Ювілеї та пам’ятні дати

Native mass spectrometry is emerging as a powerful tool for the characterization of intact antibodies and antibody-based therapeutics. Here, we demonstrate new possibilities provided by the implementation of a high mass quadrupole mass selector on the recently introduced Orbitrap Exactive EMR mass spectrometer. This configuration allows precursor ion selection, and thus tandem mass spectrometry experiments, even on analytes with masses in the hundreds of kilodaltons. We apply tandem mass spectrometry to localize the drug molecules in the therapeutic antibody-drug conjugate brentuximab vedotin, which displays a heterogeneous drug load. Our tandem MS data reveal that drug conjugation takes place nonhomogeneously to cysteine residues both on the light and heavy chains. Next, we analyzed how many antigens bind to IgG hexamers, based on a recently described antibody mutant IgG1-RGY that forms hexamers and activates complement in solution. The fully saturated IgG1-RGY-antigen complexes displayed a stoichiometry of IgG:CD38 of 6:12, possessing a molecular weight of about 1.26 MDa and demonstrating that IgG assembly does not hamper antigen binding. Through tandem MS experiments, we retrieve information about the spatial arrangement and stoichiometry of the subunits within this complex. These examples underscore the potential of this further modified Orbitrap-EMR instrument especially for the in-depth characterization by native tandem mass spectrometry of antibodies and antibody-based constructs

An optimized method for 15N R1 relaxation rate measurements in non-deuterated proteins

15N longitudinal relaxation rates are extensively used for the characterization of protein dynamics; however, their accurate measurement is hindered by systematic errors. 15N CSA/1H-15N dipolar cross-correlated relaxation (CC) and amide proton exchange saturation transfer from water protons are the two main sources of systematic errors in the determination of 15N R1 rates through 1H-15N HSQC-based experiments. CC is usually suppressed through a train of 180° proton pulses applied during the variable 15N relaxation period (T), which can perturb water magnetization. Thus CC cancellation is required in such a way as to minimize water saturation effects. Here we examined the level of water saturation during the T period caused by various types of inversion proton pulses to suppress CC: (I) amide-selective IBURP-2; (II) cosine-modulated IBURP-2; (III) Watergate-like blocks; and (IV) non-selective hard. We additionally demonstrate the effect of uncontrolled saturation of aliphatic protons on 15N R1 rates. In this paper we present an optimized pulse sequence that takes into account the crucial effect of controlling also the saturation of the aliphatic protons during 15N R1 measurements in non-deuterated proteins. We show that using cosine-modulated IBURP-2 pulses spaced 40 ms to cancel CC in this optimized pulse program is the method of choice to minimiz

Molecular recognition in gas phase: theoretical and experimental study of non-covalent protein-ligand complexes by mass-spectrometry

