Combined chemical shift changes and amino acid specific chemical shift mapping of protein–protein interactions

Protein–protein interactions are often studied by chemical shift mapping using solution NMR spectroscopy. When heteronuclear data are available the interaction interface is usually predicted by combining the chemical shift changes of different nuclei to a single quantity, the combined chemical shift perturbation In this paper different procedures (published and non-published) to calculate are examined that include a variety of different functional forms and weighting factors for each nucleus. The predictive power of all shift mapping methods depends on the magnitude of the overlap of the chemical shift distributions of interacting and non-interacting residues and the cut-off criterion used. In general, the quality of the prediction on the basis of chemical shift changes alone is rather unsatisfactory but the combination of chemical shift changes on the basis of the Hamming or the Euclidian distance can improve the result. The corrected standard deviation to zero of the combined chemical shift changes can provide a reasonable cut-off criterion. As we show combined chemical shifts can also be applied for a more reliable quantitative evaluation of titration data.     

Use of chemical ionization for GC–MS metabolite profiling

Metabolite profiling is commonly performed by GC–MS of methoximated trimethylsilyl derivatives. The popularity of this technique owes much to the robust, library searchable spectra produced by electron ionization (EI). However, due to extensive fragmentation, EI spectra of trimethylsilyl derivatives are commonly dominated by trimethylsilyl fragments (e.g. m/z 73 and 147) and higher m/z fragment ions with structural information are at low abundance. Consequently different metabolites can have similar EI spectra, and this presents problems for identification of “unknowns” and the detection and deconvolution of overlapping peaks. The aim of this work is to explore use of positive chemical ionization (CI) as an adjunct to EI for GC–MS metabolite profiling. Two reagent gases differing in proton affinity (CH₄ and NH₃) were used to analyse 111 metabolite standards and extracts from plant samples. NH₃-CI mass spectra were simple and generally dominated by [MH]⁺ and/or the adduct [M+NH₄]⁺. For the 111 metabolite standards, m/z 73 and 147 were less than 3% of basepeak in NH₃-CI and less than 30% of basepeak in CH₄-CI. With CH₄-CI, [MH]⁺ was generally present but at lower relative abundance than for NH₃-CI. CH₄-CI spectra were commonly dominated by losses of CH₄ [M+1-16]⁺, 1–3 TMSOH [M+1-nx90]⁺, and combinations of CH₄ and TMSOH losses [M+1-nx90-16]⁺. CH₄-CI and NH₃-CI mass spectra are presented for 111 common metabolites, and CI is used with real samples to help identify overlapping peaks and aid identification via determination of the pseudomolecular ion with NH₃-CI and structural information with CH₄-CI.     

Use of chemical ionization for GC–MS metabolite profiling

Metabolite profiling is commonly performed by GC–MS of methoximated trimethylsilyl derivatives. The popularity of this technique owes much to the robust, library searchable spectra produced by electron ionization (EI). However, due to extensive fragmentation, EI spectra of trimethylsilyl derivatives are commonly dominated by trimethylsilyl fragments (e.g. m/z 73 and 147) and higher m/z fragment ions with structural information are at low abundance. Consequently different metabolites can have similar EI spectra, and this presents problems for identification of “unknowns” and the detection and deconvolution of overlapping peaks. The aim of this work is to explore use of positive chemical ionization (CI) as an adjunct to EI for GC–MS metabolite profiling. Two reagent gases differing in proton affinity (CH₄ and NH₃) were used to analyse 111 metabolite standards and extracts from plant samples. NH₃-CI mass spectra were simple and generally dominated by [MH]⁺ and/or the adduct [M+NH₄]⁺. For the 111 metabolite standards, m/z 73 and 147 were less than 3% of basepeak in NH₃-CI and less than 30% of basepeak in CH₄-CI. With CH₄-CI, [MH]⁺ was generally present but at lower relative abundance than for NH₃-CI. CH₄-CI spectra were commonly dominated by losses of CH₄ [M+1-16]⁺, 1–3 TMSOH [M+1-nx90]⁺, and combinations of CH₄ and TMSOH losses [M+1-nx90-16]⁺. CH₄-CI and NH₃-CI mass spectra are presented for 111 common metabolites, and CI is used with real samples to help identify overlapping peaks and aid identification via determination of the pseudomolecular ion with NH₃-CI and structural information with CH₄-CI.

