Bella L. Grigorenko

Photoinduced reactions play an important role in the photocycle of fluorescent proteins from the green fluorescent protein (GFP) family. Among such processes are photoisomerization, photooxidation/photoreduction, breaking and making of covalent bonds, and excited-state proton transfer (ESPT). Many of these transformations are initiated by electron transfer (ET). The quantum yields of these processes vary significantly, from nearly 1 for ESPT to 10–4–10–6 for ET. Importantly, even when quantum yields are relatively small, at the conditions of repeated illumination the overall effect is significant. Depending on the task at hand, fluorescent protein photochemistry is regarded either as an asset facilitating new applications or as a nuisance leading to the loss of optical output. The phenomena arising due to phototransformations include (i) large Stokes shifts, (ii) photoconversions, photoactivation, and photoswitching, (iii) phototoxicity, (iv) blinking, (v) permanent bleaching, and (vi) formation of long-lived intermediates. The focus of this review is on the most recent experimental and theoretical work on photoinduced transformations in fluorescent proteins. We also provide an overview of the photophysics of fluorescent proteins, highlighting the interplay between photochemistry and other channels (fluorescence, radiationless relaxation, and intersystem crossing). The similarities and differences with photochemical processes in other biological systems and in dyes are also discussed.

The unique properties of green fluorescent protein (GFP) have been harnessed in a variety of bioimaging techniques, revolutionizing many areas of the life sciences. Molecular-level understanding of the underlying photophysics provides an advantage in the design of new fluorescent proteins (FPs) with improved properties; however, because of its complexity, many aspects of the GFP photocycle remain unknown. In this Account, we discuss computational studies of FPs and their chromophores that provide qualitative insights into mechanistic details of their photocycle and the structural basis for their optical properties. In a reductionist framework, studies of well-defined model systems (such as isolated chromophores) help to understand their intrinsic properties, while calculations including protein matrix and/or solvent demonstrate, on the atomic level, how these properties are modulated by the environment. An interesting feature of several anionic FP chromophores in the gas phase is their low electron detachment energy. For example, the bright excited ππ* state of the model GFP chromophore (2.6 eV) lies above the electron detachment continuum (2.5 eV). Thus, the excited state is metastable with respect to electron detachment. This autoionizing character needs to be taken into account in interpreting gas-phase measurements and is very difficult to describe computationally. Solvation (and even microsolvation by a single water molecule) stabilizes the anionic states enough such that the resonance excited state becomes bound. However, even in stabilizing environments (such as protein or solution), the anionic chromophores have relatively low oxidation potentials and can act as light-induced electron donors. Protein appears to affect excitation energies very little (<0.1 eV), but alters ionization or electron detachment energies by several electron volts. Solvents (especially polar ones) have a pronounced effect on the chromophore's electronic states; for example, the absorption wavelength changes considerably, the ground-state barrier for cis-trans isomerization is reduced, and fluorescence quantum yield drops dramatically. Calculations reveal that these effects can be explained in terms of electrostatic interactions and polarization, as well as specific interactions such as hydrogen bonding. The availability of efficient computer implementations of predictive electronic structure methods is essential. Important challenges include developing faster codes (to enable better equilibrium sampling and excited-state dynamics modeling), creating algorithms for properties calculations (such as nonlinear optical properties), extending standard excited-state methods to autoionizing (resonance) states, and developing accurate QM/MM schemes. The results of sophisticated first-principle calculations can be interpreted in terms of simpler, qualitative molecular orbital models to explain general trends. In particular, an essential feature of the anionic GFP chromophore is an almost perfect resonance (mesomeric) interaction between two Lewis structures, giving rise to charge delocalization, bond-order scrambling, and, most importantly, allylic frontier molecular orbitals spanning the methine bridge. We demonstrate that a three-center Hückel-like model provides a useful framework for understanding properties of FPs. It can explain changes in absorption wavelength upon protonation or other structural modifications of the chromophore, the magnitude of transition dipole moment, barriers to isomerization, and even non-Condon effects in one- and two-photon absorption.

The recently discovered photoreceptor proteins containing BLUF (sensor of blue light using FAD) domains mediate physiological responses to blue light in bacteria and euglena. In BLUF domains, blue light activates the flavin chromophore yielding a signaling state characterized by a approximately 10 nm red-shifted absorption. We developed molecular models for the dark and light states of the BLUF domain of the Rhodobacter sphaeroides AppA protein, which are based on the crystal structures and quantum-mechanical simulations. According to these models, photon absorption by the flavin results in a tautomerization and 180 degree rotation of the Gln side chain that interacts with the flavin cofactor. This chemical modification of the Gln residue induces alterations in the hydrogen bond network in the core of the photoreceptor domain, which were observed in numerous spectroscopic experiments. The calculated electronic transition energies and vibrational frequencies of the proposed dark and light states are consistent with the optical and IR spectral changes observed during the photocycle. Light-induced isomerization of an amino acid residue instead of a chromophore represents a feature that has not been described previously in photoreceptors.

We present the results of quantum chemical calculations of the electronic properties of the anionic form of the green fluorescent protein chromophore in the gas phase. The vertical detachment energy of the chromophore is found to be 2.4-2.5 eV, which is below the strongly absorbing ππ* state at 2.6 eV. The vertical excitation of the lowest triplet state is around 1.9 eV, which is below the photodetachment continuum. Thus, the lowest bright singlet state is a resonance state embedded in the photodetachment continuum, whereas the lowest triplet state is a regular bound state. Based on our estimation of the vertical detachment energy, we attribute a minor feature in the action spectrum as due to the photodetachment transition. The benchmark results for the bright ππ* state demonstrated that the scaled opposite-spin method yields vertical excitation within 0.1 eV (20 nm) from the experimental maximum at 2.59 eV (479 nm). We also report estimations of the vertical excitation energy obtained with the equation-of-motion coupled cluster with the singles and doubles method, a multireference perturbation theory corrected approach MRMP2 as well as the time-dependent density functional theory with range-separated functionals. Expanding the basis set with diffuse functions lowers the ππ* vertical excitation energy by 0.1 eV at the same time revealing a continuum of "ionized" states, which embeds the bright ππ* transition.

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.

The effect of the protein environment on the electronic structure of the green fluorescent protein (GFP) chromophore is investigated by QM/MM (quantum mechanics/molecular mechanics) calculations. The protein has very small effect on the excitation energy of the bright absorbing and the lowest triplet states of the anionic GFP chromophore, deprotonated 4-hydroxybenzylidene-2,3-dimethylimidazolinone (HBDI) anion, however, it increases vertical detachment energy from 2.5 eV (gas-phase deprotonated HBDI anion) to 5.0 eV (solvated protein). We also investigated possible existence of the charge-transfer-to-solvent (CTTS) states associated with the GFP chromophore. Although precursors of such states appear in cluster calculations, a tightly packed structure of the protein prevents the formation of the CTTS states in this system. Motivated by a recently discovered new type of photoconversion, oxidative redding, we characterized the redox properties of GFP. The computed standard reduction potential of the anionic form of GFP is 0.47 V (for the GFP• + 1e → GFP– reaction), and the reduction potential at physiological conditions (pH 7, T = 25 °C) is 0.06 V.

Organophosphorus agents (OPs) are irreversible inhibitors of acetylcholinesterase (AChE). OP poisoning causes major cholinergic syndrome. Current medical counter-measures mitigate the acute effects but have limited action against OP-induced brain damage. Bioscavengers are appealing alternative therapeutic approach because they neutralize OPs in bloodstream before they reach physiological targets. First generation bioscavengers are stoichiometric bioscavengers. However, stoichiometric neutralization requires administration of huge doses of enzyme. Second generation bioscavengers are catalytic bioscavengers capable of detoxifying OPs with a turnover. High bimolecular rate constants (kcat/Km>106 M-1min-1) are required, so that low enzyme doses can be administered. Cholinesterases (ChE) are attractive candidates because OPs are hemi-substrates. Moderate OP hydrolase (OPase) activity has been observed for certain natural ChEs and for G117H-based human BChE mutants made by site-directed mutagenesis. However, before mutated ChEs can become operational catalytic bioscavengers their dephosphylation rate constant must be increased by several orders of magnitude. New strategies for converting ChEs into fast OPase are based either on combinational approaches or on computer redesign of enzyme. The keystone for rational conversion of ChEs into OPases is to understand the reaction mechanisms with OPs. In the present work we propose that efficient OP hydrolysis can be achieved by re-designing the configuration of enzyme active center residues and by creating specific routes for attack of water molecules and proton transfer. Four directions for nucleophilic attack of water on phosphorus atom were defined. Changes must lead to a novel enzyme, wherein OP hydrolysis wins over competing aging reactions. Kinetic, crystallographic and computational data have been accumulated that describe mechanisms of reactions involving ChEs. From these studies, it appears that introducing new groups that create a stable H-bonded network susceptible to activate and orient water molecule, stabilize transition states and intermediates may determine whether dephosphylation is favored over aging. Mutations on key residues (L286, F329, F398) were considered. QM/MM calculations suggest that mutation L286H combined to other mutations favors water attack from apical position. However, the aging reaction is competing. Axial direction of water attack is not favorable to aging. QM/MM calculation shows that F329H+F398H-based multiple mutants display favorable energy barrier for fast reactivation without aging.

Abstract The mechanism of the hydrolysis reaction of guanosine triphosphate (GTP) by the protein complex Ras–GAP (p21 ras – p120 GAP ) has been modeled by the quantum mechanical—molecular mechanical (QM/MM) and ab initio quantum calculations. Initial geometry configurations have been prompted by atomic coordinates of a structural analog (PDBID:1WQ1). It is shown that the minimum energy reaction path is consistent with an assumption of two‐step chemical transformations. At the first stage, a unified motion of Arg789 of GAP, Gln61, Thr35 of Ras, and the lytic water molecule results in a substantial spatial separation of the γ‐phosphate group of GTP from the rest of the molecule (GDP). This phase of hydrolysis process proceeds through the low‐barrier transition state TS1. At the second stage, Gln61 abstracts and releases protons within the subsystem including Gln61, the lytic water molecule and the γ‐phosphate group of GTP through the corresponding transition state TS2. Direct quantum calculations show that, in this particular environment, the reaction GTP + H 2 O → GDP + H 2 PO can proceed with reasonable activation barriers of less than 15 kcal/mol at every stage. This conclusion leads to a better understanding of the anticatalytic effect of cancer‐causing mutations of Ras, which has been debated in recent years. Proteins 2005. © 2005 Wiley‐Liss, Inc.