[spa]Las biomoléculas de los organismos vivos realizan sus funciones principalmente a través de interacciones débiles reversibles entre ellas. La transducción de señal, la replicación de ADN/ARN, otros procesos enzimáticos y, virtualmente, cualquier otro proceso involucrado en las funciones vitales de cualquier organismo vivo (de las simples amebas, al complejo ser humano), requiere que las moléculas “hablen” entre ellas. Dicho lenguaje se basa en interacciones no covalentes. La flexibilidad conformacional es una propiedad esencial de las grandes biomoléculas, y muchas de las funciones desempeñadas por proteínas se basan en su capacidad para cambiar de conformación en respuesta a un factor externo. Geométricamente hablando, la presencia de flexibilidad en una proteína obstaculiza el diseño racional de medicamentos porque posibilita la existencia de un número muy elevado de conformaciones de dicha proteína. Por este motivo, cualquier información sobre la flexibilidad de una proteína es sumamente valiosa para la comprensión de PPI y PLI y para el diseño racional de medicamentos. Los capítulos 1-3 de la presente tesis versan sobre la solvatación, mientras que la flexibilidad se estudiara en el capitulo 4.[eng]In the present thesis we have explored different factors that impede accurate quantitative description of non-covalent protein-protein and protein-ligand interactions and design of new potent and specific binders from the scratch. Firstly, we addressed the role of solvent in the mechanism of non-covalent interactions. Secondly, we tackled the question about the intrinsic conformational flexibility of the protein molecules and the part it plays in weak interactions between proteins. In the first part of the thesis we studied the interactions of vascular endothelial growth factor (VEGF) protein with five cyclic peptides in solution and gas phase. The results showed that affinities of five ligands to VEGF in solution and gas phase are ranked in inversed order. That is, the that has the highest affinity in solution (as shown by chemical shift perturbation NMR and isothermal titration calorimetry) forms the weakest complex with VEGF in gas phase, and vice versa. We compared gas-phase and solution binding affinities of of five peptides and made qualitative conclusions about the role of the solvent in protein-ligand interactions. In order to obtain more quantitative information about the gas-phase behavior of non-covalent complexes we have developed a combined experimental/theoretical approach to study the energetics of collisional activation of the ion prior to dissociation. We applied developed strategy to model CID in traveling wave ion guide (TWIG) collision cell. We validated the model on the CID of leu-enkephalin peptide and then applied developed strategy to five non-covalent protein-peptide complexes and found activation energies of their dissociation reactions. Next we applied ESI native MS to study the allosteric interactions between the molecular chaperonin GroEL and ATP. The obtained data allowed to construct a scheme of conformational transition of GroEL upon binding of ATP and distinguish between two different cooperativity models, providing strong arguments in favor of Monod-Wyman-Changeux (MWC) model. Finally, be studied the backbone dynamics of VEGF with a combination of NMR relaxation and all-atom force-field based normal mode analysis (NMA). We showed that combination of experimental and computational approach allows to identify flexible zones with higher level of confidence. We also found out that residues, that are involved VEGF-receptor interactions, reside in or close to the flexible zones, suggesting the critical role conformational plasticity plays in the non-covalent protein-protein interactions

An assessment of the dynamic interaction of the rolling stock and the long-span bridges on high-speed railways

When the high-speed railway traffic is being organized, it becomes necessary to elaborate bridge design standards for high-speed railways. To ensure safety of traffic and reliability of bridges one needs to resolve the dynamic problems of various types of high-speed trains moving along the structures. The article analyzes the magnitude of inertial response as the function of the external stress parameters. A simplified structure assessment technique is offered based on the criterion of passenger comfort while crossing the bridges. The usefulness of the given research arises from the reduction of complexity of the complicated dynamic calculations needed to describe a high-speed train travelling along the artificial structures. Dependences of relative elastic deflection values from the span length are proposed, which allows designing and calculating long-span structures using the simplified dynamic models

Interactions of Coherent Structures on the Surface of Deep Water

We numerically investigate pairwise collisions of solitary wave structures on the surface of deep water—breathers. These breathers are spatially localised coherent groups of surface gravity waves which propagate so that their envelopes are stable and demonstrate weak oscillations. We perform numerical simulations of breather mutual collisions by using fully nonlinear equations for the potential flow of ideal incompressible fluid with a free surface written in conformal variables. The breather collisions are inelastic. However, the breathers can still propagate as stable localised wave groups after the interaction. To generate initial conditions in the form of separate breathers we use the reduced model—the Zakharov equation. We present an explicit expression for the four-wave interaction coefficient and third order accuracy formulas to recover physical variables in the Zakharov model. The suggested procedure allows the generation of breathers of controlled phase which propagate stably in the fully nonlinear model, demonstrating only minor radiation of incoherent waves. We perform a detailed study of breather collision dynamics depending on their relative phase. In 2018 Kachulin and Gelash predicted new effects of breather interactions using the Dyachenko−Zakharov equation. Here we show that all these effects can be observed in the fully nonlinear model. Namely, we report that the relative phase controls the process of energy exchange between breathers, level of energy loses, and space positions of breathers after the collision

An assessment of the dynamic interaction of the rolling stock and the long-span bridges on high-speed railways