     

Combined biological–chemical procedure for the mineralization of TNT

Contamination of ground and surface water with 2,4,6-trinitrotoluene (TNT) and its biological and chemical transformation products are a persisting problem at former TNT production sites. We have investigated the photochemical degradation of TNT and its aminodinitro-(ADNT) and diaminonitrotoluene (DANT) metabolites using OH-radical generating systems like Fenton and hydrogen peroxide irradiated with UV, in order to compare the degradation and mineralization rate of ADNT- and DANT-isomers with TNT itself. As a result, we find that the aminoderivatives were mineralized much faster than TNT. Consequently, as ADNTs and DANTs are the known dead-end products of biological TNT degradations, we have combined our photochemical procedure with a preceding biological treatment of TNT by a mixed culture from sludge of a sewage plant. This consecutive degradation procedure, however, shows a reduced mineralization rate of the ADNTa and DANTs in the biologically derived supernatant as compared to the pure substances, suggesting that during the biological TNT treatment by sludge competing substrates are released into the solution, and that a more defined biological procedure would be necessary in order to achieve an effective, ecologically and economically acceptable mineralization of TNT from aqueous systems.     

DGGE analysis of 16S rDNA of ammonia-oxidizing bacteria in chemical–biological flocculation and chemical coagulation systems

Microbial community DNA was extracted from activated sludge samples taken from a chemical bioflocculation process and a chemical coagulation process in Shanghai, China. 16S rDNA of ammonia-oxidizing bacteria (AOB)was amplified by nested polymerase chain reaction and fingerprinted by denaturing gradient gel electrophoresis for microbial structure analysis. The Shannon diversity index of each sample was determined. The results indicated that the microbial structure of AOB in chemical bioflocculation process was comparable at two operational conditions. The ammonia-oxidizing bacterial communities were similar in three channels of the chemical bioflocculation process and in three serial tanks in the chemical coagulation process at the same condition. The diversity of microbial structures in the chemical bioflocculation process was higher than in the chemical coagulation process, in which the microbial structure was similar to that in the influent. Although the microbial study provides insights to the nitrification removal, higher microbial diversity of AOB does not necessarily mean higher ammonia oxidization. Molecular analysis should be combined with chemical assays to optimize operational conditions.     

EGCG inhibit chemical reactivity of iron through forming an Ngal–EGCG–iron complex

Accumulated evidence indicates that the interconversion of iron between ferric (Fe³⁺) and ferrous (Fe²⁺) can be realized through interaction with reactive oxygen species in the Fenton and Haber–Weiss reactions and thereby physiologically effects redox cycling. The imbalance of iron and ROS may eventually cause tissue damage such as renal proximal tubule injury and necrosis. Many approaches were exploited to ameliorate the oxidative stress caused by the imbalance. (?)-Epigallocatechin-3-gallate, the most active and most abundant catechin in tea, was found to be involved in the protection of a spectrum of renal injuries caused by oxidative stress. Most of studies suggested that EGCG works as an antioxidant. In this paper, Multivariate analysis of the LC–MS data of tea extracts and binding assays showed that the tea polyphenol EGCG can form stable complex with iron through the protein Ngal, a biomarker of acute kidney injury. UV–Vis and Luminescence spectrum methods showed that Ngal can inhibit the chemical reactivity of iron and EGCG through forming an Ngal–EGCG–iron complex. In thinking of the interaction of iron and ROS, we proposed that EGCG may work as both antioxidant and Ngal binding siderphore in protection of kidney from injuries.     

Quantum chemical molecular dynamics simulation of carbon nanotube–graphene fusion

Abstract

Here we report a quantum mechanical molecular dynamics (QM/MD) study of a fusion process of an open-ended carbon nanotube on a graphene hole, which results in the formation of a so-called pillared graphene structure – a three-dimensional nanomaterial consisting entirely of sp²-carbons. The self-consistent-charge density-functional tight-binding potential was adopted in this study. Two different sizes of graphene holes with 12 or 24 central carbon atoms removed from a graphene flake, and a (6,6) carbon nanotube with a compatible diameter were adopted. Formations of 6–7–6/5–8–5 defect structures were found on the fusion border between tube and graphene hole. The 6–7–6 structure was found to bear less curvature-induced strain energy and therefore to be more stable and much easier to form than the 5–8–5 structure.     

