Theoretical insights into the formation and stability of radical oxygen species in cryptochrome

International audienceCryptochrome is a blue-light absorbing flavoprotein containing a flavin adenine dinucleotide (FAD) cofactor. FAD can accept up to two electrons and two protons, which can be subsequently transferred to substrates present in the binding pocket. It is well known that reactive oxygen species are generated when triplet molecular oxygen is present in the cavity. Here, we investigate the formation and stability of radical oxygen species in Drosophila melanogaster cryptochrome using molecular dynamics simulations and electronic structure calculations. We find that superoxide and hydroxyl radicals in doublet spin states are stabilized in the pocket due to attractive electrostatic interactions and hydrogen bonding with partially reduced FAD. These finding validate from a molecular dynamics perspective that [FAD •−-HO • 2 ] or [FADH •-O •− 2 ] can be alternative radical pairs at the origin of magnetoreception

Multipolar Force Fields for Amide-I Spectroscopy from Conformational Dynamics of the Alanine-Trimer

The dynamics and spectroscopy of N-methyl-acetamide (NMA) and trialanine in solution is characterized from molecular dynamics (MD) simulations using different energy functions, including a conventional point charge (PC)-based force field, one based on a multipolar (MTP) representation of the electrostatics, and a semiempirical DFT method. For the 1-d infrared spectra, the frequency splitting between the two amide-I groups is 10 cm$^{-1}$ from the PC, 13 cm$^{-1}$ from the MTP, and 47 cm$^{-1}$ from SCC-DFTB simulations, compared with 25 cm$^{-1}$ from experiment. The frequency trajectory required for determining the frequency fluctuation correlation function (FFCF) is determined from individual (INM) and full normal mode (FNM) analyses of the amide-I vibrations. The spectroscopy, time-zero magnitude of the FFCF $C(t=0)$, and the static component $\Delta_0^2$ from simulations using MTP and analysis based on FNM are all consistent with experiments for (Ala)$_3$. Contrary to that, for the analysis excluding mode-mode coupling (INM) the FFCF decays to zero too rapidly and for simulations with a PC-based force field the $\Delta_0^2$ is too small by a factor of two compared with experiments. Simulations with SCC-DFTB agree better with experiment for these observables than those from PC-based simulations. The conformational ensemble sampled from simulations using PCs is consistent with the literature , whereas that covered by the MTP-based simulations is dominated by P$_{\rm II}$ which agrees with and confirms recently reported, Bayesian-refined populations based on 1-dimensional infrared experiments. Full normal mode analysis together with a MTP representation provides a meaningful model to correctly describe the dynamics of hydrated trialanine

Insights into Serotonin-Receptor Binding and Stability via Molecular Dynamics Simulations: Key Residues for Electrostatic Interactions and Signal Transduction

Serotonin-receptor binding plays a key role in several neurological and biological processes, including mood, sleep, hunger, cognition, learning, and memory. In this article, we performed molecular dynamics simulation to examine the key residues that play an essential role in the binding of serotonin to the G-protein-coupled 5-HT$_{1B}$ receptor (5HT$_{1B}$R) via electrostatic interactions. Key residues for electrostatic interactions were identified via bond distance analysis and frustration analysis method. An end-point free energy calculation method determines the stability of the 5-HT$_{1B}$R due to serotonin binding. The single-point mutation of the polar/charged amino acid residues (Asp129, Thr134) on the binding sites and the calculation of binding free energy validates the quantitative contribution of these residues in the stability of the serotonin-receptor complex. The principal component analysis reflects that the serotonin-bound 5-HT$_{1B}$R is more stabilized than the apo-receptor regarding dynamical changes. The difference dynamic cross-correlations map shows the correlation between the transmembranes and mini-G$_{o}$, which indicates that the signal transduction happens between mini-G$_{o}$ and the receptor. Allosteric pathway analysis reveals the key nodes for signal transduction in 5-HT$_{1B}$R. These results provide useful insights into the study of signal transduction pathways and mutagenesis to regulate the functionality of the complex. The developed protocols can be applied to study local non-covalent interactions and long-range allosteric communications in any protein-ligand system for computer-aided drug design

Electronic substitution effect on the ground and excited state properties of indole chromophore: A computational study

