the vascular wall and degradable polymeric matrix

Today the most popular approach for the prevention of the restenosis consists in the use of the drug eluting stents. The stent acts as a source of drug, from a coating or from a reservoir, which is transported into and through the artery wall. In this study, the behaviour of a model of a hydrophilic drug (heparin) released from a coronary stent into the arterial wall is investigated. The presence of the specific binding site action is modelled using a reversible chemical reaction that explains the prolonged presence of drug in the vascular tissue. An axi-symmetric model of a single stent strut is considered. First an advection–diffusion problem is solved using the finite element method. Then a simplified model with diffusion only in the arterial wall is compared with: (i) a model including the presence of reversible binding sites in the vascular wall and (ii) a model featuring a drug reservoir made of a degradable polymeric matrix. The results show that the inclusion of a reversible binding for the drug leads to delayed release curves and that the polymer erosion affects the drug release showing a quicker elution of the drug from the stent.     

Mimicking the germinal center reaction in hybridoma cells to isolate temperature-selective anti-PEG antibodies

Modification of antibody class and binding properties typically requires cloning of antibody genes, antibody library construction, phage or yeast display and recombinant antibody expression. Here, we describe an alternative “cloning-free” approach to generate antibodies with altered antigen-binding and heavy chain isotype by mimicking the germinal center reaction in antibody-secreting hybridoma cells. This was accomplished by lentiviral transduction and controllable expression of activation-induced cytidine deaminase (AID) to generate somatic hypermutation and class switch recombination in antibody genes coupled with high-throughput fluorescence-activated cell sorting (FACS) of hybridoma cells to detect altered antibody binding properties. Starting from a single established hybridoma clone, we isolated mutated antibodies that bind to a low-temperature structure of polyethylene glycol (PEG), a polymer widely used in nanotechnology, biotechnology and pharmaceuticals. FACS of AID-infected hybridoma cells also facilitated rapid identification of class switched variants of monoclonal IgM to monoclonal IgG. Mimicking the germinal center reaction in hybridoma cells may offer a general method to identify and isolate antibodies with altered binding properties and class-switched heavy chains without the need to carry out DNA library construction, antibody engineering and recombinant protein expression.     

2-(1H-Pyrazol-4-yl)acetic acids as CRTh2 antagonists

High throughput screening identified the pyrazole-4-acetic acid substructure as CRTh2 receptor antagonists. Optimisation of the compounds uncovered a tight SAR but also identified some low nanomolar inhibitors.     

Determination of the biochemical properties of full-length human PIF1 ATPase

The PIF1 helicase family performs many cellular functions. To better understand the functions of the human PIF1 helicase, we characterized the biochemical properties of its ATPase. PIF1 is very sensitive to temperature, whereas it is not affected by pH, and the ATPase activity of human PIF1 is dependent on the divalent cations Mg²⁺ and Mn²⁺ but not Ca²⁺ and Zn²⁺. Inhibition was observed when single-stranded DNA was coated with RPA or SSB. Moreover, the ATPase activity of PIF1 proportionally decreased with decreasing oligonucleotide length due to a decreased binding ability. A minimum of 10 oligonucleotide bases are required for PIF1 binding and the hydrolysis of ATP. The analysis of the biochemical properties of PIF1 together with numerous genetic observations should aid in the understanding of its cellular functions.     

Metal ion interactions with mAbs: Part 1

Fragmentation in the hinge region of an IgG1 monoclonal antibody (mAb) can affect product stability, potentially causing changes in potency and efficacy. Metals ions, such as Cu²⁺, can bind to the mAb and undergo hydrolysis or oxidation, which can lead to cleavage of the molecule. To better understand the mechanism of Cu²⁺-mediated mAb fragmentation, hinge region cleavage products and their rates of formation were studied as a function of pH with and without Cu²⁺. More detailed analysis of the chemical changes was investigated using model linear and cyclic peptides (with the sequence of SCDKTHTC) derived from the upper hinge region of the mAb. Cu²⁺ mediated fragmentation was determined to be predominantly via a hydrolytic pathway in solution. The sites and products of hydrolytic cleavage are pH and strain dependent. In more acidic environments, rates of Cu²⁺ induced hinge fragmentation are significantly slower than at higher pH. Although the degradation reaction rates between the linear and cyclic peptides are not significantly different, the products of degradation vary. mAb fragmentation can be reduced by modifying His, which is a potential metal binding site and a known ligand in other metalloproteins. These results suggest that a charge may contribute to stabilization of a specific molecular structure involved in hydrolysis, leading to the possible formation of a copper binding pocket that causes increased susceptibility of the hinge region to degradation.     

Identification of the binding site of Rlp7 on assembling 60S ribosomal subunits in Saccharomyces cerevisiae

Eukaryotic ribosome assembly requires over 200 assembly factors that facilitate rRNA folding, ribosomal protein binding, and pre-rRNA processing. One such factor is Rlp7, an essential RNA binding protein required for consecutive pre-rRNA processing steps for assembly of yeast 60S ribosomal subunits: exonucleolytic processing of 27SA₃ pre-rRNA to generate the 5′ end of 5.8S rRNA and endonucleolytic cleavage of the 27SB pre-rRNA to initiate removal of internal transcribed spacer 2 (ITS2). To better understand the functions of Rlp7 in 27S pre-rRNA processing steps, we identified where it crosslinks to pre-rRNA. We found that Rlp7 binds at the junction of ITS2 and the ITS2-proximal stem, between the 3′ end of 5.8S rRNA and the 5′ end of 25S rRNA. Consistent with Rlp7 binding to this neighborhood during assembly, two-hybrid and affinity copurification assays showed that Rlp7 interacts with other assembly factors that bind to or near ITS2 and the proximal stem. We used in vivo RNA structure probing to demonstrate that the proximal stem forms prior to Rlp7 binding and that Rlp7 binding induces RNA conformational changes in ITS2 that may chaperone rRNA folding and regulate 27S pre-rRNA processing. Our findings contradict the hypothesis that Rlp7 functions as a placeholder for ribosomal protein L7, from which Rlp7 is thought to have evolved in yeast. The binding site of Rlp7 is within eukaryotic-specific RNA elements, which are not found in bacteria. Thus, we propose that Rlp7 coevolved with these RNA elements to facilitate eukaryotic-specific functions in ribosome assembly and pre-rRNA processing.     

The central role of protein S12 in organizing the structure of the decoding site of the ribosome

The ribosome decodes mRNA by monitoring the geometry of codon–anticodon base-pairing using a set of universally conserved 16S rRNA nucleotides within the conformationally dynamic decoding site. By applying single-molecule FRET and X-ray crystallography, we have determined that conditional-lethal, streptomycin-dependence mutations in ribosomal protein S12 interfere with tRNA selection by allowing conformational distortions of the decoding site that impair GTPase activation of EF-Tu during the tRNA selection process. Distortions in the decoding site are reversed by streptomycin or by a second-site suppressor mutation in 16S rRNA. These observations encourage a refinement of the current model for decoding, wherein ribosomal protein S12 and the decoding site collaborate to optimize codon recognition and substrate discrimination during the early stages of the tRNA selection process.