[In silico specificity determination of neoantigen-reactive T-lymphocytes]. / In silico opredelenie spetsifichnosti neoantigen-reaktivnykh T-limfotsitov.

Effective personalized immunotherapies of the future will need to capture not only the peculiarities of the patient's tumor but also of his immune response to it. In this study, using results of in vitro high-throughput specificity assays, and combining comparative models of pMHCs and TCRs using molecular docking, we have constructed all-atom models for the putative complexes of all their possible pairwise TCR-pMHC combinations. For the models obtained we have calculated a dataset of physics-based scores and have trained binary classifiers that perform better compared to their solely sequence-based counterparts. These structure-based classifiers pinpoint the most prominent energetic terms and structural features characterizing the type of protein-protein interactions that underlies the immune recognition of tumors by T cells.

[Molecular mechanism of chromogenic substrate hydrolysis in the active site of human carboxylesterase-1]. / Molekuliarnyi mekhanizm reaktsii gidroliza khromogennogo substrata para-nitrofenilatsetata v aktivnom tsentre karboksilésterazy-1 cheloveka.

Human carboxylesterases are involved in the protective processes of detoxification during the hydrolytic metabolism of xenobiotics. Knowledge of the molecular mechanisms of substrates hydrolysis in the enzymes active site is necessary for the rational drug design. In this work, the molecular mechanism of the hydrolysis reaction of para-nitrophenyl acetate in the active site of human carboxylesterase was determined using modern methods of molecular modeling. According to the combined method of quantum mechanics/molecular mechanics calculations, the chemical reaction occurs within four elementary steps, including two steps of the acylation stage, and two steps of the deacylation stage. All elementary steps have low energy barriers, with the gradual lowering of the intermediate energies that stimulates reaction in the forward direction. The molecular docking was used to estimate the binding constants of the enzyme-substrate complex and the dissociation constant of enzyme-product complexes. The effective kinetic parameters of the enzymatic hydrolysis in the active site of carboxylesterase are determined by numerical solution of the differential kinetic equations.

Supercomputer simulation of the covalent inhibition of the main protease of SARS-CoV-2.

Molecular modeling tools were applied to design a potential covalent inhibitor of the main protease (Mpro) of the SARS-CoV-2 virus and to investigate its interaction with the enzyme. The compound includes a benzoisothiazolone (BZT) moiety of antimalarial drugs and a 5-fluoro-6-nitropyrimidine-2,4(1.H,3H)-dione (FNP) moiety mimicking motifs of inhibitors of other cysteine proteases. The BZT moiety provides a fair binding of the ligand on the protein surface, whereas the warhead FNP is responsible for efficient nucleophilic aromatic substitution reaction with the catalytic cysteine residue in the Mpro active site, leading to a stable covalent adduct. According to supercomputer calculations of the reaction energy profile using the quantum mechanics/molecular mechanics method, the energy of the covalent adduct is 21 kcal mol-1 below the energy of the reactants, while the highest barrier along the reaction pathway is 9 kcal mol-1. These estimates indicate that the reaction can proceed efficiently and can block the Mpro enzyme. The computed structures along the reaction path illustrate the nucleophilic aromatic substitution (SNAr) mechanism in enzymes. The results of this study are important for the choice of potential drugs blocking the development of coronavirus infection.

Computer Modeling of N-Acetylglutamate Synthase: From Primary Structure to Elemental Stages of Catalysis.

Three-dimensional full-atom model of the enzyme complex with acetyl-CoA and substrate was constructed on the basis of the primary sequence of amino acid residues of N-acetyl glutamate synthase. Bioinformatics approaches of computer modeling were applied, including multiple sequence alignment, prediction of co-evolutionary contacts, and ab initio folding. On the basis of the results of calculations by classical molecular dynamics and combined quantum and molecular mechanics (QM/MM) methods, the structure of the active site and the reaction mechanism of N-acetylglutamate formation are described. Agreement of the structures of the enzyme-product complexes obtained in computer modeling and in the X-ray studies validates the reliability of modeling predictions.

Photoinduced electron transfer facilitates tautomerization of the conserved signaling glutamine side chain in BLUF protein light sensors.