Plant–insect chemical communication in ecological communities: An information theory perspective

Cross-species communication, where signals are sent by one species and perceived by others, is one of the most intriguing types of communication that functionally links different species to form complex ecological networks. Global change and human activity can affect communication by increasing fluctuations in species composition and phenology, altering signal profiles and intensity, and introducing noise. So far, most studies on cross-species communication have focused on a few specific species isolated from ecological communities. Scaling up investigations of cross-species communication to the community level is currently hampered by a lack of conceptual and practical methodologies. Here, we propose an interdisciplinary framework based on information theory to investigate mechanisms shaping cross-species communication at the community level. We use plants and insects, the cornerstones of most ecosystems, as a showcase and focus on chemical communication as the key communication channel. We first introduce some basic concepts of information theory, then we illustrate information patterns in plant–insect chemical communication, followed by a further exploration of how to integrate information theory into ecological and evolutionary processes to form testable mechanistic hypotheses. We conclude by highlighting the importance of community-level information as a means to better understand the maintenance and workings of ecological systems, especially during rapid global change.     

Protein–ligand structure guided by backbone and side-chain proton chemical shift perturbations

The fragment-based drug design approach consists of screening libraries of fragment-like ligands, to identify hits that typically bind the protein target with weak affinity ( \(100\,\upmu \hbox {M}\) –5 mM). The determination of the protein–fragment complex 3D structure constitutes a crucial step for uncovering the key interactions responsible for the protein–ligand recognition, and for growing the initial fragment into potent active compounds. The vast majority of fragments are aromatic compounds that induce chemical shift perturbations (CSP) on protein NMR spectra. These experimental CSPs can be quantitatively used to guide the ligand docking, through the comparison between experimental CSPs and CSP back-calculation based on the ring current effect. Here we implemented the CSP back-calculation into the scoring function of the program PLANTS. We compare the results obtained with CSPs measured either on amide or aliphatic protons of the human peroxiredoxin 5. We show that the different kinds of protons lead to different results for resolving the 3D structures of protein–fragment complexes, with the best results obtained with the \(\hbox {H}_{\alpha }\) protons.     

Salicylate–urea-based soluble epoxide hydrolase inhibitors with high metabolic and chemical stabilities

We investigated N-adamantyl-N′-phenyl urea derivatives as simple sEH inhibitors. Salicylate ester derivatives have high inhibitory activities against human sEH, while the free benzoic acids are less active. The methyl salicylate derivative is a potent sEH inhibitor, which also has high metabolic and chemical stabilities; suggesting that such inhibitors are potential lead molecule for bioactive compounds acting in vivo.     

Streptavidin–biotin technology: improvements and innovations in chemical and biological applications

Streptavidin and its homologs (together referred to as streptavidin) are widely used in molecular science owing to their highly selective and stable interaction with biotin. Other factors also contribute to the popularity of the streptavidin–biotin system, including the stability of the protein and various chemical and enzymatic biotinylation methods available for use with different experimental designs. The technology has enjoyed a renaissance of a sort in recent years, as new streptavidin variants are engineered to complement native proteins and novel methods of introducing selective biotinylation are developed for in vitro and in vivo applications. There have been notable developments in the areas of catalysis, cell biology, and proteomics in addition to continued applications in the more established areas of detection, labeling and drug delivery. This review summarizes recent advances in streptavidin engineering and new applications based on the streptavidin–biotin interaction.     

Accuracy and precision of protein–ligand interaction kinetics determined from chemical shift titrations

NMR-monitored chemical shift titrations for the study of weak protein?Cligand interactions represent a rich source of information regarding thermodynamic parameters such as dissociation constants (K _D ) in the micro- to millimolar range, populations for the free and ligand-bound states, and the kinetics of interconversion between states, which are typically within the fast exchange regime on the NMR timescale. We recently developed two chemical shift titration methods wherein co-variation of the total protein and ligand concentrations gives increased precision for the K _D value of a 1:1 protein?Cligand interaction (Markin and Spyracopoulos in J Biomol NMR 53: 125?C138, 2012). In this study, we demonstrate that classical line shape analysis applied to a single set of ¹H?C¹⁵N 2D HSQC NMR spectra acquired using precise protein?Cligand chemical shift titration methods we developed, produces accurate and precise kinetic parameters such as the off-rate (k _off ). For experimentally determined kinetics in the fast exchange regime on the NMR timescale, k _off ?~?3,000?s^?1 in this work, the accuracy of classical line shape analysis was determined to be better than 5?% by conducting quantum mechanical NMR simulations of the chemical shift titration methods with the magnetic resonance toolkit GAMMA. Using Monte Carlo simulations, the experimental precision for k _off from line shape analysis of NMR spectra was determined to be 13?%, in agreement with the theoretical precision of 12?% from line shape analysis of the GAMMA simulations in the presence of noise and protein concentration errors. In addition, GAMMA simulations were employed to demonstrate that line shape analysis has the potential to provide reasonably accurate and precise k _off values over a wide range, from 100 to 15,000?s^?1. The validity of line shape analysis for k _off values approaching intermediate exchange (~100?s^?1), may be facilitated by more accurate K _D measurements from NMR-monitored chemical shift titrations, for which the dependence of K _D on the chemical shift difference (????) between free and bound states is extrapolated to ?????=?0. The demonstrated accuracy and precision for k _off will be valuable for the interpretation of biological kinetics in weakly interacting protein?Cprotein networks, where a small change in the magnitude of the underlying kinetics of a given pathway may lead to large changes in the associated downstream signaling cascade.     

