Calculation of chromophore excited state energy shifts in response to molecular dynamics of pigment-protein complexes

The absorption and energy transfer properties of photosynthetic pigments are strongly influenced by their local environment or “site.” Local electrostatic fields vary in time with protein and chromophore molecular movement and thus transiently influence the excited state transition properties of individual chromophores. Site-specific information is experimentally inaccessible in many light-harvesting pigment–proteins due to multiple chromophores with overlapping spectra. Full quantum mechanical calculations of each chromophores excited state properties are too computationally demanding to efficiently calculate the changing excitation energies along a molecular dynamics trajectory in a pigment–protein complex. A simplified calculation of electrostatic interactions with each chromophores ground to excited state transition, the so-called charge density coupling (CDC) for site energy, CDC, has previously been developed to address this problem. We compared CDC to more rigorous quantum chemical calculations to determine its accuracy in computing excited state energy shifts and their fluctuations within a molecular dynamics simulation of the bacteriochlorophyll containing light-harvesting Fenna–Mathews–Olson (FMO) protein. In most cases CDC calculations differed from quantum mechanical (QM) calculations in predicting both excited state energy and its fluctuations. The discrepancies arose from the inability of CDC to account for the differing effects of charge on ground and excited state electron orbitals. Results of our study show that QM calculations are indispensible for site energy computations and the quantification of contributions from different parts of the system to the overall site energy shift. We suggest an extension of QM/MM methodology of site energy shift calculations capable of accounting for long-range electrostatic potential contributions from the whole system, including solvent and ions.     

Molecular dynamics study of early picosecond events in the bacteriorhodopsin photocycle: dielectric response, vibrational cooling and the J, K intermediates.

Molecular dynamics simulations have been carried out to study the J625 and K590 intermediates of bacteriorhodopsin's (bRs) photocycle starting from a refined structure of bR568. The coupling between the electronic states of retinal and the protein matrix is characterized by the energy difference delta E(t) between the excited state and the ground state to which the protein contributes through the Coulomb interaction. Our simulations indicate that the J625 intermediate is related to a polarization of the protein matrix due to the brief (200 fs) change of retinal's charge distribution in going to the excited state and back to the ground state, and that the rise time of the K590 intermediate is determined by vibrational cooling of retinal.     

Mechanisms for Benzene Dissociation through the Excited State of T4 Lysozyme L99A Mutant

The atomic-level mechanisms that coordinate ligand release from protein pockets are only known for a handful of proteins. Here, we report results from accelerated molecular dynamics simulations for benzene dissociation from the buried cavity of the T4 lysozyme Leu99Ala mutant (L99A). In these simulations, benzene is released through a previously characterized, sparsely populated room-temperature excited state of the mutant, explaining the coincidence for experimentally measured benzene off rate and apo protein slow-timescale NMR relaxation rates between ground and excited states. The path observed for benzene egress is a multistep ligand migration from the buried cavity to ultimate release through an opening between the F/G-, H-, and I-helices and requires a number of cooperative multiresidue and secondary-structure rearrangements within the C-terminal domain of L99A. These rearrangements are identical to those observed along the ground state to excited state transitions characterized by molecular dynamic simulations run on the Anton supercomputer. Analyses of the molecular properties of the residues lining the egress path suggest that protein surface electrostatic potential may play a role in the release mechanism. Simulations of wild-type T4 lysozyme also reveal that benzene-egress-associated dynamics in the L99A mutant are potentially exaggerations of the substrate-processivity-related dynamics of the wild type.     

Photochemical Reaction Dynamics of the Primary Event of Vision Studied by Means of a Hybrid Molecular Simulation

The photoisomerization reaction dynamics of a retinal chromophore in the visual receptor rhodopsin was investigated by means of hybrid quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) simulations. The photoisomerization reaction of retinal constitutes the primary step of vision and is known as one of the fastest reactions in nature. To elucidate the molecular mechanism of the high efficiency of the reaction, we carried out hybrid ab initio QM/MM MD simulations of the complete reaction process from the vertically excited state to the photoproduct via electronic transition in the entire chromophore-protein complex. An ensemble of reaction trajectories reveal that the excited-state dynamics is dynamically homogeneous and synchronous even in the presence of thermal fluctuation of the protein, giving rise to the very fast formation of the photoproduct. The synchronous nature of the reaction dynamics in rhodopsin is found to originate from weak perturbation of the protein surroundings and from dynamic regulation of volume-conserving motions of the chromophore. The simulations also provide a detailed view of time-dependent modulations of hydrogen-out-of-plane vibrations during the reaction process, and identify molecular motions underlying the experimentally observed dynamic spectral modulations.     