Theoretical and matrix-isolation studies of intermolecular complexes of HXeOH with water molecules are presented. The structures and possible decomposition routes of the HXeOH-(H(2)O)(n)(n = 0, 1, 2, 3) complexes are analyzed theoretically. It is concluded that the decay of these metastable species may proceed through the bent transition states (TSs), leading to the global minima on the respective potential energy surfaces, Xe + (H(2)O)(n+1). The respective barrier heights are 39.6, 26.6, 11.2, and 0.4 kcal/mol for n = 0, 1, 2, and 3. HXeOH in larger water clusters is computationally unstable with respect to the bending coordinate, representing the destabilization effect. Another decomposition channel of HXeOH-(H(2)O)(n), via a linear TS, leads to a direct break of the H-Xe bond of HXeOH. In this case, the attached water molecules stabilize HXeOH by strengthening the H-Xe bond. Due to the stabilization, a large blue shift of the H-Xe stretching mode upon complexation of HXeOH with water molecules is featured in calculations. On the basis of this computational result, the IR absorption bands at 1681 and 1742 cm(-1) observed after UV photolysis and annealing of multimeric H(2)O/Xe matrixes are assigned to the HXeOH-H(2)O and HXeOH-(H(2)O)(2) complexes. These bands are blue-shifted by 103 and 164 cm(-1) from the known monomeric HXeOH absorption.

Abstract The hydrolysis reaction of guanosine triphosphate (GTP) by p21 ras (Ras) has been modeled by using the ab initio type quantum mechanical–molecular mechanical simulations. Initial geometry configurations have been prompted by atomic coordinates of the crystal structure (PDBID: 1QRA) corresponding to the prehydrolysis state of Ras in complex with GTP. Multiple searches of minimum energy geometry configurations consistent with the hydrogen bond networks have been performed, resulting in a series of stationary points on the potential energy surface for reaction intermediates and transition states. It is shown that the minimum energy reaction path is consistent with an assumption of a two‐step mechanism of GTP hydrolysis. At the first stage, a unified action of the nearest residues of Ras and the nearest water molecules results in a substantial spatial separation of the γ‐phosphate group of GTP from the rest of the molecule (GDP). This phase of hydrolysis process proceeds through the low barrier (16.7 kcal/mol) transition state TS1. At the second stage, the inorganic phosphate is formed in consequence of proton transfers mediated by two water molecules and assisted by the Gln61 residue from Ras. The highest transition state at this segment, TS3, is estimated to have an energy 7.5 kcal/mol above the enzyme–substrate complex. The results of simulations are compared to the previous findings for the GTP hydrolysis in the Ras‐GAP (p21 ras –p120 GAP ) protein complex. Conclusions of the modeling lead to a better understanding of the anticatalytic effect of cancer causing mutation of Gln61 from Ras, which has been debated in recent years. Proteins 2007. © 2006 Wiley‐Liss, Inc.

The intrinsic chemical reaction of adenosine triphosphate (ATP) hydrolysis catalyzed by myosin is modeled by using a combined quantum mechanics and molecular mechanics (QM/MM) methodology that achieves a near ab initio representation of the entire model. Starting with coordinates derived from the heavy atoms of the crystal structure (Protein Data Bank ID code 1VOM ) in which myosin is bound to the ATP analog ADP·VO 4 − , a minimum-energy path is found for the transformation ATP + H 2 O → ADP + P i that is characterized by two distinct events: ( i ) a low activation-energy cleavage of the P γ O βγ bond and separation of the γ-phosphate from ADP and ( ii ) the formation of the inorganic phosphate as a consequence of proton transfers mediated by two water molecules and assisted by the Glu-459–Arg-238 salt bridge of the protein. The minimum-energy model of the enzyme–substrate complex features a stable hydrogen-bonding network in which the lytic water is positioned favorably for a nucleophilic attack of the ATP γ-phosphate and for the transfer of a proton to stably bound second water. In addition, the P γ O βγ bond has become significantly longer than in the unbound state of the ATP and thus is predisposed to cleavage. The modeled transformation is viewed as the part of the overall hydrolysis reaction occurring in the closed enzyme pocket after ATP is bound tightly to myosin and before conformational changes preceding release of inorganic phosphate.

Structures and optical spectra of the green fluorescent protein (GFP) forms along the proton transfer route A→I→B are characterized by first-principles calculations. We show that in the ground electronic state the structure representing the wild-type (wt) GFP with the neutral chromophore (A-form) is lowest in energy, whereas the systems with the anionic chromophore (B- and I-forms) are about 1 kcal/mol higher. In the S65T mutant, the structures with the anionic chromophore are significantly lower in energy than the systems with the neutral chromophore. The role of the nearby amino acid residues in the chromophore-containing pocket is re-examined. Calculations reveal that the structural differences between the I- and B-forms (the former has a slightly red-shifted absorption relative to the latter) are based not on the Thr203 orientation, but on the Glu222 position. In the case of wt-GFP, the hydrogen bond between the chromophore and the His148 residue stabilizes the structures with the deprotonated phenolic ring in the I- and B-forms. In the S65T mutant, concerted contributions from the His148 and Thr203 residues are responsible for a considerable energy gap between the lowest energy structure of the B type with the anionic chromophore from other structures.

Molecular mechanisms of the hydrolysis of guanosine triphosphate (GTP) to guanosine diphosphate (GDP) and inorganic phosphate (Pi) by the Ras·GAP protein complex are fully investigated by using modern modeling tools. The previously hypothesized stages of the cleavage of the phosphorus-oxygen bond in GTP and the formation of the imide form of catalytic Gln61 from Ras upon creation of Pi are confirmed by using the higher-level quantum-based calculations. The steps of the enzyme regeneration are modeled for the first time, providing a comprehensive description of the catalytic cycle. It is found that for the reaction Ras·GAP·GTP·H2O → Ras·GAP·GDP·Pi, the highest barriers correspond to the process of regeneration of the active site but not to the process of substrate cleavage. The specific shape of the energy profile is responsible for an interesting kinetic mechanism of the GTP hydrolysis. The analysis of the process using the first-passage approach and consideration of kinetic equations suggest that the overall reaction rate is a result of the balance between relatively fast transitions and low probability of states from which these transitions are taking place. Our theoretical predictions are in excellent agreement with available experimental observations on GTP hydrolysis rates.

Remarkable success in engineering novel efficient biomarkers based on fluorescent and photoactive proteins provokes a question of whether computational modeling of their properties can contribute to this important field. In this Feature Article, we analyze selected papers devoted to computer simulations of three types of photoactive systems: the green fluorescent protein and its derivatives, the flavin-binding proteins, and the phytochrome domains. The main emphasis is on structures, optical spectra, and chemical reactions in the chromophore-containing pockets. Quantum chemistry, quantum mechanics/molecular mechanics, and molecular dynamics methods are effective tools in these simulations. We highlight both the success stories and the persisting challenges, discussing the ways of elevating theoretical approaches to the level of testable predictions.

The mechanism of the hydrolysis reaction of the unprotonated methyl triphosphate (MTP) ester in water clusters has been modeled. The effective fragment potential based quantum mechanical−molecular mechanical (QM/MM) approach has been applied in the simulations. It is shown that the minimum energy reaction path is consistent with an assumption of a two-step dissociative-type process similar to the case of the guanosine triphosphate (GTP) hydrolysis in the Ras−GAP protein complex (Grigorenko, B. L.; Nemukhin, A. V.; Topol, I. A.; Cachau, R. E.; Burt, S. K. Proteins: Struct., Funct., Bioinf. 2005, 60, 495). At the first stage, a unified action of environmental molecular groups and the catalytic water molecule leads to a substantial spatial separation of the γ-phosphate group from the rest of the molecule. At the second stage, inorganic phosphate H2PO4- is formed from water and the metaphosphate anion PO3- through the chain of proton transfers along hydrogen bonds. The estimated activation barriers for MTP in aqueous solution at both stages (20 and 14 kcal/mol) are substantially higher than the corresponding barriers for the GTP hydrolysis in the protein.

Cancer-associated point mutations in Ras, in particular, at glycine 12 and glycine 13, affect the normal cycle between inactive GDP-bound and active GTP-bound states. In this work, the role of G12V and G13V replacements in the GAP-stimulated intrinsic GTP hydrolysis reaction in Ras is studied using molecular dynamics (MD) simulations with quantum mechanics/molecular mechanics (QM/MM) potentials. A model molecular system was constructed by motifs of the relevant crystal structure (Protein Data Bank entry 1WQ1). QM/MM optimization of geometry parameters in the Ras-GAP-GTP complex and QM/MM–MD simulations were performed with a quantum subsystem comprising a large fraction of the enzyme active site. For the system with wild-type Ras, the conformations fluctuated near the structure ready to be involved in the efficient chemical reaction leading to the cleavage of the phosphorus–oxygen bond in GTP upon approach of the properly aligned catalytic water molecule. Dynamics of the system with the G13V mutant is characterized by an enhanced flexibility in the area occupied by the γ-phosphate group of GTP, catalytic water, and the side chains of Arg789 and Gln61, which should somewhat hinder fast chemical steps. Conformational dynamics of the system with the G12V mutant shows considerable displacement of the Gln61 side chain and catalytic water from their favorable arrangement in the active site that may lead to a marked reduction in the reaction rate. The obtained computational results correlate well with the recent kinetic measurements of the Ras-GAP-catalyzed hydrolysis of GTP.

We report the first complete theoretical description of the chain of elementary reactions resulting in chromophore maturation in the green fluorescent protein (GFP). All reaction steps including cyclization, dehydration, and oxidation are characterized at the uniform quantum mechanics/molecular mechanics (QM/MM) computational level using density functional theory in quantum subsystems. Starting from a structure of the wild-type protein with the noncyclized Ser65-Tyr66-Gly67 tripeptide, we modeled cyclization and dehydration reactions. We then added molecular oxygen to the system and modeled the oxidation reaction resulting in the mature protein-bound chromophore. Computationally derived structures of the reaction product and several reaction intermediates agree well with the relevant crystal structures, validating the computational protocol. The highest computed energy barriers at the cyclization–dehydration (17 kcal/mol) and oxidation (21 kcal/mol) steps agree well with the values derived from the kinetics measurements (20.7 and 22.7 kcal/mol, respectively). The simulations provide strong support to the mechanism involving the cyclization–dehydration–oxidation sequence of the chromophore’s maturation reactions. The results also establish a solid basis for predictions of maturation mechanisms in other fluorescent proteins.