The BLUF domain (sensor of blue light using flavin adenine dinucleotide) from a bacterial photoreceptor protein AppA undergoes a cascade of chemical transformations, including hydrogen bond rearrangements around the flavin adenine dinucleotide (FAD) chromophore, in response to light illumination. These transformations are initiated by photoinduced electron and proton transfer from a tyrosine residue to the photoexcited flavin which is assisted by a glutamine residue. According to the recent studies, the proton-coupled electron transfer leads to formation of a radical-pair intermediate Tyr•···FADH• and a tautomeric EE form of glutamine in the ground electronic state. This intermediate is a precursor of the light-induced state of the BLUF photoreceptor implicated in biological signaling. In order to describe evolution of the radical pair, we computed reaction pathways on the ground state potential energy surface employing quantum-chemical calculations in the DFT PBE0/cc-pVDZ approximation for a molecular cluster mimicking the chromophore containing pocket of the AppA BLUF protein. We found a minimum-energy pathway comprised of the following consecutive reaction steps: (1) rotation of the imidic group of the EE glutamine side chain around the Cγ-Cδ bond; (2) flip of the OεH group and formation of the ZE form of the glutamine side chain; and (3) biradical recombination via coupled proton and electron transfer, leading to the ZZ form of the glutamine side chain. The potential-energy barriers for stages 1-3 do not exceed 9 kcal/mol. Energy barrier 3 describing the ZE to ZZ glutamine tautomerization is significantly smaller in the BLUF model than in isolated glutamine, since tautomerization in BLUF is facilitated by electron transfer and radical recombination. Thus, our study shows that tautomerization of the conserved glutamine is coupled to the light-induced electron transfer process in BLUF and, thus, is a viable candidate for the photoactivation mechanism which at present is very much debated.

Computational strategy for tuning spectral properties of red fluorescent proteins.

Computational methods of quantum chemistry are used to characterize structures and vertical excitation energies of the S(0)-S(1) optical transitions in the chromophore binding pockets of the red fluorescent proteins DsRed and of its artificial mutant mCherry. As previously shown, optimizing the equilibrium geometry configurations with B3LYP density functional theory, followed by ZINDO calculations of the electronic excitations, yields positions of the optical bands in good agreement with experimental data. These large scale quantum calculations elucidate the role of the hydrogen bonded network as well as point mutations in the absorption spectra of the DsRed and mCherry proteins. The effect of an external electric field applied to the fluorescent protein chromophores is examined and shows that such fields may result in large shifts in spectral bands. These strategies can be applied for rational design of the fluorescent proteins by site-directed mutagenesis.

The effect of oxidation on the electronic structure of the green fluorescent protein chromophore.

Electronic structure calculations of the singly and doubly ionized states of deprotonated 4(')-hydroxybenzylidene-2,3-dimethylimidazolinone (HBDI anion) are presented. One-electron oxidation produces a doublet radical that has blueshifted absorption, whereas the detachment of two electrons yields a closed-shell cation with strongly redshifted (by about 0.6 eV) absorption relative to the HBDI anion. The results suggest that the doubly oxidized species may be responsible for oxidative redding of green fluorescent protein. The proposed mechanism involves two-step oxidation via electronically excited states and is consistent with the available experimental information [A. M. Bogdanov, A. S. Mishin, I. V. Yampolsky, et al., Nat. Chem. Biol. 5, 459 (2009)]. The spectroscopic signatures of the ionization-induced structural changes in the chromophore are also discussed.

Quantum Chemistry Calculations Provide Support to the Mechanism of the Light-Induced Structural Changes in the Flavin-Binding Photoreceptor Proteins.

The proposed mechanisms of photoinduced reactions in the blue light using flavin chromophore photoreceptor proteins are primarily based on the results of X-ray crystallography and spectroscopy studies. Of particular value are the observed band shifts in optical and vibrational spectra upon formation of the signaling (light-induced) state. However, the same set of experimental data has given rise to contradictory interpretations suggesting different structures of the dark and signaling states. To verify the specific mechanism of light-induced changes involving the rotation/tautomerization transformations with the conserved Gln residue near the flavin chromophore, we performed accurate quantum chemical calculations of the equilibrium structures, vibrational and absorption bands of the model systems mimicking the BLUF domain of flavoprotein AppA. Geometry optimization and calculations of vibrational frequencies were carried out with the QM(B3LYP/cc-pVDZ)/MM(AMBER) approach starting from the representative molecular dynamics (MD) snapshots. The MD simulations were initiated from the available crystal structures of the AppA protein. Calculations of the vertical excitation energies were performed with the scaled opposite spin configuration interaction with single substitutions SOS-CIS(D) method that enables efficient treatment of excited states in large molecular systems. The computed molecular structures as well as the spectral shifts (the red shift by 12÷16 nm in absorption and the downshift by 25 cm(-1) for the C4═O flavin vibrational mode) are in excellent agreement with the experimental results, lending a strong support to the mechanism proposed by Domratcheva et al. (Biophys. J. 2008, 94, 3872).

