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PubMed1999

Molecular dynamics study of substance P peptides in a biphasic membrane mimic.

Two neuropeptides, substance P (SP) and SP-tyrosine-8 (SP-Y8), have been studied by molecular dynamics (MD) simulation in a TIP3P water/CCl4 biphasic solvent system as a mimic for the water-membrane system. Initially, distance restraints derived from NMR nuclear Overhauser enhancements (NOE) were incorporated in the restrained MD (RMD) in the equilibration stage of the simulation. The starting orientation/position of the peptides for the MD simulation was either parallel to the water/CCl4 interface or in a perpendicular/insertion mode. In both cases the peptides equilibrated and adopted a near-parallel orientation within approximately 250 ps. After equilibration, the conformation and orientation of the peptides, the solvation of both the backbone and the side chain of the residues, hydrogen bonding, and the dynamics of the peptides were analyzed from trajectories obtained in the RMD or the subsequent free MD (where the NOE restraints were removed). These analyses showed that the peptide backbone of nearly all residues are either solvated by water or are hydrogen-bonded. This is seen to be an important factor against the insertion mode of interaction. Most of the interactions with the hydrophobic phase come from the hydrophobic interactions of the side chains of Pro-4, Phe-7, Phe-8, Leu-10, and Met-11 for SP, and Phe-7, Leu-10, Met-11 and, to a lesser extent, Tyr-8 in SP-Y8. Concerted conformational transitions took place in the time frame of hundreds of picoseconds. The concertedness of the transition was due to the tendency of the peptide to maintain the necessary secondary structure to position the peptide properly with respect to the water/CCl4 interface.

Amino Acid Sequence↗
PubMed2005

Towards data warehousing and mining of protein unfolding simulation data.

OBJECTIVES: The prediction of protein structure and the precise understanding of protein folding and unfolding processes remains one of the greatest challenges in structural biology and bioinformatics. Computer simulations based on molecular dynamics (MD) are at the forefront of the effort to gain a deeper understanding of these complex processes. Currently, these MD simulations are usually on the order of tens of nanoseconds, generate a large amount of conformational data and are computationally expensive. More and more groups run such simulations and generate a myriad of data, which raises new challenges in managing and analyzing these data. Because the vast range of proteins researchers want to study and simulate, the computational effort needed to generate data, the large data volumes involved, and the different types of analyses scientists need to perform, it is desirable to provide a public repository allowing researchers to pool and share protein unfolding data. METHODS: To adequately organize, manage, and analyze the data generated by unfolding simulation studies, we designed a data warehouse system that is embedded in a grid environment to facilitate the seamless sharing of available computer resources and thus enable many groups to share complex molecular dynamics simulations on a more regular basis. RESULTS: To gain insight into the conformational fluctuations and stability of the monomeric forms of the amyloidogenic protein transthyretin (TTR), molecular dynamics unfolding simulations of the monomer of human TTR have been conducted. Trajectory data and meta-data of the wild-type (WT) protein and the highly amyloidogenic variant L55P-TTR represent the test case for the data warehouse. CONCLUSIONS: Web and grid services, especially pre-defined data mining services that can run on or 'near' the data repository of the data warehouse, are likely to play a pivotal role in the analysis of molecular dynamics unfolding data.

Computational Biology↗
PubMed2003

Molecular dynamics of the long neurotoxin LSIII.

Long neurotoxins bind tightly and specifically to the nicotinic acetylcholine receptor (AChR) in postsynaptic membranes and are useful for exploring the biology of synapses. In crystallographic studies of long neurotoxins the principal binding loop appears disordered, but the NMR solution structure of the long neurotoxin LSIII revealed significant local order, even though the loop is disordered with respect to the globular core. A possible mechanism for conferring global disorder while preserving local order is rigid-body motion of the loop about a hinge region. Here we report investigations of LSIII dynamics based on (13)C(alpha) magnetic relaxation rates and molecular dynamics simulation. The relaxation rates and MD simulation both confirm the hypothesis of rigid-body motion of the loop and place bounds on the extent and time scale of the motion. The bending motion of the loop is slow compared to the rapid fluctuations of individual dihedral angles, reflecting the collective nature and largely entropic free energy profile for hinge bending. The dynamics of the central binding loop in LSIII illustrates two distinct mechanisms by which molecular dynamics directly impacts biological activity. The relative rigidity of key residues involved in recognition at the tip of the central binding loop lowers the otherwise substantial entropic cost of binding. Large excursions of the loop hinge angle may endow the protein with structural plasticity, allowing it to adapt to conformational changes induced in the receptor.