A computer-designed mutant of human butyrylcholinesterase (BChE), N322E/E325G, with a novel catalytic triad was made. The catalytic triad of the wild-type enzyme (S198·H438·E325) was replaced by S198·H438·N322E in silico. Molecular dynamics for 1.5 μs and Markov state model analysis showed that the new catalytic triad should be operative in the mutant enzyme, suggesting functionality. QM/MM modeling performed for the reaction of wild-type BChE and double mutant with echothiophate showed high reactivity of the mutant towards the organophosphate. A truncated monomeric (L530 stop) double mutant was expressed in Expi293 cells. Non-purified transfected cell culture medium was analyzed. Polyacrylamide gel electrophoresis under native conditions followed by activity staining with BTC as the substrate provided evidence that the monomeric BChE mutant was active. Inhibition of the double mutant by echothiophate followed by polyacrylamide gel electrophoresis and activity staining showed that this enzyme slowly self-reactivated. However, because Expi293 cells secrete an endogenous BChE tetramer and several organophosphate-reacting enzymes, catalytic parameters and self-reactivation constants after phosphorylation of the new mutant were not determined in the crude cell culture medium. The study shows that the computer-designed double mutant (N322E/E325G) with a new catalytic triad (S198·H438·N322E) is a suitable template for design of novel active human BChE mutants that display an organophosphate hydrolase activity.

The ability to detect proteins through gating conductance by their unique surface electrostatic signature holds great potential for improving biosensing sensitivity and precision. Two challenges are: (1) defining the electrostatic surface of the incoming ligand protein presented to the conductive surface; (2) bridging the Debye gap to generate a measurable response. Herein, we report the construction of nanoscale protein-based sensing devices designed to present proteins in defined orientations; this allowed us to control the local electrostatic surface presented within the Debye length, and thus modulate the conductance gating effect upon binding incoming protein targets. Using a β-lactamase binding protein (BLIP2) as the capture protein attached to carbon nanotube field effect transistors in different defined orientations. Device conductance had influence on binding TEM-1, an important β-lactamase involved in antimicrobial resistance (AMR). Conductance increased or decreased depending on TEM-1 presenting either negative or positive local charge patches, demonstrating that local electrostatic properties, as opposed to protein net charge, act as the key driving force for electrostatic gating. This, in turn can, improve our ability to tune the gating of electrical biosensors toward optimized detection, including for AMR as outlined herein.

We report the results of computational modeling of the reactions of the SARS-CoV-2 main protease (MPro) with four potential covalent inhibitors. Two of them, carmofur and nirmatrelvir, have shown experimentally the ability to inhibit MPro. Two other compounds, X77A and X77C, were designed computationally in this work. They were derived from the structure of X77, a non-covalent inhibitor forming a tight surface complex with MPro. We modified the X77 structure by introducing warheads capable of reacting with the catalytic cysteine residue in the MPro active site. The reaction mechanisms of the four molecules with MPro were investigated by quantum mechanics/molecular mechanics (QM/MM) simulations. The results show that all four compounds form covalent adducts with the catalytic cysteine Cys 145 of MPro. From the chemical perspective, the reactions of these four molecules with MPro follow three distinct mechanisms. The reactions are initiated by a nucleophilic attack of the thiolate group of the deprotonated cysteine residue from the catalytic dyad Cys145–His41 of MPro. In the case of carmofur and X77A, the covalent binding of the thiolate to the ligand is accompanied by the formation of the fluoro-uracil leaving group. The reaction with X77C follows the nucleophilic aromatic substitution SNAr mechanism. The reaction of MPro with nirmatrelvir (which has a reactive nitrile group) leads to the formation of a covalent thioimidate adduct with the thiolate of the Cys145 residue in the enzyme active site. Our results contribute to the ongoing search for efficient inhibitors of the SARS-CoV-2 enzymes.

The conjecture that limited basis diatomics-in-molecules type potentials may serve as an accurate representation of many-body interactions is explored through molecular dynamics simulations of ArnHF (n=1–12,62). The important ingredient in the constructed potentials is the inclusion of ionic configurations of HF. Once the admixture between ionic and covalent configurations is calibrated by reference to an ab initio surface of the ArHF dimer, a single three-body potential energy surface is defined, and used in subsequent simulations of larger clusters. The vibrational frequencies of HF, which are computed from velocity–velocity autocorrelation functions, quantitatively reproduce the cluster size dependent redshifts.

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.

Development and applications of a new approach to hybrid quantum mechanical and molecular mechanical (QM/MM) theory based on the effective fragment potential (EFP) technique for modeling properties and reactivity of large molecular systems of biochemical significance are described. It is shown that a restriction of frozen internal coordinates of effective fragments in the original formulation of the theory (Gordon, M. S.; Freitag, M. A.; Bandyopadhyay, P.; Jensen, J. H.; Kairys, V.; Stevens, W. J. J. Phys. Chem. A 2001, 105, 293) can be removed by introducing a set of small EFs and replacing the EFP−EFP interactions by the customary MM force fields. The concept of effective fragments is also utilized to solve the QM/MM boundary problem across covalent bonds. The buffer fragment, which is common for both subsystems, is introduced and treated specially when energy and energy gradients are computed. An analysis of conformations of dipeptide−water complexes, as well as of dipepties with His and Lys residues, confirms the reliability of the theory. By using the Hartree−Fock and MP2 quantum chemistry methods with the OPLS-AA molecular mechanical force fields, we calculated the energy difference between the enzyme−substrate complex and the first tetrahedral intermediate for the model active site of the serine protease catalytic system. In another example, the multiconfigurational complete active space self-consistent field (CASSCF) method was used to model the homolytic dissociation of the peptide helix over the central C−N bonds. Finally, the potentials of internal rotation of the water dimer considered as a part of the water wire inside a polyglycine analogue of the ion channel gramicidin A were computed. In all cases, an importance of the peptide environment from MM subsystems on the computed properties of the quantum parts is demonstrated.

We present quantum chemical calculations of the properties of the anionic form of the green fluorescent protein (GFP) chromophore that can be directly compared to the results of experimental measurements: the cis-trans isomerization energy profile in water. Calculations of the cis-trans chromophore isomerization pathway in the gas phase and in water reveal a problematic behavior of density functional theory and scaled opposite-spin-MP2 due to the multiconfigurational character of the wave function at twisted geometries. The solvent effects treated with the continuum solvation models, as well as with the water cluster model, are found to be important and can reduce the activation energy by more than 10 kcal/mol. Strong solvent effects are explained by the change in charge localization patterns along the isomerization coordinate. At the equilibrium, the negative charge is almost equally delocalized between the phenyl and imidazolin rings due to the interaction of two resonance structures, whereas at the transition state the charge is localized on the imidazolin moiety. Our best estimate of the barrier obtained in cluster calculations employing the effective fragment potential-based quantum mechanics/molecular mechanics method with the complete active space self-consistent field description of the chromophore augmented by perturbation theory correction and the TIP3P water model is 14.8 kcal/mol, which is in excellent agreement with the experimental value of 15.4 kcal/mol. This result helps to resolve previously reported disagreements between experimental measurements and theoretical estimates.

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).

This work explores the level of transparency in reporting the details of computational protocols that is required for practical reproducibility of quantum mechanics/molecular mechanics (QM/MM) simulations. Using the reaction of an essential SARS-CoV-2 enzyme (the main protease) with a covalent inhibitor (carmofur) as a test case of chemical reactions in biomolecules, we carried out QM/MM calculations to determine the structures and energies of the reactants, the product, and the transition state/intermediate using analogous QM/MM models implemented in two software packages, NWChem and Q-Chem. Our main benchmarking goal was to reproduce the key energetics computed with the two packages. Our results indicate that quantitative agreement (within the numerical thresholds used in calculations) is difficult to achieve. We show that rather minor details of QM/MM simulations must be reported in order to ensure the reproducibility of the results and offer suggestions toward developing practical guidelines for reporting the results of biosimulations.

We present results of the modeling for the hydrolysis reaction of guanosine triphosphate (GTP) in the RAS-GAP protein complex using essentially ab initio quantum chemistry methods. One of the approaches considers a supermolecular cluster composed of 150 atoms at a consistent quantum level. Another is a hybrid QM/MM method based on the effective fragment potential technique, which describes interactions between quantum and molecular mechanical subsystems at the ab initio level of the theory. Our results show that the GTP hydrolysis in the RAS-GAP protein complex can be modeled by a substrate-assisted catalytic mechanism. We can locate a configuration on the top of the barrier corresponding to the transition state of the hydrolysis reaction such that the straightforward descents from this point lead either to reactants GTP+H(2)O or to products guanosine diphosphate (GDP)+H(2)PO(4)(-). However, in all calculations such a single-step process is characterized by an activation barrier that is too high. Another possibility is a two-step reaction consistent with formation of an intermediate. Here the Pgamma-O(Pbeta) bond is already broken, but the lytic water molecule is still in the pre-reactive state. We present arguments favoring the assumption that the first step of the GTP hydrolysis reaction in the RAS-GAP protein complex may be assigned to the breaking of the Pgamma-O(Pbeta) bond prior to the creation of the inorganic phosphate.

The advantages and disadvantages of various methods of theoretical modelling of mechanisms of enzymatic reactions based on quantum theory are discussed. Molecular mechanical, quantum mechanical and hybrid approaches are considered. Detailed analysis is performed in relation to a superfamily of enzymes, serine hydrolases, which include cholinesterases playing a crucial role in higher nervous activity. As another example, the results of modelling of enzymatic hydrolysis of nucleoside phosphates are considered. The bibliography includes 177 references.

Photobleaching and photostability of proteins of the green fluorescent protein (GFP) family are crucially important for practical applications of these widely used biomarkers. On the basis of simulations, we propose a mechanism for irreversible bleaching in GFP-type proteins under intense light illumination. The key feature of the mechanism is a photoinduced reaction of the chromophore with molecular oxygen (O2) inside the protein barrel leading to the chromophore’s decomposition. Using quantum mechanics/molecular mechanics (QM/MM) modeling we show that a model system comprising the protein-bound Chro– and O2 can be excited to an electronic state of the intermolecular charge-transfer (CT) character (Chro•···O2–•). Once in the CT state, the system undergoes a series of chemical reactions with low activation barriers resulting in the cleavage of the bridging bond between the phenolic and imidazolinone rings and disintegration of the chromophore.