When the high-speed railway traffic is being organized, it becomes necessary to elaborate bridge design standards for high-speed railways. To ensure safety of traffic and reliability of bridges one needs to resolve the dynamic problems of various types of high-speed trains moving along the structures. The article analyzes the magnitude of inertial response as the function of the external stress parameters. A simplified structure assessment technique is offered based on the criterion of passenger comfort while crossing the bridges. The usefulness of the given research arises from the reduction of complexity of the complicated dynamic calculations needed to describe a high-speed train travelling along the artificial structures. Dependences of relative elastic deflection values from the span length are proposed, which allows designing and calculating long-span structures using the simplified dynamic models

Development of a purification technology for treatment of medium- and low-activity radioactive waste of radiochemical production from Co-60 and Cs-137

The technological flowchart of purification of medium- and low-activity waste from Co-60 and Cs-137 is developed and introduced. The developed purification scheme has been successfully tested using genuine medium- and low-level liquid radioactive waste of radiochemical production containing complexing and colloid forming components complexons, surfactants. The optimal conditions of the presented method of purification ensure reduction of the residual specific activity of 60Co and 137Cs radionuclides in the solution to less than 0,9 Bq per litre

Development of a purification technology for treatment of medium- and low-activity radioactive waste of radiochemical production from Co-60 and Cs-137

The technological flowchart of purification of medium- and low-activity waste from Co-60 and Cs-137 is developed and introduced. The developed purification scheme has been successfully tested using genuine medium- and low-level liquid radioactive waste of radiochemical production containing complexing and colloid forming components complexons, surfactants. The optimal conditions of the presented method of purification ensure reduction of the residual specific activity of 60Co and 137Cs radionuclides in the solution to less than 0,9 Bq per litre

Distinct Stabilities of the Structurally Homologous Heptameric Co-Chaperonins GroES and gp31

The GroES heptamer is the molecular co-chaperonin that partners with the tetradecamer chaperonin GroEL, which assists in the folding of various nonnative polypeptide chains in Escherichia coli. Gp31 is a structural and functional analogue of GroES encoded by the bacteriophage T4, becoming highly expressed in T4-infected E. coli, taking over the role of GroES, favoring the folding of bacteriophage proteins. Despite being slightly larger, gp31 is quite homologous to GroES in terms of its tertiary and quaternary structure, as well as in its function and mode of interaction with the chaperonin GroEL. Here, we performed a side-by-side comparison of GroES and gp31 heptamer complexes by (ion mobility) tandem mass spectrometry. Surprisingly, we observed quite distinct fragmentation mechanisms for the GroES and gp31 heptamers, whereby GroES displays a unique and unusual bimodal charge distribution in its released monomers. Not only the gas-phase dissociation but also the gas-phase unfolding of GroES and gp31 were found to be very distinct. We rationalize these observations with the similar discrepancies we observed in the thermal unfolding characteristics and surface contacts within GroES and gp31 in the solution. From our data, we propose a model that explains the observed simultaneous dissociation pathways of GroES and the differences between GroES and gp31 gas-phase dissociation and unfolding. We conclude that, although GroES and gp31 exhibit high homology in tertiary and quaternary structure, they are quite distinct in their solution and gas-phase (un)folding characteristics and stability. Graphical Abstract

Data analysis using system modeling

Expert systems are increasingly being used to format safe operations. But the functions of expert systems can perform not only assistance in making decisions, but also analyze processes and help at various stages. These actions are possible when considering a system with fuzzy data. The work is devoted to solving the problem of fuzzy inference knowledge in intelligent systems based on the use of fuzzy logic. The scheme of construction of continuous logic, the computation of values of membership functions of linguistic variables of output knowledge. The proposed approach is based on the use of continuous logic, which allows for a more accurate representation of fuzzy data compared to traditional logic. The construction of the continuous logic scheme involves the use of fuzzy sets for both input and output variables, which are then used to compute the values of membership functions of linguistic variables of output knowledge. In this approach, the expert system is able to analyze processes and assist at various stages by making use of fuzzy inference knowledge. The fuzzy inference mechanism is based on the use of fuzzy logic, which allows for a more nuanced understanding of complex systems and processes. The expert system is able to analyze data from various sources and make informed recommendations based on the available information. Overall, the use of expert systems based on fuzzy logic is becoming increasingly popular in a variety of industries. By improving the accuracy of data analysis and decision-making, these systems can help to ensure safe and efficient operations