Indole, being the main chromophore of amino acid tryptophan and several other biologically relevant molecules like serotonin, melatonin, has prompted considerable theoretical and experimental interest. The current work focuses on the investigation of photophysical and photochemical properties of indole and indole derivatives e.g. tryptophan, serotonin and melatonin using theoretical and computational methodologies. Having three close-lying excited electronic states, the vibronic coupling effect becomes extremely important yet challenging for the photophysics and photochemistry of indole. Here, we have used density functional theory (DFT) extensively and evaluated the performance of DFT in compared to available experimental and ab initio results from literature. The benchmarking of the method is followed by investigation of the chemical and geometrical effects of ring substitution in indole. A bathochromic shift has been observed in the HOMO-LUMO gap as well as vertical excitation energy from indole to melatonin. While the contribution of the in-plane small adjacent groups increases the electron density of the indole ring, the out-of-plane long substituent groups have minor effect. The comparison of singlet-triplet gaps suggests highest probability of inter-system crossing for tryptophan which is in line with previous experiment. The absorption spectra calculated including the vibronic coupling are in good agreement with experiment. These results can be used to estimate the error in photophysical observables of indole derivatives calculated considering indole as prototypical system. This study also demonstrates the merits and demerits of using DFT functionals to compute the photophysical properties of indole derivatives

Vibrational Stark spectroscopy for assessing ligand-binding strengths in a protein

Nitrile groups are potentially useful spectroscopic probes in the infrared to characterize the binding and dynamics of ligands in proteins. This opens the possibility of locating and determining the binding mode of suitably labelled ligands in proteins based on optical spectroscopy, without the need for determining an X-ray structure. However, relating structure and spectroscopy requires means to accurately compute infrared spectra. This is investigated for benzonitrile (PhCN) in water, wild type (WT) and two lysozyme mutants in solution. The force field is validated by comparing with experimental data for benzonitrile in water which is the basis for computing the Stark shift and time scale for spectral diffusion of PhCN in WT and the L99A and L99G mutants of T4 lysozyme. The 1-d spectra for PhCN in WT and the two mutant proteins differ in their maximum absorption by up to 4 cm −1 , which reflects the modified electrostatic environments in the three proteins. It is also tested whether extending from 1-d to 2-d infrared spectroscopy provides further discrimination in the ligand-binding modes. First, for PhCN in solution the frequency fluctuation correlation function (FFCF) decays to zero at short times whereas in the protein a pronounced static inhomogeneous component is found. Secondly, the decay time of the FFCF for the mutant to which PhCN binds most strongly has the longest decay time. It is demonstrated explicitly that the ligand-binding free energy with respect to the three protein variants correlates with the Stark shift. This makes 1-d infrared spectroscopy together with computations a valuable tool for characterizing binding modes and potentially binding locations in proteins

Solvent Composition Drives the Rebinding Kinetics of Nitric Oxide to Microperoxidase

Abstract The rebinding kinetics of NO after photodissociation from microperoxidase (Mp-9) is studied in different solvent environments. In mixed glycerol/water (G/W) mixtures the dissociating ligand rebinds with a yield close to 1 due to the cavities formed by the solvent whereas in pure water the ligand can diffuse into the solvent after photodissociation. In the G/W mixture, only geminate rebinding on the sub-picosecond and 5 ps time scales was found and the rebinding fraction is unity which compares well with available experiments. Contrary to that, simulations in pure water find two time scales – ~10 ps and ~200 ps - indicating that both, geminate rebinding and rebinding after diffusion of NO in the surrounding water contribute. The rebinding fraction is around 0.63 within 1 ns which is in stark contrast with experiment. Including ions (Na and Cl) at 0.15 M concentration in water leads to rebinding kinetics tending to that in the glycerol/water mixture and yields agreement with experiments. The effect of temperature is also probed and found to be non-negligible. The present simulations suggest that NO rebinding in Mp is primarily driven by thermal fluctuations which is consistent with recent resonance Raman spectroscopy experiments and simulations on MbNO

Theoretical insights into the formation and stability of radical oxygen species in cryptochromes