Potential Energy Landscape of the Electronic States of the GFP Chromophore in Different Protonation Forms: Electronic Transition Energies and Conical Intersections.

We present the results of quantum chemical calculations of the transition energies and conical intersection points for the two lowest singlet electronic states of the green fluorescent protein chromophore, 4'-hydroxybenzylidene-2,3-dimethylimidazolinone, in the vicinity of its cis conformation in the gas phase. Four protonation states of the chromophore, i.e., anionic, neutral, cationic, and zwitterionic, were considered. Energy differences were computed by the perturbatively corrected complete active space self-consistent field (CASSCF)-based approaches at the corresponding potential energy minima optimized by density functional theory and CASSCF (for the ground and excited states, respectively). We also report the EOM-CCSD and SOS-CIS(D) results for the excitation energies. The minimum energy S0/S1 conical intersection points were located using analytic state-specific CASSCF gradients. The results reproduce essential features of previous ab initio calculations of the anionic form of the chromophore and provide an extension for the neutral, cationic, and zwitterionic forms, which are important in the protein environment. The S1 PES of the anion is fairly flat, and the barrier separating the planar bright conformation from the dark twisted one as well as the conical intersection point with the S0 surface is very small (less than 2 kcal/mol). On the cationic surface, the barrier is considerably higher (∼13 kcal/mol). The PES of the S1 state of the zwitterionic form does not have a planar minimum in the Franck-Condon region. The S1 surface of the neutral form possesses a bright planar minimum; the energy barrier of about 9 kcal/mol separates it from the dark twisted conformation as well as from the conical intersection point leading to the cis-trans chromophore isomerization.

Modeling reaction routes from rhodopsin to bathorhodopsin.

The quantum mechanical-molecular mechanical (QM/MM) theory was applied to calculate accurate structural parameters, vibrational and optical spectra of bathorhodopsin (BATHO), one of the primary photoproducts of the functional cycle of the visual pigment rhodopsin (RHO), and to characterize reaction routes from RHO to BATHO. The recently resolved crystal structure of BATHO (PDBID: 2G87) served as an initial source of coordinates of heavy atoms. Protein structures in the ground electronic state and vibrational frequencies were determined by using the density functional theory in the PBE0/cc-pVDZ approximation for the QM part and the AMBER force field parameters in the MM part. Calculated and assigned vibrational spectra of both model protein systems, BATHO and RHO, cover three main regions referring to the hydrogen-out-of-plan (HOOP) motion, the C==C ethylenic stretches, and the C--C single-bond stretches. The S(0)-S(1) electronic excitation energies of the QM part, including the chromophore group in the field of the protein matrix, were estimated by using the advanced quantum chemistry methods. The computed structural parameters as well as the spectral bands match perfectly the experimental findings. A structure of the transition state on the S(0) potential energy surface for the ground electronic state rearrangement from RHO to BATHO was located proving a possible route of the thermal protein activation to the primary photoproduct.

Simulated 18O kinetic isotope effects in enzymatic hydrolysis of guanosine triphosphate.

We compare the computed on the base of quantum mechanical-molecular mechanical (QM/MM) modeling kinetic isotope effects (KIEs) for a series of the (18)O-labeled substrates in enzymatic hydrolysis of guanosine triphosphate (GTP) with those measured experimentally. Following the quantitative structure-activity relationship concept, we introduce the correlation between KIEs and structure of substrates with the help of a labeling index, which also aids better imaging of presentation of both experimental and theoretical data. An evident correlation of the computed and measured KIEs provides support to the predominantly dissociative-type reaction mechanism of enzymatic GTP hydrolysis predicted in QM/MM simulations.

[Studies on the conformational state of the chromophore group (11-cis-retinal) in rhodopsin by computer molecular simulation methods].