The complete catalytic cycle of aspartoacylase (ASPA), a zinc-dependent enzyme responsible for cleavage of N-acetyl-l-aspartate, is characterized by the methods of molecular modeling. The reaction energy profile connecting the enzyme–substrate (ES) and the enzyme–product (EP) complexes is constructed by the quantum mechanics/molecular mechanics (QM/MM) method assisted by the molecular dynamics (MD) simulations with the QM/MM potentials. Starting from the crystal structure of ASPA complexed with the intermediate analogue, the minimum-energy geometry configurations and the corresponding transition states are located. The stages of substrate binding to the enzyme active site and release of the products are modeled by MD calculations with the replica-exchange umbrella sampling technique. It is shown that the first reaction steps, nucleophilic attack of a zinc-bound nucleophilic water molecule at the carbonyl carbon and the amide bond cleavage, are consistent with the glutamate-assisted mechanism hypothesized for the zinc-dependent hydrolases. The stages of formation of the products, acetate and l-aspartate, and regeneration of the enzyme are characterized for the first time. The constructed free energy diagram from the reactants to the products suggests that the enzyme regeneration, but not the nucleophilic attack of the catalytic water molecule, corresponds to the rate-determining stage of the full catalytic cycle of ASPA.

Amide-imide tautomerization presents a pervasive class of chemical transformations in organic chemistry of natural compounds. In this Perspective, we describe two distinctively different protein systems, in which the amide-imide tautomerization in the glutamine side chain takes place in enzymatic or photochemical reactions. First, hydrolysis of guanosine triphosphate (GTP) catalyzed by the Ras-GAP protein complex suggests the occurrence of the imide tautomer of glutamine in reaction intermediates. Second, photoexcitation of flavin-binding protein domains (BLUFs) initiates a chain of reactions in the chromophore-binding pocket, including amide-imide tautomerization of glutamine. Mechanisms of these reactions at the atomic level have been revealed in quantum mechanics/molecular mechanics (QM/MM) simulations. To reinforce conclusions on the critical role of amide-imide tautomerization of glutamine in these reactions we describe results of new quantum chemistry and QM/MM calculations for relevant molecular model systems. We reexamine results of the recent IR spectroscopy studies of BLUF domains, which provide experimental evidences of Gln tautomerization in proteins. We also propose to validate the glutamine-assisted mechanism of enzymatic GTP hydrolysis by using IR spectroscopy in a proper range of wavenumbers.

We present the results of high-level electronic structure and dynamics simulations of the photoactive protein Dreiklang. With the goal of understanding the details of the Dreiklang photocycle, we carefully characterize the excited states of the ON- and OFF-forms of Dreiklang. The key finding of our study is the existence of a low-lying excited state of a charge-transfer character in the neutral ON form and that population of this state, which is nearly isoenergetic with the locally excited bright state, initiates a series of steps that ultimately lead to the formation of the hydrated dark chromophore (OFF state). These results allow us to refine the mechanistic picture of Dreiklang’s photocycle and photoactivation.

The dynamics of Hen+, n=3–13, clusters formed by electron impact ionization of the neutral is studied theoretically using mixed quantum/classical dynamics by both mean-field and surface hopping methods. Potential energy surfaces and nonadiabatic couplings among them are determined from a semiempirical, minimal basis DIM Hamiltonian. The dynamics of hole hopping, hole localization, and cluster fragmentation are described through trajectory data. He3+ clusters, with initial conditions given by the zero-energy quantum distribution of nuclear coordinates, dissociate through two-channels, He+He+He+ and He+He2+ with relative yields of 20% and 80%. The motif of hole localization on a pair of atoms, and subsequent dissociation of the initial pair with hole hop to a new pair is observed in trimers, and repeats in larger clusters. In the larger clusters, hole hopping among He2 pairs provides an additional, less important mechanism of charge migration. The coupled electronic-nuclear dynamics of triatomic units describes the mechanism of energy loss, by transfer of vibrational to translational energy. This leads to ejection of energetic neutral atoms as well as the ejection of He2+ prior to evaporative cooling of the cluster. He2+ is the exclusive charged unit produced in the fragmentation of He13+ clusters. In bulk He the same dynamics should lead to fast vibrational relaxation t&amp;lt;10 ps and formation of He3+ as the positive ion core.

Geometry configurations of a large fraction of the kindling fluorescent protein asFP595 around the chromophore region were optimized by using the effective fragment potential quantum mechanical−molecular mechanical (QM/MM) method. The initial coordinates of heavy atoms were taken from the structure 1XMZ from the Protein Data Bank archive corresponding to the dark-adapted state of the Ala143 → Gly mutant of asFP595. Optimization of geometry parameters was performed for all internal coordinates in the QM part composed of the chromophore unit and the side chains of His197, Glu215, and Arg92 as well as for positions of effective fragments constituting the MMpart. The structures corresponding to the anion trans, anion cis, and zwitterion trans moieties were considered among various alternatives for the chromophore unit inside the protein matrix. The QM/MM simulations show that the protein environment provides stabilization for the trans-zwitterion isomer compared to the gas-phase conditions. By using the multiconfigurational CASSCF and the time-dependent density functional theory calculations, we estimated positions of spectral bands corresponding to vertical S0−S1 transitions. The results of simulations support the assumption that the dark state of asFP595 corresponds to the anionic or zwitterionic trans-conformation, while the kindled state corresponds to the anionic cis-conformation.

Conformation dictates many physical and chemical properties of molecules. The importance of conformation in the selectivity and function of biologically active molecules is widely accepted. However, clear examples of conformation-dependent bimolecular chemical reactions are lacking. Here we consider a case of formic acid (HCOOH) that is a valuable model system containing the −COOH carboxyl functional group, similar to many biomolecules including the standard amino acids. We have found a strong case of conformation-dependent reaction between formic acid and atomic oxygen obtained in cryogenic matrices. The reaction surprisingly leads to peroxyformic acid only from the ground-state trans conformer of formic acid, and it results in the hydrogen-bonded complex for the higher-energy cis conformer.

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.

The minimum energy reaction profiles corresponding to two possible reaction mechanisms of adenosine triphosphate (ATP) hydrolysis in myosin are computed in this work within the framework of the quantum mechanics-molecular mechanics (QM/MM) method by using the same partitioning of the model system to the QM and MM parts and the same computational protocol. On the first reaction route, one water molecule performs nucleophilic attack at the phosphorus center P(γ) from ATP while the second water molecule in the closed protein cleft serves as a catalytic base assisted by the Glu residue from the myosin salt bridge. According to the present QM/MM calculations consistent with the results of kinetic studies this reaction pathway is characterized by a low activation energy barrier about 10 kcal/mol. The computed activation energy barrier for the second mechanism, which assumes the penta-coordinated oxyphosphorane transition state upon involvement of single water molecule in the reaction, is considerably higher than that for the two-water mechanism.

Among fluorescent proteins (FPs) used as genetically encoded fluorescent tags, the red-emitting FPs are of particular importance as suitable markers for deep tissue imaging. Using electronic structure calculations, we predict a new structural motif for achieving red-shifted absorption and emission in FPs from the GFP family. By introducing four point mutations, we arrive to the structure with the conventional anionic GFP chromophore sandwiched between two tyrosine residues. Contrary to the existing red FPs in which the red shift is due to extended conjugation of the chromophore, in the triple-decker motif, the chromophore is unmodified and the red shift is due to π-stacking interactions. The absorption/emission energies of the triple-decker FP are 2.25/2.16 eV, respectively, which amounts to shifts of ∼40 (absorption) and ∼25 nm (emission) relative to the parent species, the I form of wtGFP. Using a different structural motif based on a smaller chromophore may help to improve optical output of red FPs by reducing losses due to radiationless relaxation and photobleaching.

Penicillin acylase from Escherichia coli is a unique enzyme that belongs to the recently discovered superfamily of N-terminal nucleophile hydrolases. It catalyzes selective hydrolysis of the side chain amide bond of penicillins and cephalosporins while leaving the labile amide bond in the β-lactam ring intact. Despite wide applications of penicillin acylase in the industry of β-lactam antibiotics and production of chiral amino compounds, its catalytic mechanism at atomic resolution has not yet been characterized. The complete cycle of chemical transformations of the most specific substrate of the enzyme, penicillin G, leading to formation of 6-aminopenicillanic and phenylacetic acids was modeled following quantum mechanics–molecular mechanics (QM/MM) calculations of the minimum energy reaction profile. The active site residues and the substrate were included in the QM part, and the rest of the system was treated applying molecular mechanics and classical force field parameters. The 3D structures in the enzyme active site corresponding to the noncovalent enzyme–substrate complex, the covalent acylenzyme intermediate, the noncovalent enzyme–product complex, the tetrahedral intermediates, and the respective transition states have been identified. QM/MM studies have shown that the α-amino group of the N-terminal catalytic βSer1 plays a key role in the catalytic machinery and directly assists its hydroxyl group in a proton relay at major stages of penicillin acylase catalytic mechanism, formation and hydrolysis of the covalent acylenzyme intermediate, which are characterized by close energy barriers. The βSer1 residue together with the oxyanion hole residues βAla69 and βAsn241 as well as βArg263 and βGln23 constitute a buried active site interaction network responsible for stabilization of tetrahedral intermediates, transition states, orientation of substrate and catalytic residues. βArg263 and βGln23 maintain the integrity of the catalytic machinery: βArg263 participates in orientation of the substrate as well as the α-amino group of βSer1 and coordinates the oxyanion hole residue βAsn241 across the whole catalytic cycle, whereas the backbone of βGln23 is responsible for orientation of both the βSer1 and the substrate. These results deliver insight into the earlier unknown ability of N-terminal amino acid to activate its own nucleophilic group directly as well as into organization of the stabilizing interaction network in penicillin acylase’s active site and will be used to design more effective enzyme variants for synthesis of new penicillins and cephalosporins.