Solvent-assisted excited state proton transfer and photoacidity of 2-hydroxypyridine. A nonadiabatic dynamics study

Reactions involving proton transfer, especially those taking place in excited states (ESPT), received considerable attention. These reactions underlie many biological and biochemical processes that are vital to life. DNA mutations excited state funnelling and transport phenomena are just examples. Among many molecules undergoing this type of photoreactions, phenols show a unique property, the so-called photoacidity. In the present communication, solvent (methanol and ammonia) assisted excited state proton transfer and photo acidity of 2-hydroxypyridine (2HP) are investigated at the DFT M06-2X / def2pvv level of theory. Excited states of 2HP and its complexes with methanol and ammonia were examined at two levels. First, sampling a Wigner distribution of 300 points, the photoabsorption spectra were simulated within the nuclear ensemble approximation. Second, simulation of the dynamics of the excited states with the surface hopping method. Several separate spectral windows with three hundred trajectories each, were considered. Lifetime of excited states and photochemical channels observed were discussed.     

Electronic excited states and excitation transfer kinetics in the Fenna-Matthews-Olson protein of the photosynthetic bacterium Prosthecochloris aestuarii at low temperatures

The molecular structure-function relationship of the Fenna-Matthews-Olson light-harvesting complex of the photosynthetic green bacterium Prosthecochloris aestuarii has been investigated. It has been assumed that the electronic excited states responsible for the function (transfer of electronic excitation energy) result from the dipole-dipole interactions between the bacteriochlorophyll molecules bound to the polypeptide chain of the complex at a specific three-dimensional geometry. The molecular structure-electronic excited states relationship has been addressed on the basis of simultaneous simulations of several spectroscopic observations. Current electronic excited state models for the Fenna-Matthews-Olson complex have generally been based on obtaining an optimal match between the information contents of the optical steady-state spectra and the bacteriochlorophyll organization. Recent kinetic and spectral information gathered from ultrafast time-resolved measurements have not yet been used effectively for further refinement of the excited state models and for quantification of the relation between the excited states and the energy transfer processes. In this study, we have searched for a model that not only can explain the key features of several steady-state spectra but also the temporal and spectral evolution observed in a recent absorption difference experiment and we have discussed the implications of this model for equilibration of the electronic excitation energy in systems at low temperatures. Received: 12 June 1998 / Revised version: 19 October 1998 / Accepted: 30 November 1998     

How environment supports a state: molecular dynamics simulations of two states in bacteriorhodopsin suggest lipid and water compensation

The light-driven proton pump bacteriorhodopsin (bR) is a transmembrane protein that uses large conformational changes for proton transfer from the cytoplasmic to the extracellular regions. Crystal structures, due to their solvent conditions, do not resolve the effect of lipid molecules on these protein conformational changes. To begin to understand the molecular details behind such large conformational changes, we simulated two conformations of wild-type bacteriorhodopsin, one of the dark-adapted state and the second of an intermediate (M(O)) state, each within an explicit dimyristoyl-phosphatidylcholine (DMPC) lipid bilayer. The simulations included all-hydrogen and all-atom representations of protein, lipid, and water and were performed for 20 ns. We investigate the equilibrium properties and the dynamic motions of the two conformations in the lipid setting. We note that the conformational state of the M(O) intermediate bR remains markedly different from the dark-adapted bR state in that the M(O) intermediate shows rearrangement of the cytoplasmic portions of helices C, F, and G, and nearby loops. This difference in the states remained throughout the simulations, and the results are stable on the molecular dynamics timescale and provide an illustration of the changes in both lipid and water that help to stabilize a particular state. Our analysis focuses on how the environment adjusts to these two states and on how the dynamics of the helices, loops, and water molecules can be related to the pump mechanism of bacteriorhodopsin. For example, water generally behaves in the same manner on the extracellular sides of both simulations but is decreased in the cytoplasmic region of the M(O) intermediate. We suspect that the different water behavior is closely related to the fluctuations of microcavities volume in the protein interior, which is strongly coupled to the collective motion of the protein. Our simulation result suggests that experimental observation can be useful to verify a decreased number of waters in the cytoplasmic regions of the late-intermediate stages by measuring the rate of water exchange with the interior of the protein.     