Computer Simulation↗
PubMed2000

Effect of coordinated ions on structure and flexiblity of parallel G-quandruplexes: a molecular dynamics study.

Single tract guanine residues can associate to form stable parallel quadruplex structures in the presence of certain cations. Nanosecond scale molecular dynamics simulations have been performed on fully solvated fibre model of parallel d(G7) quadruplex structures with Na+ or K+ ions coordinated in the cavity formed by the 06 atoms of the guanine bases. The AMBER 4.1 force field and Particle Mesh Ewald technique for electrostatic interactions have been used in all simulations. These quadruplex structures are stable during the simulation, with the middle four base tetrads showing root mean square deviation values between 0.5 to 0.8 A from the initial structure as well the high resolution crystal structure. Even in the absence of any coordinated ion in the initial structure, the G-quadruplex structure remains intact throughout the simulation. During the 1.1 ns MD simulation, one Na+ counter ion from the solvent as well as several water molecules enter the central cavity to occupy the empty coordination sites within the parallel quadruplex and help stabilize the structure. Hydrogen bonding pattern depends on the nature of the coordinated ion, with the G-tetrad undergoing local structural variation to accommodate cations of different sizes. In the absence of any coordinated ion, due to strong mutual repulsion, 06 atoms within G-tetrad are forced farther apart from each other, which leads to a considerably different hydrogen bonding scheme within the G-tetrads and very favourable interaction energy between the guanine bases constituting a G-tetrad. However, a coordinated ion between G-tetrads provides extra stacking energy for the G-tetrads and makes the quadruplex structure more rigid. Na+ ions, within the quadruplex cavity, are more mobile than coordinated K+ ions. A number of hydrogen bonded water molecules are observed within the grooves of all quadruplex structures.

DNA↗
PubMed2004

Molecular dynamics simulations of beta-turn forming tetra- and hexapeptides.

It was previously shown that the structural ensemble of model peptides DDKG and GKDG (H. Ishii et al. Biopolymers 24, 2045-2056, 1985), DEKS (A. Otter et al. J. Biomol. Struct. Dyn. 7, 455-476, 1989) NPGQ (F. R. Carbone et al. Int. J. Pept. Protein. Res. 26, 498-508, 1985), SALN (H. Santa et al. J. Biomol. Struct. Dyn. 16, 1033-1041, 1999), SYPFDV and SYPYDV (J. Yao et al. J. Mol. Biol. 243, 736-753, 1994), VP(D)AH and VP(D)SH (B. Imperiali et al. J. Am. Chem. Soc. 114, 3182-3188, 1992) in solution contains a significant - or in some cases dominant - proportion of beta-turn conformation. In this study, a protein database was searched for the above, unprotected sequences which incorporate only L-amino acid residues. Simulated annealing and 25 ns MD simulations of structures were also performed. The DSSP and STRIDE secondary structure-assigning algorithms and clustering were used to analyze trajectories and i, i+3 hydrogen bonds were also sought. The DSSP analysis showed a fluctuation between beta-turn and random meander structure, although bend structures were not detected because of the insufficient length of peptide chains. This alternating trend was confirmed when the STRIDE algorithm was used to analyze trajectories, but STRIDE assigned more turn structures. The population of the strongest clusters was above 40% and the middle structures adopted beta-turn structure for most sequences. These results are in good agreement with previous experimental results and support the idea of the ultra-marginal stability of turns in the absence of stabilizing long-range interactions of the neighboring segments of a polypeptide chain. However, interactions between the side-chains in tetrapeptides could also contribute to turn stability and result in unusual stability in some cases. Our observations suggest that such interactions are the consequence rather than the driving force of turn formation.

Amino Acid Sequence↗
PubMed2006

Predictive modelling of topology and loop variations in dimeric DNA quadruplex structures.

We have used a combination of simulated annealing (SA), molecular dynamics (MD) and locally enhanced sampling (LES) methods in order to predict the favourable topologies and loop conformations of dimeric DNA quadruplexes with T2 or T3 loops. This follows on from our previous MD simulation studies on the influence of loop lengths on the topology of intramolecular quadruplex structures [P. Hazel et al. (2004) J. Am. Chem. Soc., 126, 16 405-16 415], which provided results consistent with biophysical data. The recent crystal structures of d(G4T3G4)2 and d(G4BrUT2G4) (P. Hazel et al. (2006) J. Am. Chem. Soc., in press) and the NMR-determined topology of d(TG4T2G4T)2 [A.T. Phan et al. (2004) J. Mol. Biol., 338, 93-102] have been used in the present study for comparison with simulation results. These together with MM-PBSA free-energy calculations indicate that lateral T3 loops are favoured over diagonal loops, in accordance with the experimental structures; however, distinct loop conformations have been predicted to be favoured compared to those found experimentally. Several lateral and diagonal loop conformations have been found to be similar in energy. The simulations suggest an explanation for the distinct patterns of observed dimer topology for sequences with T3 and T2 loops, which depend on the loop lengths, rather than only on G-quartet stability.

