NMR spectroscopy techniques for screening and identifying ligand binding to protein receptors

Binding events of ligands to receptors are the key for an understanding of biological processes. Gaining insight into protein-protein and protein-ligand interactions in solution has recently become possible on an atomic level by new NMR spectroscopic techniques. These experiments identify binding events either by looking at the resonance signals of the ligand or the protein. Ideally, both techniques together deliver a complete picture of ligand binding to a receptor. The approaches discussed in this review allow screening of compound libraries as well as a detailed identification of the groups involved in the binding events. Also, characterization of the binding strength and kinetics is possible, competitive binding as well as allosteric effects can be identified, and it has even been possible to identify ligand binding to intact viruses and membrane-bound proteins.     

Genomic variation and point mutations analysis of Indian COVID-19 patient samples submitted in GISAID database

Corona virus disease 2019 (COVID-19) endemic has havoc on the world; the causative virus of the pandemic is SARS CoV-2. Pharmaceutical companies and academic institutes are in continuous efforts to identify anti-viral therapy or vaccines, but the most significant challenge faced is the highly evolving genome of SARS CoV-2, which is imparting evolutionary selective benefits to the virus. To understand the viral mutations, we have retrieved nine hundred and thirty-four samples from different states of India via the GISAID database and analyzed the frequency of all types of point mutation in all structural, non-structural proteins, and accessory factors of SARS CoV-2. Spike glycol protein, nsp3, nsp6, nsp12, N and NS3 were the most evolving proteins. High frequency point mutations were Q496P (nsp2), A380V (nsp4), A994D (nsp3), L37F (nsp6), P323L & A97V (nsp12), Q57H (ns3), D614G (S), P13L (N), R203K (N), G204R (N) and S194L (N).     

SPECIFIC PROTEIN OCCURRENCE RELATED WITH PHOTOPERIODIC FLOWER INDUCTION IN THE COTYLEDONS OF Pharbitis nil CV. VIOLET

In our experiment, three groups of seedlings of SDP Pharbitis nil cv. violet were sepa-rately treated with three different photoperiods (1,16 h dark period--SD; 2, continuous illumi-nation--CL; 3, 16 h dark treatment with 10 min white light in the middle of the dark period--NB). By analysing proteins in the cotyledons from three groups with 2-D PAGE, we found nodifference in protein pattern between the three groups at 0 or 8 h after photoperiodic treatments.At 24 h after the treatments, a specific protein(MW:19 kD; pI: 4.5)appeared only in the cotyledonsof the seedlings which endured SD. This protein disappeared at 72 h after SD. ActinomycinD could inhibit flowering and the specific protein occurrence when applied to cotyledonsprior to SD, but it had no inhibition effect on flowering as well as the specific proteinoccurrence when applied to cotyledons after SD. Chloroamphenicol, a protein synthesisinhibitor, inhibited flowering when applied to cotyledons prior to or immediately after SD,but it did not i     

In situ determination of adsorption kinetics of proteins in a finite bath

A method for fast in situ measurement of adsorption kinetics based on a finite bath was developed. We modified the conventional finite bath by replacing the external loop by a dip probe which enables in situ measurement of the concentration change in the contactor. Deposition of adsorbent particles on the reflection surface of the dip probe compromised measurements. Different membranes, a polyamide, a polypropylene and a nylon membrane were tested to protect the internal reflection surface of the dip probe from fouling with adsorbent particles. The nylon membrane provided efficient protection and high mass transfer evaluated by response time experiments. Unspecific adsorption of the model protein on the membrane could also be excluded. To corroborate the measurements of the dip probe the results were compared to a conventional finite bath and to a shallow-bed. The uptake curves for human polyclonal IgG at different concentrationes (0.1-3 g/l) on rProtein A Sepharose FF and MabSelect were used as model system. The effective diffusion coefficients were determined using a pore diffusion model. These values were in good agreement for all methods.     