Irradiation of the green fluorescent protein (GFP) by intense violet or UV light leads to decarboxylation of the Glu222 side chain in the vicinity of the chromophore (Chro). This phenomenon is utilized in optical highlighters, such as photoactivatable GFP (PA-GFP). Using state-of-the-art quantum chemical calculations, we investigate the feasibility of the mechanism proposed in the experimental studies [van Thor et al. Nature Struct. Biol.2002, 9, 37–41; Bell et al. J. Am. Chem. Soc.2003, 125, 37–41]. It was hypothesized that a primary event of this photoconversion involves population of a charge-transfer (CT) state via either the first excited state S1 when using longer wavelength (404 and 476 nm) or a higher excited state when using higher energy radiation (254 and 280 nm). Based on the results of electronic structure calculations, we identify these critical CT states (produced by electron transfer from Glu to electronically excited Chro) and show that they are accessible via different routes, i.e., either directly, by one-photon absorption, or through a two-step excitation via S1. The calculations are performed for model systems representing the chromophore and the key nearby residues using two complementary approaches: (i) the multiconfigurational quasidegenerate perturbation theory of second order with the occupation restricted multiple active space scheme for configuration selection in the multiconfigurational self-consistent field reference; and (ii) the single-reference configuration interaction singles method with perturbative doubles that does not involve active space selection. We examined electronic transitions with nonzero oscillator strengths in the UV and visible range between the electronic states involving the Chro and Glu residues. Both methods predict the existence of CT states with nonzero oscillator strength in the UV range and a local excited state of the chromophore accessible via S1 that may lead to the target CT state. The results suggest several possible scenarios for the primary photoconversion event. We also demonstrate that the point mutation Thr203His exploited in PA-GFP results in shifting the light wavelength to access the CT up to 20 nm, which suggests a possibility of a rational design of photoactivatable proteins in silico.

An application of the hybrid quantum mechanical and molecular mechanical (QM/MM) model to calculations of the structures of the hydrogen-bonded complex of the dipeptide N-acetyl-l-alanine N′-methylamide with water molecules is described. This particular approach is essentially based on the effective fragment potential theory [J. Phys. Chem. A 105 (2001) 293]. We discuss both options in the QM/MM treatment of the system, considering once the dipeptide as a quantum part and water molecules as a MM subsystem, and vice versa, taking the dipeptide as a collection of effective fragments, while water molecules constitute a QM part. The first method is realized in the gamess program, in the second case a new version, combining quantum chemical and molecular mechanical packages is required. Our approach assumes that the MM subsystem is viewed as a flexible composition of effective fragments while fragment–fragment interactions are replaced by the MM force fields. It is shown that these QM/MM models correctly describe the conformational properties of dipeptide, namely, the changes in the backbone angles φ and ψ due to complexation with water.

The energy profiles for the reaction OH- + CO2 → HCO3- are analyzed following the results of calculations carried out using both a continuum solvation model and a cluster approach. The minimum energy path, computed with the quantum chemistry LMP2 and B3LYP approximations, corresponds to the activation-less process in the gas phase but shows a barrier on the way from the reactants to the product in the dielectric continuum medium. In the cluster approach, the reacting species were completely surrounded by 30 water molecules, each considered as an effective fragment potential (EFP) acting on the quantum system. Positions of all particles were optimized along the reaction coordinate in this quantum mechanical−molecular mechanical (QM/MM) approximation. The energy profile obtained with the QM/MM(EFP) approach is in remarkable agreement with the results of the continuum model, showing the barrier in the same region. An analysis of the arrangements of the water molecules around the reacting species, as well as changes in geometry configurations and electronic distributions of the solute species, allows us to conclude that on the segment of the reaction path close to the potential barrier a considerable fraction of the negative charge on OH- transfers to CO2, accompanied by a sharp bending of the O−C−O species. As a result, the hydroxide anion loses water molecules from its hydration shell. We show that the height of the barrier on the free energy curve for the reaction OH- + CO2 → HCO3- in water can be estimated within the limits 8−13 kcal/mol, and its precise quantity depends on the reference value of experimental free energy of solvation of OH-.

Abstract The AppA protein with the BLUF (blue light using flavin adenine dinucleotide) domain is a blue light photoreceptor that cycle between dark‐adapted and light‐induced functional states. We characterized possible reaction intermediates in the photocycle of AppA BLUF. Molecular dynamics (MD), quantum chemical and quantum mechanical–molecular mechanical (QM/MM) calculations were carried out to describe several stable structures of a molecular system modeling the protein. The coordinates of heavy atoms from the crystal structure (PDB code 2IYG) of the protein in the dark state served as starting point for 10 ns MD simulations. Representative MD frames were used in QM(B3LYP/cc‐pVDZ)/MM(AMBER) calculations to locate minimum energy configurations of the model system. Vertical electronic excitation energies were estimated for the molecular clusters comprising the quantum subsystems of the QM/MM optimized structures using the SOS‐CIS(D) quantum chemistry method. Computational results support the occurrence of photoreaction intermediates that are characterized by spectral absorption bands between those of the dark and light states. They agree with crystal structures of reaction intermediates (PDB code 2IYI) observed in the AppA BLUF domain. Transformations of the Gln63 side chain stimulated by photo‐excitation and performed with the assistance of the chromophore and the Met106 side chain are responsible for these intermediates.

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.

Different mechanisms of GTP hydrolysis by Ras–GAP are revealed in QM/MM simulations depending on molecular groups at position 61 in Ras.

We report results of a computational study of the reaction mechanism of guanosine triphosphate (GTP) hydrolysis catalyzed by the GTP-binding (GTPase) enzyme rat sarcoma-related nuclear (Ran) in complex with its activating protein RanGAP. According to structural investigations, Ran-RanGAP operates without the so-called arginine finger, an arginine residue from a distinct promoter (GTPase-accelerating protein (GAP)) that completes the active site in many GTPases. In this work, we construct model systems for Ran with and without GAP by motifs of the crystal structure of Ran-RanGAP with the GTP analogue and simulate the GTP hydrolysis reaction in these systems. Enzyme–substrate and enzyme–product complexes and reaction intermediates are obtained in quantum mechanics/molecular mechanics (QM/MM) simulations. Calculations of the free-energy reaction profiles are performed at the molecular dynamics level with the ab initio-type QM(DFT(PBE0-D3)/6-31G**)/MM potentials. We show that the computed activation barriers on the pathways for Ran catalysis with and without GAP are in line with the experimentally estimated rate constants. We demonstrate that mapping the Laplacian of the electron density provides easily visible images of substrate activation, which help distinguish between the reactive and nonreactive enzyme–substrate complexes and explain qualitative features of the enzyme-catalyzed GTP hydrolysis reactions consistent with the computed free-energy profiles. A comparison of reactions in Ran and Ran-RanGAP allows us to characterize the role of GAP operating without an arginine finger.

We report the results of a computational study of the mechanism of the light-induced chemical reaction of chromophore hydration in the fluorescent protein Dreiklang, responsible for its switching from the fluorescent ON-state to the dark OFF-state. We explore the relief of the charge-transfer excited-state potential energy surface in the ON-state to locate minimum energy conical intersection points with the ground-state energy surface. Simulations of the further evolution of model systems allow us to characterize the ground-state reaction intermediate tentatively suggested in the femtosecond studies of the light-induced dynamics in Dreiklang and finally to arrive at the reaction product. The obtained results clarify the details of the photoswitching mechanism in Dreiklang, which is governed by the chemical modification of its chromophore.

A photochemical transformation to arylnitrene occurs in GFP variants with a non-canonical amino acid residue, p -azidophenylalanine, that replaces Tyr66. The arylnitrene reduction is coupled with the oxidation of the nearby His148 side chain.

It is shown that the inclusion of excited ionic configurations in the diatomics-in-molecules (DIM) Hamiltonian serves as a natural means to account for main features of non-additivity in three-body potential energy surfaces of HeCl2 and ArCl2 van der Waals complexes. For ground state Cl2(1Σg), while consideration of only neutral configurations leads toT-shaped isomers, inclusion of the excited Cl+Cl−1 configuration stabilizes the linear isomer and destabilizes the T-shaped isomer. Within the same formalism, the excited Cl2(3Π) only sustains minima in the T-shaped isomer. Potential energy surfaces created with a minimal DIM basis are constructed and shown to compare favorably with the most accurate ab initio surfaces and experiments. Analytical forms are given for three-body surfaces, meant for fitting purposes and as a convenience in simulations of dynamics.

beta-carotene 15,15'-monooxygenase (BCMO1) catalyzes the crucial first step in vitamin A biosynthesis in animals. We wished to explore the possibility that a carbocation intermediate is formed during the cleavage reaction of BCMO1, as is seen for many isoprenoid biosynthesis enzymes, and to determine which residues in the substrate binding cleft are necessary for catalytic and substrate binding activity. To test this hypothesis, we replaced substrate cleft aromatic and acidic residues by site-directed mutagenesis. Enzymatic activity was measured in vitro using His-tag purified proteins and in vivo in a beta-carotene-accumulating E. coli system.Our assays show that mutation of either Y235 or Y326 to leucine (no cation-pi stabilization) significantly impairs the catalytic activity of the enzyme. Moreover, mutation of Y326 to glutamine (predicted to destabilize a putative carbocation) almost eliminates activity (9.3% of wt activity). However, replacement of these same tyrosines with phenylalanine or tryptophan does not significantly impair activity, indicating that aromaticity at these residues is crucial. Mutations of two other aromatic residues in the binding cleft of BCMO1, F51 and W454, to either another aromatic residue or to leucine do not influence the catalytic activity of the enzyme. Our ab initio model of BCMO1 with beta-carotene mounted supports a mechanism involving cation-pi stabilization by Y235 and Y326.Our data are consistent with the formation of a substrate carbocation intermediate and cation-pi stabilization of this intermediate by two aromatic residues in the substrate-binding cleft of BCMO1.

We present results of theoretical studies of the photoabsorption band corresponding to the vertical electronic transition S(0)-S(1) between first two singlet states of the model chromophore from the green fluorescent protein (GFP) in its neutral form. Predictions of quantum chemical approaches including ab initio and semi-empirical approximations are compared for the model systems which mimic the GFP chromophore in different environments. We provide evidences that the protein matrix in GFP accounts for a fairly large shift of about 40 nm in the S(0)-S(1) absorption band as compared to the gas phase.

We re-visited the results of quantum mechanics – molecular mechanics (QM/MM) approaches aiming to construct the reaction energy profile for the acylation stage of acetylcholine hydrolysis by acetylcholinesterase. The main emphasis of this study was on the energy of the first tetrahedral intermediate (TI) relative to the level of the enzyme–substrate (ES) complex for which contradictory data from different works had been reported. A new series of stationary points on the potential energy surface was calculated by using electronically embedding QM/MM schemes when starting from the crystal structure mimicking features of the reaction intermediate (PDB ID: 2VJA). A thoughtful analysis allows us to conclude that the energy of TI should be lower than that of ES, and a proper treatment of contributions from the oxyanion hole residues accounts for their relative positions.