Concerted motions in copper plastocyanin and azurin: an essential dynamics study

Essential dynamics analysis of molecular dynamics simulation trajectories (1.1 ns) of two copper containing electron transfer proteins, plastocyanin and azurin, has been performed. The protein essential modes have been analysed in order to identify large concerted motions which could be relevant for the electron transfer function exerted by these proteins. The analysis, conducted for temporal windows of different lengths along the protein trajectories, shows a rapid convergence and indicates that for both the proteins the predominant internal motions occur in a subspace of only a few degrees of freedom. Moreover, it is found that for both the proteins the likely binding sites (i.e. the hydrophobic and negative patches) with the reaction partners move in a concerted fashion with a few structural regions far from the active site. Such results are discussed in connection with the possible involvement of large concerted motions in the recognition and binding interaction with physiological electron transfer partners.     

Conformational dynamics of a multidomain protein by neutron scattering and computational analysis

The flexible conformations of a multidomain protein are responsible for its biological functions. Although MurD, a 47-kDa protein that consists of three domains, sequentially changes its domain conformation from an open form to a closed form through a semiclosed form in its enzymatic reaction, the domain dynamics in each conformation remains unclear. In this study, we verify the conformational dynamics of MurD in the corresponding three states (apo and ATP- and inhibitor-bound states) with a combination of small-angle x-ray and neutron scattering (SAXS and SANS), dynamic light scattering (DLS), neutron backscattering (NBS), neutron spin echo (NSE) spectroscopy, and molecular dynamics (MD) simulations. Applying principal component analysis of the MD trajectories, twisting and open-closed domain modes are identified as the major collective coordinates. The deviations of the experimental SAXS profiles from the theoretical calculations based on the known crystal structures become smaller in the ATP-bound state than in the apo state, and a further decrease is evident upon inhibitor binding. These results suggest that domain motions of the protein are suppressed step by step of each ligand binding. The DLS and NBS data yield collective and self-translational diffusion constants, respectively, and we used them to extract collective domain motions in nanometer and nanosecond scales from the NSE data. In the apo state, MurD shows both twisting and open-closed domain modes, whereas an ATP binding suppresses twisting domain motions, and a further reduction of open-closed mode is seen in the inhibitor-binding state. These observations are consistent with the structure modifications measured by the small-angle scattering as well as the MD simulations. Such changes in the domain dynamics associated with the sequential enzymatic reactions should be related to the affinity and reaction efficiency with a ligand that binds specifically to each reaction state.     

Observation of terahertz vibrations in Pyrococcus furiosus rubredoxin via impulsive coherent vibrational spectroscopy and nuclear resonance vibrational spectroscopy--interpretation by molecular mechanics

We have used impulsive coherent vibrational spectroscopy (ICVS) to study the Fe(S-Cys)(4) site in oxidized rubredoxin (Rd) from Pyrococcus furiosus (Pf). In this experiment, a 15 fs visible laser pulse is used to coherently pump the sample to an excited electronic state, and a second <10 fs pulse is used to probe the change in transmission as a function of the time delay. PfRd was observed to relax to the ground state by a single exponential decay with time constants of approximately 255-275 fs. Superimposed on this relaxation are oscillations caused by coherent excitation of vibrational modes in both excited and ground electronic states. Fourier transformation reveals the frequencies of these modes. The strongest ICV mode with 570 nm excitation is the symmetric Fe-S stretching mode near 310 cm(-1), compared to 313 cm(-1) in the low temperature resonance Raman. If the rubredoxin is pumped at 520 nm, a set of strong bands occurs between 20 and 110 cm(-1). Finally, there is a mode at approximately 500 cm(-1) which is similar to features near 508 cm(-1) in blue Cu proteins that have been attributed to excited state vibrations. Normal mode analysis using 488 protein atoms and 558 waters gave calculated spectra that are in good agreement with previous nuclear resonance vibrational spectra (NRVS) results. The lowest frequency normal modes are identified as collective motions of the entire protein or large segments of polypeptide. Motion in these modes may affect the polar environment of the redox site and thus tune the electron transfer functions in rubredoxins.     

Protein dynamics: From the native to the unfolded state and back again

Simulations to study protein unfolding and folding were performed. The unfolding simulations make use of molecular dynamics and treat an atomic model of barnase in aqueous solvent. The cooperative nature of the unfolding transition and the important role of water are described. The folding simulations are based on a bead model of the protein on a cubic lattice. It is shown for the 27-mer model that a large energy gap between the lowest energy (native) state and the excited states is a necessary and sufficient condition for fast folding.     