Determination of binding constants and stoichiometries for platinum anticancer drugs and serum transport proteins by capillary electrophoresis using the Hummel-Dreyer method

A CE method has been developed to evidence and quantitatively characterize the interaction between platinum-based antitumor drugs and human serum proteins. This method is a variant of affinity CE modified regarding both experimental setup and data treatment so as to measure the peaks (or vacancies) that correspond to the bound drug when it slowly binds to the protein. Using the formalism of the Hummel-Dreyer method and cisplatin and oxaliplatin as test compounds, a protocol for determining albumin and transferrin binding constants and stoichiometries, including (and distinguished by) 48 hours of incubation of the reaction mixture, was elaborated. Relative affinities of drugs toward different proteins in aqueous solution at physiological pH, chloride concentration, and temperature were compared in terms of overall binding constants and numbers of drug molecules attached to the protein. The results indicate that both platinum drugs bind to albumin more strongly than to transferrin, supporting the concept that the albumin fraction is a major drug supply route for chemotherapeutical needs. From a comparison with the binding parameters measured previously for cisplatin by other methods, conclusions were drawn about the validity of CE as a simple and convenient method for assaying protein-drug reactions with slow kinetics.     

Probing in vitro digestion at oil–water interfaces

Interfacial layers have been widely applied to study the formation and stability of emulsion-based systems. However, the application of isolated interfaces to address digestibility of emulsions is often limited because of the complexity of experimental methods and results. This review summarizes the latest developments in analytical methods and literature data on effects of digestion on interfacial layers. Particular emphasis is given to understand the changes on interfacial magnitudes during oral, gastric, and duodenal digestion, either applied separately or sequentially. Limitations of interfacial aspects and key factors that influence emulsion microstructure in bulk and lipid digestion are identified. Understanding the behavior of interfacial layers upon gastrointestinal digestion promotes an accurate tracking of the physiological fate of emulsions.     

Scoring functions: A view from the bench

Computational approaches to drug design are presently hindered by the complexity of the physical chemistry which underlies weak, non- covalent interactions between protein targets and small molecule ligands. Although a number of programs are now available for the design of novel potential ligands, it remains a key problem to rank these rapidly and reliably by estimated binding affinity. Such a step is necessary to select only the most promising candidates for synthesis and experimental characterisation. To calculate ligand affinity quickly and reliably is an extremely difficult problem, but it may well prove possible to estimate sufficiently accurately given an appropriate set of parameters to score individual protein–ligand interactions. Improvements in the situation will require a wider set of thermodynamically characterised systems than is currently available.     

鲨鱼软骨质量的聚类分析法

用电感耦合等离子体原子发射光谱法（ＩＣＰ－ＡＥＳ）测定了１８个鲨鱼软骨真品中１３个元素含量，对测定结果进行分析和变量聚类分析，抽提５个特征指标，并以谱系数聚类建立了鉴别鲨鱼软骨真伪的数学模式，用３个假冒产品和一个鲨鱼软骨胶囊对其进行检验，结果表明，用无机元素所建立的数学模式不仅能鉴别鲨鱼软骨的真伪，还能区分鲨鱼软骨的部分及不同部位的混合物。     

Exploring bioanalytical applications of assisted protein reassembly

Reassembly of protein from its peptide fragments is a technique that can have many applications in the bioanalytical field. Typically, a reporter protein fragmented into its two peptides is employed as a label in this study. This fragments of peptide can reassemble yielding an active functional reporter. This reassembly of the protein can be assisted by non-covalently interacting peptides or proteins, which are attached to the fragmented reporter. This technique has been employed in several applications including study of protein–protein interactions, antibody screening, immunoassays, and high-throughput screening. This review focuses on different reporters employed in the study of reassembly of proteins and applications of this strategy in bioanalysis.     

Real-time onboard wind and windshear determination,part 2: Detection

This paper is concerned with windshear detection in connection with real-time wind identification (Ref. 1). It presents a comparative evaluation of two techniques, one based on the shear/downdraft factor and one based on the wind difference index. The comparison is done with reference to a particular microburst, that which caused the 1985 crash of Flight Delta 191 at Dallas-Fort Worth International Airport.The shear/downdraft factor has the merit of combining the effects of the shear and the downdraft into a single entity. However, its effectiveness is hampered by the fact that, in a real situation, the windshear is accompanied by free-stream turbulence, which tends to blur the resulting signal. In turn, this results in undesirable nuisance warnings if the magnitude of the shear factor due to free-stream turbulence is temporarily larger than that due to true windshear. Therefore, proper filtering is necessary prior to using the shear/downdraft factor in detection and guidance. One effective way for achieving this goal is to average the shear/downdraft factor over a specified time interval . The effect of on the average shear/downdraft factor is studied.