The collective monograph is devoted to discussing the history of creation, studying the properties, neutralizing and using organophosphorus neurotoxins, which include chemical warfare agents, agricultural crop protection chemical agents (herbicides and insecticides) and medicines. The monograph summarizes the results of current scientific research and new prospects for the development of this field of knowledge in the 21st century, including the use of modern physicochemical methods for experimental study and theoretical analysis of biocatalysis and its mechanisms based on molecular modeling with supercomputer power. The book is intended for specialists who are interested in the current state of research in the field of organophosphorus neurotoxins. The monograph will be useful for students, graduate students, researchers specializing in the field of physical chemistry, physicochemical biology, chemical enzymology, toxicology, biochemistry, molecular biology and genetics, biotechnology, nanotechnology and biomedicine.

The results of a computational study of the synthesis of a key brain metabolite, N-acetyl-l-aspartate, catalyzed by aspartate N-acetyltransferase, encoded by the NAT8L gene, are reported. The reaction Gibbs energy profiles were computed using molecular dynamics simulations with interaction potentials estimated on-the-fly by the quantum mechanics/molecular mechanics QM(PBE0/6-31G**)/MM(CHARMM) approach. The revealed reaction mechanism includes four elementary steps with corresponding activation energies not exceeding 14 kcal mol−1

With the proper inclusion of ion-pair configurations, the diatomics-in-molecules formalism can be used to accurately describe hydrogen bonding. This is demonstrated for the well characterized prototype, the HF dimer, the structure and entire potential energy surface of which is reproduced within its known accuracy: At the stationary points (potential minimum and saddle points) energies and bond lengths are reproduced with an accuracy of ∼1%, and the soft hydrogen bond angles are determined to within ∼5%. This is accomplished through a minimal basis Hamiltonian—19-dimensional matrix to describe the planar complex—constructed with analytic fits to accurately known or determined pair potentials. The construct includes the H+F− ion-pair states of the HF monomer units. The three-body nature of the inductive ion-pair interactions with neutrals is preserved, in the spirit of diatomic-in-ionic-systems. Based on ab initio estimates, in the limited range of interest, a Gaussian function describes the mixing between ionic and neutral states. The amplitude of this function is the only adjustable parameter in the model. The ionicity anisotropy and nonadditivity of interactions, responsible for the structure of the HF dimer, result naturally from mixing between ionic and neutral surfaces.

The ab initio QM/MM calculations are used to optimize geometry configurations of the chromophore and surrounding residues for the kindling protein asFP595. The time-dependent DFT method is applied to estimate parameters of the S0–S1 vertical transition of the chromophore at the protein geometry taking into account effects from the nearest residues. The results of simulations provide a theoretical support to the hypothesis on the possibility of trans–cis izomerization of the chromophore in the mechanism of kindling. The system can absorb light in the trans anion form of the chromophore and emit at longer wavelength in the cis anion form.

The kindling fluorescent protein (KFP), the Ala143Gly variant of the natural chromoprotein asFP595, is a prospective biomarker in live cells. Following the results of QM/MM calculations, we predict that excitation of the protein under certain conditions, favoring formation of KFP fractions with the neutral chromophore, should result in fluorescence from the cationic form of the chromophore which is unusual for the members of the green fluorescent protein family. Occurrence of the neutral form is due to a water wire connecting the chromophore with the exterior of the protein. Occurrence of the cationic form is due to the excited-state proton transfer from the conserved Glu215 to the imidazolinone ring nitrogen of the chromophore. The emission band from conformations with the trans cationic chromophore should be noticeably shifted to the blue side around 520 nm compared to the well-known red fluorescence around 600 nm arising from the cis anionic species.

Human butyrylcholinesterase (BChE) is a bioscavenger that protects the enzyme which is critical for the central nerve system, acetylcholinesterase, from poisoning by organophosphorus agents. Elucidating the details of the hydrolysis reaction mechanism is important to understand how the phosphorylated BChE can be reactivated. Application of the QM(DFTB)/MM(AMBER) method to construct the minimum energy pathways for the hydrolysis reaction of the diethylphosphorylated BChE allowed us to suggest a mechanism of reactivation of the wild-type and the G117H mutated enzyme. Unlike previous approaches assuming that either His438 or His117 serves as a general base in the catalysis, in our proposal the Glu197 residue is responsible for activation of the nucleophilic water molecule (Wat) leading to the chemical transformations that restore the catalytic Ser198 residue in BChE. In agreement with the experimental data, it is shown that the G117H mutation facilitates the reactivation of the inhibited enzyme.

We characterize structures and electronic excitation of domains from a bacteriophytochrome-based infrared fluorescent protein carrying a covalently bound biliverdin chromophore using the quantum mechanics/molecular mechanics methods. We show that geometry optimization at the density functional theory level and application of the extended multiconfigurational quasidegenerate perturbation theory in quantum subsystems allows us to achieve high calculation accuracy for the model systems mimicking the red-adsorbing and the far-red-adsorbing protein forms. Specifically, discrepancies between experimental and theoretical excitation energies are less than 0.05 eV (∼20 nm) for the Q-band in both protein forms.

We report a mechanism of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP) conversion by the mammalian type V adenylyl cyclase revealed in molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) simulations. We characterize a set of computationally derived enzyme–substrate (ES) structures showing an important role of coordination shells of magnesium ions in the solvent accessible active site. In the lowest energy ES conformation, the coordination shell of MgA2+ does not include the Oδ1 atom of the conserved Asp440 residue. Starting from this conformation, a one-step reaction mechanism is characterized that includes proton transfer from the ribose O3′H3′ group in ATP to Asp440 via a shuttling water molecule concerted with PA–O3A bond cleavage and O3′–PA bond formation. The energy profile of this route is consistent with the observed reaction kinetics. The computed energy profiles initiated from higher energy ES complexes are characterized by larger energy expenses to complete the reaction. Consistent with experimental data, we show that the Asp440Ala mutant of the enzyme should exhibit a reduced but retained activity. All considered reaction pathways include proton wires from the O3′H3′ group via shuttling water molecules.

The diatomics-in-molecules (DIM) technique is applied for a description of the low-lying states of the Rg–Hal2 van der Waals complexes correlating with the lowest states of constituent atoms Rg(1S)+Hal(2Pj)+Hal(2Pj). The important feature of this approach is the construction of polyatomic basis functions as products of the Hal2 diatomic eigenstates classified within the Hund “c” scheme and the atomic rare gas wave function. Necessary transformations to the other basis set representations are described, and finally all the matrix elements are expressed in terms of nonrelativistic adiabatic energies of Hal2 and Rg Hal fragments and spin-orbit splitting constant of the halogen atom. Our main concern is to test the DIM-based approximations of different levels taking the He–Cl2 system as an example. Namely, we have compared the results obtained within a hierarchy of approaches: (1) the simplest pairwise potential scheme as a far extreme of the DIM model, (2) the same as (1) but with the different components (Σ and Π) for He–Cl interaction, (3) the accurate DIM technique without spin-orbit terms, and (4) the highest level which takes into account all these contributions. The results have been compared to the other DIM like models as well. The shapes of two-dimensional potential surfaces for the ground (X) and excited (B) states of HeCl2, binding energies De with respect to He+Cl2, stretching and bending vibrational frequencies of the complex, binding energies D0, and spectral shifts for the B←X transition are discussed.

Quantum chemical methods are applied to study the stages of the proton transfer in water wires attached to molecular walls containing the side chains of His and Asp residues. Several molecular models of variable complexity are considered by using different techniques, and in every case, the most important reactions of cleavage and formation of chemical bonds in proton wires are treated at the ab initio level. The largest molecular model, which mimics structural elements of the M2 ion channel, is investigated with the help of a quantum mechanical−molecular mechanical (QM/MM) method with flexible effective fragments. Smaller models are considered at nonempirical levels in order to purify conclusions of the QM/MM approach. The results of calculations show that the only transition state structure found on the proton-transfer route refers to the stage of proton detachment from the Nε atom of the imidazole ring to the neighboring water molecule. The corresponding energy barriers are estimated.

Productive photochemical synthesis of hydrogen peroxide, H2O2, from the H2O···O(3P) van der Waals complex is studied in solid krypton. Experimentally, we achieve the three-step formation of H2O2 from H2O and N2O precursors frozen in solid krypton. First, 193 nm photolysis of N2O yields oxygen atoms in solid krypton. Upon annealing at ∼25 K, mobile oxygen atoms react with water forming the H2O···O complex, where the oxygen atom is in the triplet ground state. Finally, the H2O···O complex is converted to H2O2 by irradiation at 300 nm. According to the complete active space self-consistent field modeling, hydrogen peroxide can be formed through the photoexcited H2O+−O- charge-transfer state of the H2O···O complex, which agrees with the experimental evidence.

Kindling fluorescent protein (KFP) is considered as a prospective fluorophore for high-resolution nanoscopy. Analysis of pH dependence of the absorption and fluorescence spectra of KFP in aqueous solutions prompted us to assume that a shift in conformational equilibrium is responsible for substantial enhancement of red fluorescence in KFP at alkaline pH. Variations in pH also resulted in noticeable shifts in band maxima for absorption, fluorescence excitation, and fluorescence emission. These observations can be interpreted as an appearance of pH-induced fluorescent conformational states of the protein. On the basis of the available crystal structures of the protein and the results of molecular modeling, we suggest that appearance of these pH-induced fluorescent states is due to changes in the hydrogen bond network around the chromophore moiety (but not the cromophore itself), especially those associated with the side chains of Cys62 and Ser158. We hypothesize that conformational partitioning and fluctuations in protein ionization at alkali pH play an essential role in the appearance of fluorescent properties of KFP.

The unique properties of the photoswitchable protein Dreiklang are attributed to a reversible hydration/dehydration reaction at the imidazolinone ring of the chromophore. Recovery of the fluorescent state, which is associated with a chemical reaction of the chromophore's dehydration, is an important part of the photocycle of this protein. Here we characterize the fluorescent (ON) and nonfluorescent (OFF) states of Dreiklang and simulate the thermal recovery reaction OFF → ON using computational approaches. By using molecular modeling methods including the quantum mechanics/molecular mechanics (QM/MM) technique, we characterize the structures and spectra of the ON- and OFF-states. The results are consistent with available experimental data. The computed reaction profile explains the observed recovery reaction and clarifies the mechanism of chemical transformations in the chromophore-containing pocket in Dreiklang.

