Maria G. Khrenova

Effect of Protein Environment on Electronically Excited and Ionized States of the Green Fluorescent Protein Chromophore

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.

Photoinduced Electron Transfer Facilitates Tautomerization of the Conserved Signaling Glutamine Side Chain in BLUF Protein Light Sensors

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

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

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

Hydrolysis of Guanosine Triphosphate (GTP) by the Ras·GAP Protein Complex: Reaction Mechanism and Kinetic Scheme.

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.

Computational characterization of reaction intermediates in the photocycle of the sensory domain of the AppA blue light photoreceptor

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.

FLIM-FRET Imaging of Caspase-3 Activity in Live Cells Using Pair of Red Fluorescent Proteins

We report a new technique to detect enzyme activity inside cells. The method based on Fluorescence Lifetime Imaging (FLIM) technology allows one to follow sensor cleavage by proteolytic enzyme caspase-3. Specifically, we use the FLIM FRET of living cells via the confocal fluorescence microscopy. A specially designed lentivector pLVT with the DNA fragment of TagRFP-23-KFP was applied for transduction of A549 cell lines. Computer simulations are carried out to estimate FRET efficiency and to analyze possible steric restrictions of the reaction between the substrate TagRFP-23-KFP and caspase-3 dimer. Successful use of the fuse protein TagRFP-23-KFP to register the caspase-3 activation based on average life-time measurements is demonstrated. We show that the average life-time distribution is dramatically changed for cells with the modified morphology that is typical for apoptosis. Namely, the short-lived component at 1.8-2.1 ns completely disappears and the long-lived component appears at 2.4-2.6 ns. The latter is a fingerprint of the TagRFP molecule released after cleavage of the TagRFP-23-KFP complex by caspase-3. Analysis of life-time distributions for population of cells allows us to discriminate apoptotic and surviving cells within single frame and to peform statistical analysis of drug efficiency. This system can be adjusted for HTS by using special readers oriented on measurements of fluorescence life-time.

Modeling the Role of G12V and G13V Ras Mutations in the Ras-GAP-Catalyzed Hydrolysis Reaction of Guanosine Triphosphate

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.

Mechanism of proteolysis in matrix metalloproteinase-2 revealed by QM/MM modeling

The mechanism of enzymatic peptide hydrolysis in matrix metalloproteinase‐2 (MMP‐2) was studied at atomic resolution through quantum mechanics/molecular mechanics (QM/MM) simulations. An all‐atom three‐dimensional molecular model was constructed on the basis of a crystal structure from the Protein Data Bank (ID: 1QIB), and the oligopeptide Ace‐Gln‐Gly∼Ile‐Ala‐Gly‐Nme was considered as the substrate. Two QM/MM software packages and several computational protocols were employed to calculate QM/MM energy profiles for a four‐step mechanism involving an initial nucleophilic attack followed by hydrogen bond rearrangement, proton transfer, and CN bond cleavage. These QM/MM calculations consistently yield rather low overall barriers for the chemical steps, in the range of 5–10 kcal/mol, for diverse QM treatments (PBE0, B3LYP, and BB1K density functionals as well as local coupled cluster treatments) and two MM force fields (CHARMM and AMBER). It, thus, seems likely that product release is the rate‐limiting step in MMP‐2 catalysis. This is supported by an exploration of various release channels through QM/MM reaction path calculations and steered molecular dynamics simulations.

Modeling reaction routes from rhodopsin to bathorhodopsin

The quantum mechanical–molecular mechanical (QM/MM) theory was applied to calculate accurate structural parameters, vibrational and optical spectra of bathorhodopsin (BATHO), one of the primary photoproducts of the functional cycle of the visual pigment rhodopsin (RHO), and to characterize reaction routes from RHO to BATHO. The recently resolved crystal structure of BATHO (PDBID: 2G87) served as an initial source of coordinates of heavy atoms. Protein structures in the ground electronic state and vibrational frequencies were determined by using the density functional theory in the PBE0/cc‐pVDZ approximation for the QM part and the AMBER force field parameters in the MM part. Calculated and assigned vibrational spectra of both model protein systems, BATHO and RHO, cover three main regions referring to the hydrogen‐out‐of‐plan (HOOP) motion, the CC ethylenic stretches, and the CC single‐bond stretches. The S0–S1 electronic excitation energies of the QM part, including the chromophore group in the field of the protein matrix, were estimated by using the advanced quantum chemistry methods. The computed structural parameters as well as the spectral bands match perfectly the experimental findings. A structure of the transition state on the S0 potential energy surface for the ground electronic state rearrangement from RHO to BATHO was located proving a possible route of the thermal protein activation to the primary photoproduct. Proteins 2010.

Computational characterization of the chemical step in the GTP hydrolysis by Ras-GAP for the wild-type and G13V mutated Ras

The free energy profiles for the chemical reaction of the guanosine triphosphate hydrolysis GTP + H2O → GDP + Pi by Ras‐GAP for the wild‐type and G13V mutated Ras were computed by using molecular dynamics protocols with the QM(ab initio)/MM potentials. The results are consistent with the recent measurements of reaction kinetics in Ras‐GAP showing about two‐order reduction of the rate constant upon G13V mutation in Ras: the computed activation barrier on the free energy profile is increased by 3 kcal/mol upon the G13V replacement. The major reason for a higher energy barrier is a shift of the “arginine finger” (R789 from GAP) from the favorable position in the active site. The results of simulations provide support for the mechanism of the reference reaction according to which the Q61 side chain directly participates in chemical transformations at the proton transfer stage. Proteins 2015; 83:1046–1053.