Interdomain dynamics and ligand binding: molecular dynamics simulations of glutamine binding protein

Periplasmic binding proteins from Gram-negative bacteria possess a common architecture, comprised of two domains linked by a hinge region, a fold which they share with the neurotransmitter-binding domains of ionotropic glutamate receptors (GluRs). Glutamine-binding protein (GlnBP) is one such protein, whose crystal structure has been solved in both open and closed forms. Multi-nanosecond molecular dynamics simulations have been used to explore motions about the hinge region and how they are altered by ligand binding. Glutamine binding is seen to significantly reduce inter-domain motions about the hinge region. Essential dynamics analysis of inter-domain motion revealed the presence of both hinge-bending and twisting motions, as has been reported for a related sugar-binding protein. Significantly, the influence of the ligand on GlnBP dynamics is similar to that previously observed in simulations of rat glutamate receptor (GluR2) ligand-binding domain. The essential dynamics analysis of GlnBP also revealed a third class of motion which suggests a mechanism for signal transmission in GluRs.     

Excited state charge-transfer dynamics study of poplar plastocyanin by ultrafast pump-probe spectroscopy and molecular dynamics simulation

We have applied ultrafast pump-probe spectroscopy to investigate the excited state dynamics of the blue copper protein poplar plastocyanin, by exciting in the blue side of its 600-nm absorption band. The decay of the charge-transfer excited state occurs exponentially with a time constant of approximately 280 fs and is modulated by well visible oscillations. The Fourier transform of the oscillatory component, besides providing most of the vibrational modes found by conventional resonance Raman, presents additional bands in the low frequency region modes, which are reminiscent of collective motions of biological relevance. Notably, a high frequency mode at approximately 508 cm(-1), whose dynamics are consistent with that of the excited state and already observed for other blue copper proteins, is shown to be present also in poplar plastocyanin. This vibrational mode is reproduced by a molecular dynamics simulation involving the excited state of the copper site.     

Molecular dynamics simulations of hemoglobin A in different states and bound to DPG: effector-linked perturbation of tertiary conformations and HbA concerted dynamics

Recent functional studies reported on human adult hemoglobin (HbA) show that heterotropic effector-linked tertiary structural changes are primarily responsible for modulating the oxygen affinity of hemoglobin. We present the results of 6-ns molecular dynamics simulations performed to gain insights into the dynamical and structural details of these effector-linked tertiary changes. All-atom simulations were carried out on a series of models generated for T- and R-state HbA, and for 2,3-diphosphoglycerate-bound models. Cross-correlation analyses identify both intra- and intersubunit correlated motions that are perturbed by the presence of the effector. Principal components analysis was used to decompose the covariance matrix extracted from the simulations and reconstruct the trajectories along the principal coordinates representative of functionally important collective motions. It is found that HbA in both quaternary states exists as ensembles of tertiary conformations that introduce dynamic heterogeneity in the protein. 2,3-Diphosphoglycerate induces significant perturbations in the fluctuations of both HbA states that translate into the protein visiting different tertiary conformations within each quaternary state. The analysis reveals that the presence of the effector affects the most important components of HbA motions and that heterotropic effectors modify the overall dynamics of the quaternary equilibrium via tertiary changes occurring in regions where conserved functionally significant residues are located, namely in the loop regions between helices C and E, E and F, and F and G, and in concerted helix motions. The changes are not apparent when comparing the available x-ray crystal structures in the presence and absence of effector, but are striking when comparing the respective dynamic tertiary conformations of the R and T tetramers.     

Flexible fitting of atomic structures into electron microscopy maps using molecular dynamics

A novel method to flexibly fit atomic structures into electron microscopy (EM) maps using molecular dynamics simulations is presented. The simulations incorporate the EM data as an external potential added to the molecular dynamics force field, allowing all internal features present in the EM map to be used in the fitting process, while the model remains fully flexible and stereochemically correct. The molecular dynamics flexible fitting (MDFF) method is validated for available crystal structures of protein and RNA in different conformations; measures to assess and monitor the fitting process are introduced. The MDFF method is then used to obtain high-resolution structures of the E. coli ribosome in different functional states imaged by cryo-EM.     