Vibrational predissociation dynamics of ArHF and ArDF complexes is investigated theoretically for the first time owing to the use of three-dimensional potential energy surfaces (PES’s) based on the diatomics-in-molecule approach [J. Chem. Phys. 104, 5510 (1996)]. The original PES is improved empirically to yield a reasonable description of the lowest vibrational energy levels of the ArHF complex at J=0. Predissociation dynamics is studied by means of line shape and diabatic Fermi Golden Rule methods. The latter is found to provide excellent results for the total decay widths but only a qualitative estimate for the product rotational distributions. It is shown that predissociation dynamics is governed by vibrational to rotational energy transfer. The decay proceeds almost entirely into the highest accessible rotational product channel. This propensity manifests itself in the decrease of the predissociation lifetime upon increasing vibrational excitation of the diatomic fragment when the highest rotational channel appears to be closed. Another source of state specificity in the vibrational predissociation is the anisotropy of the PES. Absolute calculated lifetime values are likely too small, but exhibit some qualitative trends observed experimentally.

Abstract The structures and properties of the complexes formed upon binding the oxygen molecule to the iron sites in non‐heme 2‐oxoglutarate‐dependent enzymes are characterized by QM(CASSCF)/MM and density functional theory (DFT) calculations. Molecular models for the calculations are constructed following the crystal structure of hypoxia‐inducible factor asparaginyl hydroxylase (FIH‐1). DFT calculations for the 37‐atomic cluster have been carried out at the B3LYP(LANL2DZ dp ) level. The flexible effective fragment potential method is used as a combined quantum mechanical–molecular mechanical (QM/MM) technique to characterize the fragment of the enzymatic system, including 1,758 atoms in the MM part and 27 atoms in the QM part. In these calculations, the CASSCF(LANL2DZ dp ) approach is applied in the QM subsystem, and AMBER force field parameters are used in the MM subsystem. With both approaches, equilibrium geometry configurations have been located for different spin states of the system. In DFT calculations, the order of the states is as follows: septet, triplet (+7.7 kcal/mol), quintet (+10.7 kcal/mol). Geometry configurations correspond to the end‐on structures with no evidences of electron transfer from Fe(II) to molecular oxygen. In contrast, QM(CASSCF)/MM calculations predict the quintet state as the lowest one, while the septet structure has slightly (&lt;2 kcal/mol) higher energy, and the triplet state is considerably more energetic. In QM/MM calculations, in both quintet and septet states, the electronic configurations show considerable electron charge transfer from iron to oxygen, and the oxidation state of iron in the metal binding site can be characterized as Fe(III). © 2006 Wiley Periodicals, Inc. Int J Quantum Chem, 2006

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.

Chromoprotein asFP595 and its A143G variant called kindling fluorescent protein (KFP) are among the chronologically first species for which trans–cis chromophore isomerization has been proposed as a driving force of photoswitching. In spite of long-lasting efforts to characterize the route between protein conformations referring to the trans and cis forms of the chromophore, the molecular mechanism of this transformation is still under debate. We report the results of computational studies of the trans–cis isomerization of the anionic and neutral chromophore inside the protein matrices in the ground electronic state for both variants, asFP595 and KFP. Corresponding free energy profiles (potentials of mean force) were evaluated by using molecular dynamics simulations with the quantum mechanical – molecular mechanical (QM/MM) forces. The computed free energy barrier for the cis–trans ground state (thermal) isomerization reaction is about 2 kcal/mol higher in KFP than that in asFP595. These results provide interpretation of experimental studies on thermal relaxation from the light-induced activation of fluorescence of these proteins and correctly show that the A143G mutation in asFP595 noticeably increases the lifetime of the fluorescence species.

ABSTRACT Interpretation of the experiments showing that the Ras‐GAP protein complex maintains activity in guanosine triphosphate (GTP) hydrolysis upon replacement of Glu61 in Ras with its unnatural nitro analog, NGln, is an important issue for understanding details of chemical transformations at the enzyme active site. By using molecular modeling we demonstrate that both glutamine and its nitro analog in the aci ‐nitro form participate in the reaction of GTP hydrolysis at the stages of proton transfer and formation of inorganic phosphate. The computed structures and the energy profiles for the complete pathway from the enzyme‐substrate to enzyme‐product complexes for the wild‐type and mutated Ras suggest that the reaction mechanism is not affected by this mutation. Proteins 2015; 83:2091–2099. © 2015 Wiley Periodicals, Inc.

Fluorescent proteins (FPs) have revolutionised the life sciences, but the chromophore maturation mechanism is still not fully understood. Here we photochemically trap maturation at a crucial stage and structurally characterise the intermediate.

Oxygenase activity of the flavin-dependent enzyme RutA is commonly associated with the formation of flavin-oxygen adducts in the enzyme active site. We report the results of quantum mechanics/molecular mechanics (QM/MM) modeling of possible reaction pathways initiated by various triplet state complexes of the molecular oxygen with the reduced flavin mononucleotide (FMN) formed in the protein cavities. According to the calculation results, these triplet-state flavin-oxygen complexes can be located at both re-side and si-side of the isoalloxazine ring of flavin. In both cases, the dioxygen moiety is activated by electron transfer from FMN, stimulating the attack of the arising reactive oxygen species at the C4a, N5, C6, and C8 positions in the isoalloxazine ring after the switch to the singlet state potential energy surface. The reaction pathways lead to the C(4a)-peroxide, N(5)-oxide, or C(6)-hydroperoxide covalent adducts or directly to the oxidized flavin, depending on the initial position of the oxygen molecule in the protein cavities.

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.

The structures and vibrational spectra of the reacting species upon guanosine triphosphate (GTP) hydrolysis to guanosine diphosphate and inorganic phosphate (Pi) trapped inside the protein complex Ras-GAP were analyzed following the results of QM/MM simulations. The frequencies of the phosphate vibrations referring to the reactants and to Pi were compared to those observed in the experimental FTIR studies. A good correlation between the theoretical and experimental vibrational data provides a strong support to the reaction mechanism of GTP hydrolysis by the Ras-GAP enzyme system revealed by the recent QM/MM modeling. Evolution of the vibrational bands associated with the inorganic phosphate Pi during the elementary stages of GTP hydrolysis is predicted.

We characterize computationally a red fluorescent protein (RFP) with the chromophore (Chro) sandwiched between two aromatic tyrosine rings in a triple-decker motif. According to the original proposal [J. Phys. Chem. Lett. 2013, 4, 1743], such a tyrosine-chromophore-tyrosine π-stacked construct can be accommodated in the green fluorescent protein (GFP). A recent study [ACS Chem. Biol. 2016, 11, 508] attempted to realize the triple-decker motif and obtained an RFP variant called mRojoA-VYGV with two tyrosine residues surrounding the chromophore. The crystal structure showed that only a tyrosine-chromophore pair was involved in π-stacking, whereas the second tyrosine was oriented perpendicularly, edge-to-face with respect to the chromophore. We propose a more promising variant of this RFP with a perfect triple-decker unit achieved by introducing additional mutations in mRojoA-VYGV. The structures and optical properties of model proteins based on the structures of mCherry and mRojoA are characterized computationally by QM(DFT)/MM. The electronic transitions in the protein-bound chromophores are computed by high-level quantum chemical methods. According to our calculations, the triple-decker chromophore unit in the new RFP variant is stable within the protein and its optical bands are red-shifted with respect to the parent mCherry and mRojoA species.

The quantum mechanics/molecular mechanics approach is used to model the chain of elementary reactions involved in the backbone cyclization of the initially non-cyclized Gly65–Gly66–Gly67 tripeptide inside the protein matrix of the green fluorescent protein (GFP) Ser65Gly/Tyr66Gly mutant. The computationally characterized reaction mechanism provides support for understanding chromophore maturation in GFP-like fluorescent proteins.

Further developments of the intermolecular diatomics-in-molecules (DIM) theory towards construction of potential energy surfaces of hydrogen-bonded molecular aggregates are presented. Compared to the previously studied hydrogen fluoride clusters (HF)n [J. Chem. Phys. 111, 4442 (1999)], considerably more complicated and challenging systems, namely, water clusters (H2O)n (n=2–6) have been analyzed in this work. The present DIM, or more precisely, diatomics-in-ionic-systems, scheme is based on the balanced treatment of neutral and ionic contributions to the electronic properties of polyatomic species, and in this case takes into account the mixing of the OH and O−H+ electronic states within the valence bond description of water molecules. The potential curves of diatomic molecules required for the present application, including ionic species O−H, OH+, O2−, have been computed by ab initio quantum chemistry tools. The results of DIM calculations of equilibrium geometry configurations, binding energies, and relative energies for the low-lying isomers of (H2O)n (n=2–6) are compared to the reference data showing a good predictive power of this method.

Minimum energy pathways for the hydrolysis reaction of the cyclic guanosine monophosphate (cGMP) in water were calculated by using two versions of the quantum mechanics/molecular mechanics (QM/MM) theory. The first version corresponded to the electrostatical embedded cluster method, the second one referred to the effective fragment potential approach. In both cases the density functional theory methods (B3LYP/cc-pVDZ and PBE0/6-31G*) were applied to describe the QM subsystem composed of the cGMP substrate, the lytic water molecule and the chain of four water molecules participating in proton transfers. The shells of 301 explicit solvent water molecules (the MM subsystem) were simulated by using the TIP3P potential. Qualitatively, both QM/MM approaches resulted in similar minimum energy pathways: stationary points on the potential energy surface, corresponding to the reagents, products and reaction intermediate with the pentacoordinated phosphorus species, were located and the related saddle points separating these minimum energy structures were identified. Both computed minimum energy profiles correspond to the reaction route with a rate-limiting activation barrier much lower than that estimated previously for a direct attack of the lytic water molecule at the phosphorus center. The major difference in the profiles computed with both QM/MM approaches is due to the energy level of the reaction intermediate relative to that of the reagents.

We apply computational methods aiming to approach a full quantum mechanical treatment of chemical reactions in proteins. A combination of the quantum mechanical – molecular mechanical methodology for geometry optimization and the fragment molecular orbital approach for energy calculations is examined for an example of acetylcholinesterase catalysis. The codes based on the GAMESS(US) package operational on the ‘RSC Tornado’ computational cluster are applied to determine that the energy of the reaction intermediate upon hydrolysis of acetylcholine is lower than that of the enzyme–substrate complex. This conclusion is consistent with the experiments and it is free from the empirical force field contributions.

