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.     

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.     

The application of the genetic algorithm to the minimization of potential energy functions

We adapted the genetic algorithm to minimize the AMBER potential energy function. We describe specific recombination and mutation operators for this task. Next we use our algorithm to locate low energy conformation of three polypeptides (AGAGAGAGA, A9, and [Met]-enkephalin) which are probably the global minimum conformations. Our potential energy minima are –94.71, –98.50, and –48.94 kcal/mol respectively. Next, we applied our algorithm to the 46 amino acid protein crambin and located a non-native conformation which had an AMBER potential energy 150 kcal/mol lower than the native conformation. This is not necessarily the global minimum conformation, but it does illustrate problems with the AMBER potential energy function. We believe this occurred because the AMBER potential energy function does not account for hydration.     

Identification of formylglycine in sulfatases by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

C(alpha)-Formylglycine, the catalytic amino acid residue in the active site of sulfatases, is generated by post-translational modification of a cysteine or serine residue. We describe a highly sensitive procedure for the detection of C(alpha)-formylglycine-containing peptides in tryptic digests of sulfatase proteins. The protocol is based on the formation of hydrazone derivatives of C(alpha)-formylglycine-containing peptides when using dinitrophenylhydrazine as a matrix for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The hydrazone derivatives desorb and ionize with high efficiency and can be detected in the sub-femtomole range. The presence of C(alpha)-formylglycine is indicated by a mass increment of 180.13 u, corresponding to the hydrazone moiety, and also by a unique C-terminal fragment ion, characteristic of sulfatases, that becomes prominent in MALDI post-source decay mass spectra of the hydrazone derivatives.