Computer Simulation↗
PubMed2005

Modified replica exchange simulation methods for local structure refinement.

Parallel tempering, also known as replica exchange molecular dynamics (REMD), has recently been successfully used to study the structure and thermodynamic properties of biomolecules such as peptides and small proteins. For large systems, however, applying REMD can be costly since the number of replicas needed increases as the square root of the number of degrees of freedom in the system. Often, enhanced sampling is only needed for a subset of atoms, such as a loop region of a large protein or a small ligand binding to a receptor. In such applications, it is often reasonable to assume a weak dependence of the structure of the larger region on the instantaneous conformation of the smaller region of interest. For these cases, we derived two variant replica exchange methods, partial replica exchange molecular dynamics (PREMD) and local replica exchange molecular dynamics (LREMD). The Hamiltonian for the system is separated, with replica exchange carried out only for terms involving the subsystem of interest while the remainder of the system is maintained at a single temperature. The number of replicas required for efficient exchange thus depends on the number of degrees of freedom in the fragment needing refinement rather than on the size of the full system. The method can be applied to much larger systems than was previously practical. This also provides a means to preserve the integrity of the structure outside the refinement region without introduction of restraints. LREMD takes this weak coupling approximation a step further, employing only a single representation of the large fragment that simultaneously interacts with all of the replicas of the subsystem of interest. This is obtained by combining replica exchange with the locally enhanced sampling approximation (LES), reducing the computational expense of replica exchange simulations to near that of a single standard molecular dynamics (MD) simulation. Use of LREMD also permits the use of LES without requiring the specification of a single temperature, a known difficulty for standard LES simulations. We tested these two methods on the loop region of an RNA hairpin model system and find significant advantages over standard MD and REMD simulations.

Algorithms↗
PubMed1995

A method for characterizing transition concertedness from polymer dynamics computer simulations.

A statistical method based on classifying the transitions among a set of dihedral angles within an "energy transfer window" is developed, and used to analyze Brownian (BD) and molecular dynamics (MD) simulations of the acyl chains in a lipid bilayer, and MD of neat hexadecane. It is shown for the BD simulation that when a transition of the dihedral angle in the center of the chain occurs, a transition of a particular next nearest neighbor (or angle 2-apart) will follow concertedly with a probability of approximately 0.10 within a time window of approximately 3 ps. The MD bilayer simulations, which are based on a more flexible model of the hydrocarbon chains, yield corresponding concerted transition probabilities of approximately 0.083 and window sizes of 1-2 ps. An analysis of angles 4-apart yields concerted transition probabilities of 0.03 and 0.04 for the BD and MD bilayer simulations, respectively, and window sizes close to those of the corresponding 2-apart cases. Statistical hypothesis testing very strongly rejects the assertion that these follower transitions are occurring at random. Similar analysis reveals marginal or no evidence of concertedness between 1-apart (nearest neighbor) and between 3-apart dihedral angle transitions. The pattern of concertedness for hexadecane is qualitatively similar to that of the lipid chains, although concertedness is somewhat stronger for the 3-apart transitions and somewhat weaker for those 4-apart. Finally, it is suggested that the diffusion of small solute molecules in membranes is better facilitated by nonconcerted transitions, which are associated with relatively large displacements of the chains, than by concerted transitions, which do little to change the chain shape.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1994

Molecular dynamics simulation of the rare amino acid LL-dityrosine and a dityrosine-containing peptide: comparison with time-resolved fluorescence.