ABSTRACT We report for the first time a hydrolysis mechanism of the cyclic dimeric guanosine monophosphate (c‐di‐GMP) by the EAL domain phosphodiesterases as revealed by molecular simulations. A model system for the enzyme‐substrate complex was prepared on the base of the crystal structure of the EAL domain from the BlrP1 protein complexed with c‐di‐GMP. The nucleophilic hydroxide generated from the bridging water molecule appeared in a favorable position for attack on the phosphorus atom of c‐di‐GMP. The most difficult task was to find a pathway for a proton transfer to the O3' atom of c‐di‐GMP to promote the O3'P bond cleavage. We show that the hydrogen bond network extended over the chain of water molecules in the enzyme active site and the Glu359 and Asp303 side chains provides the relevant proton wires. The suggested mechanism is consistent with the structural, mutagenesis, and kinetic experimental studies on the EAL domain phosphodiesterases. Proteins 2016; 84:1670–1680. © 2016 Wiley Periodicals, Inc.

Argon-induced vibrational frequency shifts for the (HF)2 dimer embedded into large argon clusters have been computed with the help of molecular dynamics simulations. The potential energy surface for the (HF)2 · Arn system has been constructed with a diatomics-in-molecules potential for each ArHF triangle, a point-charge model surface for (HF)2 and pairwise ArAr interactions. When combining the computed shifts with the frequencies referring to the gas-phase complex (HF)2 we predict a vibrational spectrum of the dimer in argon matrices which is compared to experimental data.

Abstract The electronic structure of MgO is calculated in the framework of local‐density theory using the LAPW resp. cluster X α ‐SW method. The results of an improved permittivity calculation is compared with optical measurements, yielding good agreement. The effect of pressure on the electronic structure is studied for NaCl symmetry. It is found that the direct gap becomes indirect already for a compression of V/V 0 = 0.9.

We performed molecular modeling on the mechanism of serine-carboxyl peptidases, a novel class of enzymes active at acidic pH and distinguished by the conserved triad of amino acid residues Ser-Glu-Asp. Catalytic cleavage of a hexapeptide fragment of the oxidized B-chain of insulin by the Pseudomonas sedolisin, a member of the serine-carboxyl peptidases family, was simulated. Following motifs of the crystal structure of the sedolisin-inhibitor complex (PDB accession code 1NLU) we designed the model enzyme−substrate (ES) complex and performed quantum mechanical−molecular mechanical calculations of the energy profile along a reaction route up to the acylenzyme (EA) complex through the tetrahedral intermediate (TI). The energies and forces were computed by using the PBE0 exchange-correlation functional and the basis set 6-31+G** in the quantum part and the AMBER force field parameters in the molecular mechanical part. Analysis of the ES, TI, and AE structures as well as of the corresponding transition states allows us to scrutinize the chemical transformations catalyzed by sedolisin. According to the results of simulations, the reaction mechanism of serine-carboxyl peptidases should be viewed as a special case of carboxyl (aspartic) proteases, with the nucleophilic water molecule being replaced by the Ser residue. The catalytic triad Ser-Glu-Asp in sedolisin functions differently compared to the well-known triad Ser-His-Asp of serine proteases, despite the structural similarity of sedolisin and the serine proteases member, subtilisin.

The role of protonation states of the chromophore and its neighboring amino acid side chains of the reversibly switching fluorescent protein rsEGFP2 upon photoswitching is characterized by molecular modeling methods. Numerous conformations of the chromophore-binding site in computationally derived model systems are obtained using the quantum chemistry and QM/MM approaches. Excitation energies are computed using the extended multiconfigurational quasidegenerate perturbation theory (XMCQDPT2). The obtained structures and absorption spectra allow us to provide an interpretation of the observed structural and spectral properties of rsEGFP2 in the active ON and inactive OFF states. The results demonstrate that in addition to the dominating anionic and neutral forms of the chromophore, the cationic and zwitterionic forms may participate in the photoswitching of rsEGFP2. Conformations and protonation forms of the Glu223 and His149 side chains in the chromophore-binding site play an essential role in stabilizing specific protonation forms of the chromophore.

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.

Modern quantum-based methods are employed to model interaction of the flavin-dependent enzyme RutA with the uracil and oxygen molecules. This complex presents the structure of reactants for the chain of chemical reactions of monooxygenation in the enzyme active site, which is important in drug metabolism. In this case, application of quantum-based approaches is an essential issue, unlike conventional modeling of protein-ligand interaction with force fields using molecular mechanics and classical molecular dynamics methods. We focus on two difficult problems to characterize the structure of reactants in the RutA-FMN-O2 -uracil complex, where FMN stands for the flavin mononucleotide species. First, location of a small O2 molecule in the triplet spin state in the protein cavities is required. Second, positions of both ligands, O2 and uracil, must be specified in the active site with a comparable accuracy. We show that the methods of molecular dynamics with the interaction potentials of quantum mechanics/molecular mechanics theory (QM/MM MD) allow us to characterize this complex and, in addition, to surmise possible reaction mechanism of uracil oxygenation by RutA.

Potential curves of the SH, KrH and KrS molecules needed for the diatomics-in-molecules (DIM) treatment of the ground and excited states of the SH(X,A)⋯Kr complex have been computed at the SOCI/CASSCF level. The ionic and ion-pair states of these diatomic fragments which play an essential role in the DIM model of intermolecular interactions have been considered as well. The new results for the ion-pair states of SH are compared to the corresponding data for OH. The curves for KrS and XeS [M. Yamanishi, K. Hirao, K. Yamashita, J. Chem. Phys. 108 (1998) 1514] are discussed. The main features of the empirical potential surfaces of the SH⋯Kr complex are reproduced by the DIM technique.

Vibrational and electronic properties of the NBr molecule isolated in a low temperature argon matrix have been modelled within the cluster approach. Molecular dynamics simulation techniques and quantum chemical approaches have been used to study the NBr/Arn heteroclusters. The shifts in spectral features as well as changes in orbital properties of NBr due to surrounding argon atoms are discussed.

Abstract Potential curves of the OH molecule correlating to the four lowest energy dissociation limits O ( 3 P )+ H ( 2 S ), O ( 1 D )+ H ( 2 S ), O ( 1 S ) + H ( 2 S ), O − ( 2 P ) + H + have been computed at the CI/CASSCF level with the AUG-cc-pVTZ basis sets with a special emphasis on the ion-pair states 3 2 Π and C 2 Σ + . A balanced treatment of the excited state potentials is achieved by using the state-averaging MO optimization procedure. The X 1 Σ + and 1 Π potentials of OH − have been also considered. After empirical correction of the errors at the dissociation limits, the computed functions are recommended for the future use in the diatomics-in-molecules studies of the structure and dynamics of oxygen/hydrogen containing molecular systems.

The reactions of hydroxymethyl radical ˙CH2OH with glutathione tripeptide GSH and with methylthiol CH3SH in water are modeled by using the effective fragment potential (EFP) based quantum mechanical–molecular mechanical (QM/MM) methods. In the case of glutathione, the reactive part of the cysteine residue of the tripeptide and the hydroxymethyl moiety are included to the quantum part. The remaining part of GSH assigned to the MM subsystem is represented by flexible chains of small effective fragments. The energy profiles for the condensed phase reactions have been constructed by using the transition state structure of the gas-phase reference reaction of hydroxymethyl radical ˙CH2OH with methylthiol CH3SH. Optimization of geometry parameters of the systems, including coordinates of environmental particles, leads to the structures at the top of the barrier, as well as for reagent interaction complexes and product interaction complexes. The energy difference between the top of the barrier and the reagent interaction complex is considered as a measure of activation energy, which allows us to treat all systems at the uniform level. According to simulation results, the activation barrier for the aqueous reaction of hydroxymethyl radical with methylthiol should be slightly higher, and for the aqueous reaction of hydroxymethyl radical with glutathione in the lowest energy conformation should be slightly lower than that of the reference gas-phase reaction. A conclusion about higher activation barrier for aqueous reaction ˙CH2OH + CH3SH in comparison to the gas-phase process, obtained here within the supermolecular approach, is consistent with the literature results of solvation models. It is shown that conformations of peptide chain of glutathione may modify the gas-phase reaction energy profile of the reacting species to a larger extent than the influence of water molecules. The role of the peptide chain seems to be to provide either beneficial or unfavorable arrangement of the reagents in the interaction complex.

Opening the Arg-Glu salt bridge in myosin, which presumably succeeds the myosin-catalyzed hydrolysis of adenosine triphosphate, was modeled computationally on the basis of the structures corresponding to the enzyme-substrate and enzyme-product complexes found in the quantum mechanics-molecular mechanics simulations. According to the calculations of the potential of mean force, opening the bridge is considerably facilitated upon termination of the chemical reaction, but does not promote egress of inorganic phosphate by the back-door mechanism.

Quantum chemistry methods are applied to obtain numerical solutions of the Schr¨odinger equation for molecular systems. Calculations of transitions between electronic states of large molecules present one of the greatest challenges in this field which require the use of supercomputer resources. In this work we describe the results of benchmark calculations of electronic excitation in the protein domains which were designed to engineer novel fluorescent markers operating in the near-infrared region. We demonstrate that such complex systems can be efficiently modeled with the hybrid qunatum mechanics/molecular mechanics approach (QM/MM) using the modern supercomputers. More specifically, the time-dependent density functional theory (TD-DFT) method was primarily tested with respect to its performance and accuracy. GAMESS (US) and NWChem software were benchmarked in direct and storage-based TDDFT calculations with the hybrid B3LYP density functional, both showing good scaling up to 32 nodes. We note that conventional SCF calculations greatly outperform direct SCF calculations for our test system. Accuracy of TD-DFT excitation energies was estimated by a comparison to the more accurate ab initio XMCQDPT2 method.

Predictions of the diatomics-in-ionic-systems model for the variety of stationary points on the potential energy surfaces of the hydrogen fluoride clusters (HF)n (3⩽n⩽6) are compared to the results of ab initio MP2/6-311+G(2d,2p) calculations as well as to the results of the polarizable mechanics model of Hodges et al. [J. Phys. Chem. A 102, 2455 (1998)]. The diatomics-in-ionic-systems scheme which relies on the balanced treatment of neutral and ionic contributions to the electronic properties of polyatomic species within the diatomics-in-molecules theory takes into account here the mixing of the FH and F−H+ electronic states. The corresponding mixing coefficient serves as a single principal adjustable parameter of the model, finally selected by the reference value of the binding energy of (HF)3. It is shown that structures and energies of the main cyclic isomers are in a good agreement with the best estimates of Quack and Suhm [Conceptual Perspectives in Quantum Chemistry (Kluwer, Dordrecht, 1997)]. Every prediction of this model for the stationary points corresponding to 16 higher energy structures of (HF)n is confirmed by the MP2 ab initio data.