Histidine-assisted reduction of arylnitrenes upon photo-activation of phenyl azide chromophores in GFP-like fluorescent proteins.

The photochemically active sites of the proteins sfGFP and Venus, members of the green fluorescent protein (GFP) family, contain a non-canonical amino acid residue -azidophenylalanine (azF) instead of Tyr66. The light-induced decomposition of azF at these sites leads to the formation of reactive arylnitrene (nF) intermediates followed by the formation of phenylamine-containing chromophores. We report the first study of the reaction mechanism of the reduction of the arylnitrene intermediates in sfGFP and Venus using molecular modeling methods.

Multiscale Simulations of the Covalent Inhibition of the SARS-CoV-2 Main Protease: Four Compounds and Three Reaction Mechanisms.

We report the results of computational modeling of the reactions of the SARS-CoV-2 main protease (M) with four potential covalent inhibitors. Two of them, carmofur and nirmatrelvir, have shown experimentally the ability to inhibit M. Two other compounds, X77A and X77C, were designed computationally in this work.

QM/MM Modeling of the Flavin Functionalization in the RutA Monooxygenase.

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 -side and -side of the isoalloxazine ring of flavin.

Modeling Light-Induced Chromophore Hydration in the Reversibly Photoswitchable Fluorescent Protein Dreiklang.

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.

Quantum-based Modeling of Protein-ligand Interaction: The Complex of RutA with Uracil and Molecular Oxygen.

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.

How Reproducible Are QM/MM Simulations? Lessons from Computational Studies of the Covalent Inhibition of the SARS-CoV-2 Main Protease by Carmofur.

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.

Tuning Electrostatic Gating of Semiconducting Carbon Nanotubes by Controlling Protein Orientation in Biosensing Devices.

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.

Protonation States of Molecular Groups in the Chromophore-Binding Site Modulate Properties of the Reversibly Switchable Fluorescent Protein rsEGFP2.

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).

Tuning Electrostatic Gating of Semiconducting Carbon Nanotubes by Controlling Protein Orientation in Biosensing Devices.

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.

Stalling chromophore synthesis of the fluorescent protein Venus reveals the molecular basis of the final oxidation step.

Fluorescent proteins (FPs) have revolutionised the life sciences, but the mechanism of chromophore maturation is still not fully understood. Here we show that incorporation of a photo-responsive non-canonical amino acid within the chromophore stalls maturation of Venus, a yellow FP, at an intermediate stage; a crystal structure indicates the presence of O located above a dehydrated enolate form of the imidazolone ring, close to the strictly conserved Gly67 that occupies a twisted conformation. His148 adopts an "open" conformation so forming a channel that allows O access to the immature chromophore.

Modeling photophysical properties of the bacteriophytochrome-based fluorescent protein IFP1.4.

An enhanced interest in the phytochrome-based fluorescent proteins is explained by their ability to absorb and emit light in the far-red and infra-red regions particularly suitable for bioimaging. The fluorescent protein IFP1.4 was engineered from the chromophore-binding domain of a bacteriophytochrome in attempts to increase the fluorescence quantum yield.

Interplay between Locally Excited and Charge Transfer States Governs the Photoswitching Mechanism in the Fluorescent Protein Dreiklang.

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).

Structure of the Brain -Acetylaspartate Biosynthetic Enzyme NAT8L Revealed by Computer Modeling.

We report the results of computational modeling of a three-dimensional all-atom structure of the membrane-associated protein encoded by the gene, aspartate -acetyltransferase, which is essential for brain synthesis of -acetyl-l-aspartate (NAA). The lack of experimentally derived three-dimensional structures of NAT8L poses one of the obstacles in studies of the mechanism of NAA formation and understanding the precise role of NAA in neurological disorders. We apply a computational protocol employing the contact map prediction, folding, homology modeling, and refinement to obtain a structure of NAT8L with the aspartate and acetyl coenzyme A cofactors in the protein molecule.

Mechanisms of ATP to cAMP Conversion Catalyzed by the Mammalian Adenylyl Cyclase: A Role of Magnesium Coordination Shells and Proton Wires.

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 Mg does not include the O atom of the conserved Asp440 residue.

Computational Modeling Reveals the Mechanism of Fluorescent State Recovery in the Reversibly Photoswitchable Protein Dreiklang.

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.

Computational Challenges in Modeling of Representative Bioimaging Proteins: GFP-Like Proteins, Flavoproteins, and Phytochromes.

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.

Diversity of mechanisms in Ras-GAP catalysis of guanosine triphosphate hydrolysis revealed by molecular modeling.

The mechanism of the deceptively simple reaction of guanosine triphosphate (GTP) hydrolysis catalyzed by the cellular protein Ras in complex with the activating protein GAP is an important issue because of the significance of this reaction in cancer research. We show that molecular modeling of GTP hydrolysis in the Ras-GAP active site reveals a diversity of mechanisms of the intrinsic chemical reaction depending on molecular groups at position 61 in Ras occupied by glutamine in the wild-type enzyme. First, a comparison of reaction energy profiles computed at the quantum mechanics/molecular mechanics (QM/MM) level shows that an assignment of the Gln61 side chain in the wild-type Ras either to QM or to MM parts leads to different scenarios corresponding to the glutamine-assisted or the substrate-assisted mechanisms.

Computer-designed active human butyrylcholinesterase double mutant with a new catalytic triad.

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.

Amide-imide tautomerization in the glutamine side chain in enzymatic and photochemical reactions in proteins.

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.

Optimization of Cholinesterase-Based Catalytic Bioscavengers Against Organophosphorus Agents.

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.

Improving the Design of the Triple-Decker Motif in Red Fluorescent Proteins.

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.

Molecular Modeling Clarifies the Mechanism of Chromophore Maturation in the Green Fluorescent Protein.

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.

Analysis of proton wires in the enzyme active site suggests a mechanism of c-di-GMP hydrolysis by the EAL domain phosphodiesterases.

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.

Photoinduced Chemistry in Fluorescent Proteins: Curse or Blessing?

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).

Theoretical vibrational spectroscopy of intermediates and the reaction mechanism of the guanosine triphosphate hydrolysis by the protein complex Ras-GAP.

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.