The fluorescence of the rare amino acid LL-dityrosine, which is found in insoluble biological materials with structural features, was recently shown to decay non-exponentially (Kungl et al. (1992) J. Fluorescence 2, 63-74). Here we investigated the time-resolved fluorescence of a dityrosine-containing peptide (DCP) to study the influence of side chains on the fluorescence decay of the chromophore. The fluorescence decay of DCP was best fitted by three exponential terms including a sub-nanosecond rise term, the values of which are quite similar to the parameters obtained for the decay of free dityrosine. They were found to depend on the pH of the aqueous solution but not on the temperature. Analysis by an exponential series method revealed broad fluorescence lifetime distributions for DCP. Compared to the corresponding analysis of dityrosine transients, similar lifetime centers were found whereas the widths of the distributions were found broader for DCP. Molecular dyamics (MD) simulations of dityrosine at 300 K show that chi 1 and chi 2 side chain conformers (rotamers) of both tyrosine subunits interconvert on a picosecond timescale. The rates of interconversion were shown to depend critically upon the MD technique applied: in vacuo simulations yielded lower interconversion rates compared to stochastic dynamics (SD) and full MD (water explicitly included). However, MD simulations of the dityrosine-containing peptide revealed no interconversions of the chi 1 and chi 2 side chain rotamers of both tyrosine subunits within a 400 ps trajectory. Interconversions could be induced by raising the temperature of the system (DCP plus solvent) to 340 K. Side chain rotamers of dityrosine are not stable on a fluorescence time scale but are stable when a dityrosine-containing peptide is regarded. Nevertheless both molecules yield similar fluorescence decay patterns. We therefore conclude that the rotamer model proposed for the fluorescence decay of tyrosine and tryptophan cannot be applied to the fluorescence decay of dityrosine and peptides containing this chromophore. This should be of future interest when dityrosine is used as an intrinsic sensor to study complex dityrosine-containing macromolecules by fluorescence spectroscopy.

Molecular Conformation↗
PubMed1998

Diffusion of solvent around biomolecular solutes: a molecular dynamics simulation study.

Effects of the macromolecular solute on the translational mobility of surrounding solvent water, and Na+ and Cl- ions are investigated by molecular dynamics (MD) simulation. Using MD trajectories of myoglobin and d(C5T5) . d(G5A5) DNA decamer of high quality and length, we determine the average diffusion coefficients for all solvent species as a function of distance from the closest solute atom. We examine solvent mobility in the directions parallel and perpendicular to the solute surface and in proximity to three different classes of solute atoms (oxygens, nitrogens, and carbons). The nature and the magnitude of the solute effects on water diffusion appear to be very similar for protein and DNA decamer. The overall diffusion rate at the interface is lower than in the bulk. The rate is higher than the average in the direction parallel to the solute surface, and lower in the direction normal to the surface, up to 15 A away from the solute. The rate is also lower in the solvation shells of the macromolecules, producing characteristic depressions in the radial profiles of the diffusion coefficient that can be correlated with peaks in the corresponding radial distribution functions. The magnitude of these depressions is small compared to the overall change in solvent mobility at the interface. Similar features are observed in the radial profiles of the diffusion coefficient of sodium and chlorine ions as well.

Biophysical Phenomena↗
PubMed2006

Computational sampling of a cryptic drug binding site in a protein receptor: explicit solvent molecular dynamics and inhibitor docking to p38 MAP kinase.

An increasing number of structural studies reveal alternative binding sites in protein receptors that become apparent only when an inhibitor binds, and correct prediction of these situations presents a significant challenge to computer-aided drug design efforts. A striking example is provided by recent crystal structures of the p38 MAP kinase, where a 10A movement of the Phe169 side-chain creates a new binding site adjacent to the ATP binding site that is exploited by the diaryl urea inhibitor BIRB796. Here, we show that this binding site can be successfully and repeatedly identified in explicit-solvent molecular dynamics (MD) simulations of the protein that begin from an unliganded p38 crystal structure. Ligand-docking calculations performed on 5000 different structural snapshots generated during MD indicate that the conformations sampled are often surprisingly competent to bind the inhibitor BIRB796 in the crystallographically correct position and with docked energies that are generally more favorable than those of other positions. Similar docking studies with an ATP-binding site-directed inhibitor suggest that it may be possible to develop hybrid inhibitors that target both the ATP and cryptic binding sites simultaneously. Intriguingly, both inhibitors are occasionally found to dock correctly even with p38's "DFG" motif in the "wrong" conformation and BIRB796 can successfully dock, albeit infrequently, without significant displacement of the Phe169 side-chain; this suggests that the inhibitor might facilitate the latter's conformational change. Finally, two quite different conformations of p38's DFG motif are also sampled for extended periods of time during the simulations; these may provide new opportunities for inhibitor development. The MD simulations reported here, which total 390 ns in length, therefore demonstrate that existing computational methods may be of surprising utility in predicting cryptic binding sites in protein receptors prior to their experimental discovery.