WOS:000474599300024Cryptochrome is a blue-light absorbing flavoprotein containing a flavin adenine dinucleotide (FAD) cofactor. FAD can accept up to two electrons and two protons, which can be subsequently transferred to substrates present in the binding pocket. It is well known that reactive oxygen species are generated when triplet molecular oxygen is present in the cavity. Here, we investigate the formation and stability of radical oxygen species in Drosophila melanogaster cryptochrome using molecular dynamics simulations and electronic structure calculations. We find that the superoxide and hydroxyl radicals in doublet spin states are stabilized in the pocket due to the attractive electrostatic interactions and hydrogen bonding with partially reduced FAD. These findings validate from a molecular dynamics perspective that [FAD(-)-HO2] or [FADH-O-2(-)] can be alternative radical pairs at the origin of magnetoreception

Radical cation transfer in a guanine pair: an insight to the G-quadruplex structure role using constrained DFT/MM

International audienceDNA G-Quadruplex are highly sensitive to oxidation as their structures include π-stacked guanine quartets allowing fast hole transfer between the nucleobases. These transfers can be described using vertical energy gap and electronic coupling between the different diabatic states at play in a guanine pair. Using classical molecular dynamics simulation and the constrained DFT/MM implementation in deMon2k, we determine these quantity for all the interacting guanine pairs of six G-quadruplex structures including one to four quartets and corresponding to different DNA folding. We then described an uni-directional transfer within a quartet, with high electronic coupling and vertical energy gap values, which contrasts with the hole transfer between πstacked guanine, bi-directional and corresponding to smaller charge transfer parameters. The influence of the geometrical parameters on the electronic coupling is explored, while the external or internal position of the guanine may impact its oxidation probability according to the vertical energy gaps

A Computational Study on Light-Induced Spin Crossover in [Fe(Tp)(CN)3]-2 in Search of Potential Building Block for Single-Molecule Magnet

Light-induced spin crossover (LISCO) in transition metal complexes has drawn attention to the researcher due to its various application in science and technologies. The interplay of LISCO with single molecular magnetism (SMM) is interesting in view of its application towards photoregulated storage devices, magnetic photoswitches. Herein, we have studied the interplay between LISCO and SMM of a Fe(II) complex ([Fe(Tp)(CN)3]-2) which is an ultimate miniature of potential building block of SMM using density functional theory (DFT) and time-dependent DFT method. The molecular structure and energy in low-spin singlet, high-spin quintet as well as intermediate spin triplet is calculated. It is found that the molecule is stable in its LS state but can undergo spin crossover upon irradiation of UV-vis light via triplet excited states. The singlet excited states are close-lying, forming a band structure. The detailed mechanism of LISCO is proposed based on the calculated potential energy cuts and spin-orbit coupling values. While the LS state of the complex has Ms=0 and diamagnetic, the HS state has Ms=±2 and paramagnetic. The calculations suggest a positive zero field splitting parameter and a reasonably small E/D value with a high magnetic relaxation barrier of 96 cm-1. Therefore, for a good SMM, the complex has to be trapped in its HS state after the SCO and reverse spin-crossover (rSCO) has to be stopped. On the other hand, the complex can be used as photoregulated magnetic switch if both the SCO and rSCO happens at the similar time scale

Free energy simulations for protein ligand binding and stability

We summarize several computational techniques to determine relative free energies for condensed-phase systems. The focus is on practical considerations which are capable of making direct contact with experiments. Particular applications include the thermodynamic stability of apo- and holo-myoglobin, insulin dimerization free energy, ligand binding in lysozyme, and ligand diffusion in globular proteins. In addition to provide differential free energies between neighboring states, converged umbrella sampling simulations provide insight into migration barriers and ligand dissociation barriers and analysis of the trajectories yield additional insight into the structural dynamics of fundamental processes. Also, such simulations are useful tools to quantify relative stability changes for situations where experiments are difficult. This is illustrated for NO-bound myoglobin. For the dissociation of benzonitrile from lysozyme it is found that long umbrella sampling simulations are required to approximately converge the free energy profile. Then, however, the resulting differential free energy between the bound and unbound state is in good agreement with estimates from molecular mechanics with generalized Born surface area simulations. Furthermore, comparing the barrier height for ligand escape suggests that ligand dissociation contains a non-equilibrium component