Based on computer simulation methods, the molecular dynamics of the rhodopsin chromophore group (11-cis-retinal) has been analyzed. The molecular dynamics has been traced within a 3-ns time interval; thereby 3 x 10(6) discrete conformational states of opsin and rhodopsin were compared and analyzed. It was shown that, within a short time of about 0.3-0.4 ns from the start of simulation, the retinal beta-ionone ring becomes twisted around the C6-C7 bond by approximately 60 degrees compared with that of the initial configuration. The influence of retinal conformation on the positions of the maximum of the absorption band of rhodopsin at the conformational states of t=0 and t=3 ns were estimated using the ab initio methods. The results indicated that the absorption maximum for the final (3-ns) state is shifted by 10 nm toward the long wavelength region compared with the initial state. This suggests that the rhodopsin molecule with its twisted chromophore will possess a considerably lower activation energy than the rhodopsin molecule where the beta-ionone ring is in a planar orientation to the retinal polyene chain.

Computer modeling of the structure and spectra of fluorescent proteins.

Fluorescent proteins from the family of green fluorescent proteins are intensively used as biomarkers in living systems. The chromophore group based on the hydroxybenzylidene-imidazoline molecule, which is formed in nature from three amino-acid residues inside the protein globule and well shielded from external media, is responsible for light absorption and fluorescence. Along with the intense experimental studies of the properties of fluorescent proteins and their chromophores by biochemical, X-ray, and spectroscopic tools, in recent years, computer modeling has been used to characterize their properties and spectra. We present in this review the most interesting results of the molecular modeling of the structural parameters and optical and vibrational spectra of the chromophorecontaining domains of fluorescent proteins by methods of quantum chemistry, molecular dynamics, and combined quantum-mechanical-molecular-mechanical approaches. The main emphasis is on the correlation of theoretical and experimental data and on the predictive power of modeling, which may be useful for creating new, efficient biomarkers.

Mechanism of the chemical step for the guanosine triphosphate (GTP) hydrolysis catalyzed by elongation factor Tu.

Elongation factor Tu (EF-Tu), the protein responsible for delivering aminoacyl-tRNAs (aa-tRNAs) to ribosomal A site during translation, belongs to the group of guanosine-nucleotide (GTP/GDP) binding proteins. Its active 'on'-state corresponds to the GTP-bound form, while the inactive 'off'-state corresponds to the GDP-bound form. In this work we focus on the chemical step, GTP+H(2)O-->GDP+Pi, of the hydrolysis mechanism. We apply molecular modeling tools including molecular dynamics simulations and the combined quantum mechanical-molecular mechanical calculations for estimates of reaction energy profiles for two possible arrangements of switch II regions of EF-Tu. In the first case we presumably mimic binding of the ternary complex EF-Tu.GTP.aa-tRNA to the ribosome and allow the histidine (His85) side chain of the protein to approach the reaction active site. In the second case, corresponding to the GTP hydrolysis by EF-Tu alone, the side chain of His85 stays away from the active site, and the chemical reaction GTP+H(2)O-->GDP+Pi proceeds without participation of the histidine but through water molecules. In agreement with the experimental observations which distinguish rate constants for the fast chemical reaction in EF-Tu.GTP.aa-tRNA.ribosome and the slow spontaneous GTP hydrolysis in EF-Tu, we show that the activation energy barrier for the first scenario is considerably lower compared to that of the second case.

Conformation dependence of pKa's of the chromophores from the purple asFP595 and yellow zFP538 fluorescent proteins.

Two members of the green fluorescent protein family, the purple asFP595 and yellow zFP538 proteins, are perspective fluorescent markers for use in multicolor imaging and resonance energy-transfer applications. We report the results of quantum based calculations of the solution pKa values for selected protonation sites of the denatured asFP595 and zFP538 chromophores in the trans- and cis-conformations in order to add in the interpretation of photophysical properties of these proteins. The pKa values were determined from the theromodynamic cycle based on B3LYP/6-311++G(2df,2p) calculations of the gas phase free energies of the molecules and the B3LYP/6-311++G(d,p) calculations of solvation energies. The results show that the pKa's of the protonation sites of the chromophore from asFP595 noticeably depend on the isomer conformation (cis- or trans-), while those of zFP538 are much less sensitive to isomerization.

Quantum chemical modeling of the reduction of cis-diammineplatinum(IV) tetrachloride [Pt(NH3)2Cl4] by methyl thiolate anion.