Animals↗
PubMed2006

Hydration, swelling, interlayer structure, and hydrogen bonding in organolayered double hydroxides: insights from molecular dynamics simulation of citrate-intercalated hydrotalcite.

Molecular dynamics (MD) simulation of the Mg/Al (3:1) layered double hydroxide (LDH), hydrotalcite (HT), containing citrate, C6H5O7(3-), as the charge balancing interlayer anion provides new molecular scale insight into the interlayer structure, hydrogen bonding, and energetics of the hydration and consequent swelling of LDH compounds containing organic and biomolecules. Citrate-HT exhibits affinity for water up to very high hydration levels, in contrast to the preferred low hydration states of most LDHs intercalated with small, inorganic anions. This result is consistent with the recent experimental observation of the delamination of lactate-HT. The high water affinity is rationalized in terms of the preference of citrate ion for hydrogen bonds (H-bonds) donated from water molecules rather than from the hydroxyl groups of the metal hydroxide layer and the need to develop an integrated interlayer H-bond network among the citrate ions, water, and -OH groups of the hydroxide layers. The changes in the orientation of citrate molecules with progressive hydration are also intimately related to its preference to accept hydrogen bonds from water.

Journal Article↗
PubMed1997

Molecular dynamics simulations of the docking of substituted N5-deazapterins to dihydrofolate reductase.

Orientations of the deazapterin ring and the conformational preferences of groups appended to the deazapterin ring in a set of 8-substituted deazapterin cations docked into the dihydrofolate reductase (DHFR) binding site have been investigated using a methodology based on the simulated annealing technique within molecular dynamics (MD) simulations. Of five possible binding pockets for the 8-substituents, identified from a preliminary manual docking study, one has been definitively eliminated after an analysis of MD trajectories, while another remains uncertain. Using a new method based on standard thermodynamic cycles and a linear approximation of polar and non-polar free energy contributions from MD averages, binding affinities of the different ligands in each binding site have been correlated with experimental dissociation constants. The study has provided insights into structure-activity relationships for use in the design of modified inhibitors of DHFR.

Binding Sites↗
PubMed2005

Nature of minor-groove binders-DNA complexes in the gas phase.

The structure of noncovalent complexes of DNA duplex with minor groove binders (mG-binders) has been analyzed by state of the art molecular dynamics (MD) simulations. More than 3.3 micros of MD trajectories (including 4 x 0.5 micros trajectories) were collected for the Dickerson's dodecamer bound to DAPI, Hoechst 33258, and Netropsin. Comparison of these trajectories with control simulations in water allowed us to determine that the extreme dehydration and partial neutralization occurring during electrospray experiments does not produce the disruption of the DNA:mG-binder complexes or the dissociation of the two strands of the duplex. Irrespective of the drug and the simulation conditions the mG-binders remains bound to the DNA near the preferential binding position in aqueous conditions. Large distortions appear in the two DNA strands, which maintain however a memory of the original DNA duplex structure in water, and a general helical-like conformation.

Binding Sites↗
PubMed2005

Atomistic-scale simulations of the initial chemical events in the thermal initiation of triacetonetriperoxide.

To study the initial chemical events related to the detonation of triacetonetriperoxide (TATP), we have performed a series of molecular dynamics (MD) simulations. In these simulations we used the ReaxFF reactive force field, which we have extended to reproduce the quantum mechanics (QM)-derived relative energies of the reactants, products, intermediates, and transition states related to the TATP unimolecular decomposition. We find excellent agreement between the QM-predicted reaction products and those observed from 100 independent ReaxFF unimolecular MD cookoff simulations. Furthermore, the primary reaction products and average initiation temperature observed in these 100 independent unimolecular cookoff simulations match closely with those observed from a TATP condensed-phase cookoff simulation, indicating that unimolecular decomposition dominates the thermal initiation of the TATP condensed phase. Our simulations demonstrate that thermal initiation of condensed-phase TATP is entropy-driven (rather than enthalpy-driven), since the initial reaction (which mainly leads to the formation of acetone, O(2), and several unstable C(3)H(6)O(2) isomers) is almost energy-neutral. The O(2) generated in the initiation steps is subsequently utilized in exothermic secondary reactions, leading finally to formation of water and a wide range of small hydrocarbons, acids, aldehydes, ketones, ethers, and alcohols.

Journal Article↗
PubMed2001

Unrestrained 5 ns molecular dynamics simulation of a cisplatin-DNA 1,2-GG adduct provides a rationale for the NMR features and reveals increased conformational flexibility at the platinum binding site.