Uncoupling of osteoblast–osteoclast regulation in a chemical murine model of Gaucher disease

Gaucher disease (GD) is caused by mutations in the GBA gene that confer a deficient level of activity of glucocerebrosidase (GCase). This deficiency leads to accumulation of the glycolipid glucocerebroside in the lysosomes of cells of monocyte/macrophage system. Type I GD is the mildest form and is characterized by the absence of neuronopathic affection. Bone compromise in Gaucher disease patients is the most disabling aspect of the disease. However, pathophysiological aspects of skeletal alterations are still poorly understood.     

Physical–chemical principles underlying RTK activation,and their implications for human disease

RTKs, the second largest family of membrane receptors, exert control over cell proliferation, differentiation and migration. In recent years, our understanding of RTK structure and activation in health and disease has skyrocketed. Here we describe experimental approaches used to interrogate RTKs, and we review the quantitative biophysical frameworks and structural considerations that shape our understanding of RTK function. We discuss current knowledge about RTK interactions, focusing on the role of different domains in RTK homodimerization, and on the importance and challenges in RTK heterodimerization studies. We also review our understanding of pathogenic RTK mutations, and the underlying physical–chemical causes for the pathologies. This article is part of a Special Issue entitled: Protein Folding in Membranes.     

Bioremediation of synthetic high–chemical oxygen demand wastewater using microalgal species Chlorella pyrenoidosa

The microalgal species Chlorella pyrenoidosa was cultivated in synthetic wastewater of initial chemical oxygen demand (COD), nitrate, and phosphate concentrations of 5000, 100, and 40 mg/L, respectively. The aim of the study was to find out the tolerance of microalgae to different COD concentrations and the extent of COD degradation at those concentrations. Three dilutions of wastewater (initial COD concentrations 5000, 3000, and 1000 mg/L) and three inoculum sizes (0.1, 0.2, and 0.3 g/L) were considered for the study. The experimental parameters such as total organic carbon, total inorganic carbon, COD, optical density, total solids, nitrate, and phosphate were measured on a daily basis. Biodegradation kinetics was determined for all cases using first-order reaction and Monod degradation equations. Optimal results showed that up to 90% reduction in TOC was obtained for 1000 COD wastewater while only 38% reduction in total organic carbon (TOC) was achieved for 5000 COD wastewater. Over 95% reduction in nitrate and nearly 90% removal of phosphate were obtained with the lowest microalgal inoculum concentration (i.e., 0.1 g/L) for all COD dilutions. This study showed that microalgal species C. pyrenoidosa can successfully degrade the organic carbon source (i.e., acetate) with significant removal efficiencies for nitrate and phosphate.     

Wet chemical synthesis of chitosan hydrogel–hydroxyapatite composite membranes for tissue engineering applications

Chitosan, a deacetylated derivative of chitin is a commonly studied biomaterial for tissue-engineering applications due to its biocompatibility, biodegradability, low toxicity, antibacterial activity, wound healing ability and haemostatic properties. However, chitosan has poor mechanical strength due to which its applications in orthopedics are limited. Hydroxyapatite (HAp) is a natural inorganic component of bone and teeth and has mechanical strength and osteoconductive property. In this work, HAp was deposited on the surface of chitosan hydrogel membranes by a wet chemical synthesis method by alternatively soaking the membranes in CaCl₂ (pH 7.4) and Na₂HPO₄ solutions for different time intervals. These chitosan hydrogel–HAp membranes were characterized using SEM, AFM, EDS, FT-IR and XRD analyses. MTT assay was done to evaluate the biocompatibility of these membranes using MG-63 osteosarcoma cells. The biocompatibility studies suggest that chitosan hydrogel–HAp composite membranes can be useful for tissue-engineering applications.