We present here both an ab initio and quantum mechanical/molecular mechanical (QM/MM) study of the cis-[Pt(NH3)2Cl4] complex reduction by methyl thiolate anion, SCH(-3), which is used as a model of glutathione. Geometry and electronic structure of cis-[Pt(NH3)2Cl4] are determined without and in aqueous medium. The mechanism of the reaction of reduction is characterized. The calculated activation energy of the reaction compares remarkably well with the experimental value.

Hybrid diatomics-in-molecules-based quantum mechanical/molecular mechanical approach applied to the modeling of structures and spectra of mixed molecular clusters Arn(HCl)m and Arn(HF)m.

A new hybrid QM/DIM approach aimed at describing equilibrium structures and spectroscopic properties of medium size mixed molecular clusters is developed. This methodology is applied to vibrational spectra of hydrogen chloride and hydrogen fluoride clusters with up to four monomer molecules embedded in argon shells Arn(H(Cl/F))m (n = 1-62, m = 1-4). The hydrogen halide complexes (QM part) are treated at the MP2/aug-cc-pVTZ level, while the interaction between HX molecules and Ar atoms (MM part) is described in terms of the semiempirical DIM methodology, based on the proper mixing between neutral and ionic states of the system [Grigorenko et al., J. Chem. Phys. 104, 5510 (1996)]. A detailed analysis of the resulting topology of the QM/DIM potential energy (hyper-)surface in the triatomic subsystem Ar-HX reveals more pronounced nonadditive atomic induction and dispersion contributions to the total interaction energy in the case of the Ar-HCl system. An extension of the original analytical DIM-based potential in the frame of the present model as well as the current limitations of the method are discussed. A modified algorithm for the gradient geometry optimization, along with partly analytical force constant matrix evaluation, is developed to treat large cages of argon atoms around molecular clusters. Calculated frequency redshifts of HX stretching vibrations in the mixed clusters relative to the isolated hydrogen-bonded complexes are in good agreement with experimental findings.

Flexible effective fragment QM/MM method: validation through the challenging tests.

A new version of the QM/MM method, which is based on the effective fragment potential (EFP) methodology [Gordon, M. et al., J Phys Chem A 2001, 105, 293] but allows flexible fragments, is verified through calculations of model molecular systems suggested by different authors as challenging tests for QM/MM approaches. For each example, the results of QM/MM calculations for a partitioned system are compared to the results of an all-electron ab initio quantum chemical study of the entire system. In each case we were able to achieve approximately similar or better accuracy of the QM/MM results compared to those described in original publications. In all calculations we kept the same set of parameters of our QM/MM scheme. A new test example is considered when calculating the potential of internal rotation in the histidine dipeptide around the C(alpha)bond;C(beta) side chain bond.

Formation of mixed d- and f-block metal clusters in inert matrices: comparison of the observed and theoretical optical spectra of AgHo.

The UV visible spectra obtained after simultaneous cocondensation of silver and holmium atoms with argon matrices at 9 K have been studied in the 200-800 nm region. While no new feature can be observed upon deposition, selective irradiation into both silver or holmium atomic absorptions results in growth of a new band at 430 nm, associated with formation of a mixed silver holmium species, tentatively assigned as AgHo. To support the assignment of the observed bands ab initio quantum chemical calculations were carried out for the dinuclear and trinuclear silver and holmium species, using pseudopotential approaches. Results for the electronic excitation energies and corresponding transition dipole moments for the diatomic molecules Ag2, Ho2, AgHo provide evidence that the 430 nm band should be attributed to the mixed cluster AgHo (theoretical band position at 436 nm), while the doublets at 498/504 and 558/563 nm belong to the homonuclear species Ho2 (theoretical values are at 482 and 562 nm). First conclusions are drawn with respect to the formation of the metal trimers Ho3, Ag2Ho, AgHo2.

Spectroscopic study of low temperature interactions in metal-organic co-condensates.

The results on spectroscopic study of low temperature interactions of metal atoms, small clusters and nanoparticles with different organic and inorganic substances in the temperature range 12-300 K are presented. Complexation and reactions of atoms and clusters of magnesium, samarium and silver with carbon dioxide, ethylene and some mesogenic cyanophenyls were studied by the technique of matrix isolation and low temperature co-condensation of metal and ligand vapors, low temperature UV-Vis, IR- and ESR-spectroscopy in combination with quantum chemistry calculations. It was shown that cryochemical reactions of metal particles of different sizes reflected the system's redundant energy.