A 5 ns unrestrained molecular dynamics (MD) simulation of the DNA duplex d(GCCG*G*ATCGC)-d(GCGATCCGGC), bearing a cis-Pt(NH(3))(2)(2+) unit crosslinking the two G* guanine bases, is reported. The MD trajectory was a posteriori correlated with NMR data determined for the same adduct, and it is shown that interproton distances and the characteristic chemical shifts are accounted for by the simulation. The simulation and its confrontation with the NMR data have confirmed the finding derived early from static models that the cytosine complementary to the 5' G*, C17, is mobile with respect to its adjacent bases. However, in contrast to our previous description of this mobility, which included rupture of the Watson-Crick hydrogen bonds and formation of non-Watson-Crick hydrogen bonds, the MD simulation indicated that the G*4-C17 pair moves continuously along a trajectory roughly perpendicular to the local helix axis, with retention of all three Watson-Crick hydrogen bonds. The simulation indicated the reversible formation of a hydrogen bond between the 5' oriented NH(3) ligand of platinum and the C3pG*4 phosphate group, in accord with our former prediction. Furthermore, the simulation has disclosed previously undetected BI <=> BII transitions at the G*5pA6 and A6pT7 steps, connected to formation/rupture of a hydrogen bond between the 3' oriented NH(3) ligand of platinum and the N7 atom of A6. All these conformational equilibria affect the form of the minor groove and increase the conformational flexibility at the platination site, and are thus likely to facilitate recognition by cellular proteins.

Base Sequence↗
PubMed2005

Relating kinetic rates and local energetic roughness by accelerated molecular-dynamics simulations.

We show that our accelerated molecular-dynamics (MD) approach can extend the time scale in all-atom MD simulations of biopolymers. We also show that this technique allows for the kinetic rate information to be recaptured. In deducing the kinetic rates, the relationship between the local energetic roughness of the potential-energy landscape and the effective diffusion coefficient is established. These are demonstrated on a very slow but important biomolecular process: the dynamics of cis-trans-isomerization of Ser-Pro motifs. We do not only recapture the slow kinetic rates, which is difficult in traditional MD, but also obtain the underlying roughness of the energy landscape of proteins at atomistic resolution.

Algorithms↗
PubMed2002

Molecular dynamics simulations of photoactive yellow protein (PYP) in three states of its photocycle: a comparison with X-ray and NMR data and analysis of the effects of Glu46 deprotonation and mutation.

Photoactive yellow protein (PYP) is a prototype of the PAS domain superfamily of signaling proteins. The signaling process is coupled to a three-state photocycle. After the photoinduced trans-cis isomerization of the chromophore, 4-hydroxycinnamic acid (pCA), an early intermediate (pR) is formed, which proceeds to a second intermediate state (pB) on a sub-millisecond time scale. The signaling process is thought to be connected to the conformational changes upon the formation of pB and its recovery to the ground state (pG), but the exact signaling mechanism is not known. Experimental studies of PYP by solution NMR and X-ray crystallography suggest a very flexible protein backbone in the ground as well as in the signaling state. The relaxation from the pR to the pB state is accompanied by the protonation of the chromophore's phenoxyl group. This was found to be of crucial importance for the relaxation process. With the goal of gaining a better understanding of these experimental observations on an atomistic level, we performed five MD simulations on the three different states of PYP: a 1 ns simulation of PYP in its ground state [pG(MD)], a 1 ns simulation of the pR state [pR(MD)], a 2 ns simulation of the pR state with the chromophore protonated (pRprot), a 2 ns simulation of the pR state with Glu46 exchanged by Gln (pRGln) and a 2 ns simulation of PYP in its signaling state [pB(MD)]. Comparison of the pG simulation results with X-ray and NMR data, and with the results obtained for the pB simulation, confirmed the experimental observations of a rather flexible protein backbone and conformational changes during the recovery of the pG from the pB state. The conformational changes in the region around the chromophore pocket in the pR state were found to be crucially dependent on the strength of the Glu46-pCA hydrogen bond, which restricts the mobility of the chromophore in its unprotonated form considerably. Both the mutation of Glu46 with Gln and the protonation of the chromophore weaken this hydrogen bond, leading to an increased mobility of pCA and large structural changes in its surroundings. These changes, however, differ considerably during the pRGln and pRprot simulations, providing an atomistic explanation for the enhancement of the rate constant in the Gln46 mutant.

Bacterial Proteins↗