Molecular dynamics simulation and essential dynamics study of mutated plastocyanin: structural, dynamical and functional effects of a disulfide bridge insertion at the protein surface

A molecular dynamics simulation (1.1 ns) at 300 K, of fully hydrated Ile21Cys, Glu25Cys plastocyanin mutant has been performed to investigate the structural, dynamical and functional effects of a disulfide bridge insertion at the surface of the protein. A detailed analysis of the root mean square fluctuations, H-bonding pattern and dynamical cross-correlation map has been performed. An essential dynamics method has also been applied as complementary analysis to identify concerted motions (essential modes), that could be relevant to the electron transfer function. The results have been compared with those previously obtained for wild-type plastocyanin and have revealed that the mutant shows a different pattern of H-bonds, with several interactions lost and a higher flexibility, especially around the electron transfer copper site. The analysis of dynamical cross-correlation map and of essential modes, has shown that the mutant performs different functional concerted motions, which might be related to the binding recognition with its electron transfer partners in comparison with the wild-type protein.     

Evidence of protein collective motions on the picosecond timescale

We investigate the presence of structural collective motions on a picosecond timescale for the heme protein, cytochrome c, as a function of oxidation and hydration, using terahertz (THz) time domain spectroscopy and molecular dynamics simulations. The THz response dramatically increases with oxidation, with the largest increase for lowest hydrations, and highest frequencies. For both oxidation states the THz response rapidly increases with hydration saturating above ∼25% (g H₂O/g protein). Quasiharmonic vibrational modes and dipole-dipole correlation functions were calculated from molecular dynamics trajectories. The collective mode density of states alone reproduces the measured hydration dependence, providing strong evidence of the existence of these motions. The large oxidation dependence is reproduced only by the dipole-dipole correlation function, indicating the contrast arises from diffusive motions consistent with structural changes occurring in the vicinity of buried internal water molecules. This source for the observed oxidation dependence is consistent with the lack of an oxidation dependence in nuclear resonant vibrational spectroscopy measurements.     

Resolving the excited state equilibrium of peridinin in solution

The carotenoid peridinin is abundant in the biosphere, as it is the main pigment bound by the light-harvesting complexes of dinoflagellates, where it collects blue and green sunlight and transfers energy to chlorophyll a with high efficiency. Its molecular structure is particularly complex, giving rise to an intricate excited state manifold, which includes a state with charge-transfer character. To disentangle the excited states of peridinin and understand their function in vivo, we applied dispersed pump-probe and pump-dump-probe spectroscopy. The preferential depletion of population from the intramolecular charge transfer state by the dump pulse demonstrates that the S(1) and this charge transfer state are distinct entities. The ensuing dump-induced dynamics illustrates the equilibration of the two states which occurs on the time scale of a few picoseconds. Additionally, the dump pulse populates a short-lived ground state intermediate, which is suggestive of a complex relaxation pathway, probably including structural reorientation or solvation of the ground state. These findings indicate that the unique intramolecular charge transfer state of peridinin is an efficient energy donor to chlorophyll a in the peridinin-chlorophyll-protein complex and thus plays a significant role in global light harvesting.     

Population shuffling between ground and high energy excited states

Stochastic processes powered by thermal energy lead to protein motions traversing time-scales from picoseconds to seconds. Fundamental to protein functionality is the utilization of these dynamics for tasks such as catalysis, folding, and allostery. A hierarchy of motion is hypothesized to connect and synergize fast and slow dynamics toward performing these essential activities. Population shuffling predicts a “top-down” temporal hierarchy, where slow time-scale conformational interconversion leads to a shuffling of the free energy landscape for fast time-scale events. Until now, population shuffling was only applied to interconverting ground states. Here, we extend the framework of population shuffling to be applicable for a system interconverting between low energy ground and high energy excited states, such as the SH3 domain mutants G48M and A39V/N53P/V55L from the Fyn tyrosine kinase, providing another tool for accessing the structural dynamics of high energy excited states. Our results indicate that the higher energy gauche− rotameric state for the leucine χ₂ dihedral angle contributes significantly to the distribution of rotameric states in both the major and minor forms of the SH3 domain. These findings are corroborated with unrestrained molecular dynamics (MD) simulations on both the major and minor states of the SH3 domain demonstrating high correlations between experimental and back-calculated leucine χ₂ rotameric populations. Taken together, we demonstrate how fast time-scale rotameric side-chain population distributions can be extracted from slow time-scale conformational exchange data further extending the scope and the applicability of the population shuffling model.