Modeling of the structure of bacteriorhodopsin. A molecular dynamics study.

Secondary structure predictions for membrane proteins are relatively reliable and permit the construction of model structures that may serve as initial conformations for molecular dynamics simulations. This might provide a scheme to predict the three-dimensional structures of membrane proteins. The feasibility of such an approach is tested for bacteriorhodopsin. We were not able to fully predict the kidney-shaped structure of bacteriorhodopsin. However, features compatible with this structure developed in a simulation starting from a circular arrangement of the seven predicted helices. When instead we started from the kidney shape, assigning the seven predicted helices in different ways to those on the structure, we could distinguish between the different assignments on the basis of energy and tilt of the helices. In this way we could select the correct assignment from a few others. For the correct assignment, the helices spontaneously adopted a tilt that agrees remarkably well with the experimental model structure derived by others. The root-mean-square deviation between our best molecular dynamics structure and the experimental model structure is 3.8 A, caused mainly by deviations in the internal degrees of freedom of the helices.     

Distal residue-CO interaction in carbonmonoxy myoglobins: a molecular dynamics study of three distal mutants.

Six 90-ps molecular dynamics trajectories, two for each of three distal mutants of sperm whale carbonmonoxy myoglobin, are reported; solvent waters within 16 A of the active site have been included. In both His64GIn trajectories, the distal side chain remains part of the heme pocket, forming a "closed" conformation similar to that of the wild type 64N delta H tautomer. Despite a connectivity more closely resembling the N epsilon H histidine tautomer, close interactions with the carbonyl ligand similar to those observed for the wild type 64N epsilon H tautomer are prevented in this mutant by repulsive interactions between the carbonyl O and the 64O epsilon. The aliphatic distal side chain of the His64Leu mutant shows little interaction with the carbonyl ligand in either His64Leu trajectory. Solvent water molecules move into and out of the active site in the His64Gly mutant trajectories; during all the other carbonmonoxy myoglobin trajectories, including the wild type distal tautomers considered in an earlier work, solvent molecules rarely encroach closer than 6 A of the active site. These results are consistent with a recent structural interpretation of the wild type infrared spectrum, and the current reinterpretation that the distal-ligand interaction in carbonmonoxy myoglobin is largely electrostatic, not steric, in nature.     

Thermodynamic stability of water molecules in the bacteriorhodopsin proton channel: a molecular dynamics free energy perturbation study.

The proton transfer activity of the light-driven proton pump, bacteriorhodopsin (bR) in the photochemical cycle might imply internal water molecules. The free energy of inserting water molecules in specific sites along the bR transmembrane channel has been calculated using molecular dynamics simulations based on a microscopic model. The existence of internal hydration is related to the free energy change on transfer of a water molecule from bulk solvent into a specific binding site. Thermodynamic integration and perturbation methods were used to calculate free energies of hydration for each hydrated model from molecular dynamics simulations of the creation of water molecules into specific protein-binding sites. A rigorous statistical mechanical formulation allowing the calculation of the free energy of transfer of water molecules from the bulk to a protein cavity is used to estimate the probabilities of occupancy in the putative bR proton channel. The channel contains a region lined primarily by nonpolar side-chains. Nevertheless, the results indicate that the transfer of four water molecules from bulk water to this apparently hydrophobic region is thermodynamically permitted. The column forms a continuous hydrogen-bonded chain over 12 A between a proton donor, Asp 96, and the retinal Schiff base acceptor. The presence of two water molecules in direct hydrogen-bonding association with the Schiff base is found to be strongly favorable thermodynamically. The implications of these results for the mechanism of proton transfer in bR are discussed.     

Immunoglobulin kappa light chain and its amyloidogenic mutants: a molecular dynamics study

AL amyloidosis and LCDD are pathological conditions caused by extracellural deposition of monoclonal Ig light chain variable domains. In the former case, deposits have a form of amyloid fibrils, in the latter, amorphous aggregates. 1REI kappa light chain variable domain and its two point mutants, R61N and D82I, were chosen for the analysis in this work. Wild 1REI does not create deposits in vitro, while R61N aggregates as amyloid fibrils and D82I creates amorphous aggregates. Both mutated residues create a conserved salt bridge; thus, substitution of any of them should decrease V(L) domain stability. For these three proteins, 5 ns MD simulations were conducted in temperatures of 300 K and 400 K, with protonated and unprotonated acidic residues, mimicking acidic and neutral experimental pH conditions (3 sets: N300, N400, and A400). The analysis of trajectories focused on characterization of changes in conformational behavior and stability of Ig kappa light chain variable domain caused by single aminoacid substitutions that were experimentally proved to enhance aggregation propensity, both in the form of amyloid and amorphous aggregates. Residue D82 turns out to be involved not only in R61-D82 but also in K45-D82 interaction, which was not observed in the X-ray structure, but frequently populated simulations of 1REI. The substitution D82I excludes both interactions, resulting in substantial destabilization (i.e., easier aggregation). Examination of behavior of edge regions of V(L) beta-sandwich reveals significant alterations in D82I mutant compared to wild 1REI, while relatively small changes occur in R61N. This suggests that mild and slow destabilization is the reason of the conversion of V(L) to partially folded amyloidogenic intermediate structure.     

pK(a) calculations along a bacteriorhodopsin molecular dynamics trajectory

Electrostatic calculations of pK(a-values) are reported along a 400 ps molecular dynamics trajectory of bacteriorhodopsin. The sensitivity of calculated pK(a) values to a number of structural factors and factors related to the modelling of the electrostatics are also studied. The results are very sensitive to the choice of internal dielectric constant of the protein (in the interval 2-4). Moreover it is important to include internal water molecules and to average over a long enough portion ( approximately 100 ps) of an equilibrium molecular dynamics trajectory. The internal waters are necessary to get an ion-counter ion complex with the Schiff base and Arg 82 protonated and the aspartic groups (85 and 212) deprotonated. The fluctuations along the MD-trajectory do not change the protonation state of internal residues at neutral pH. However, at other pH values the averaging along a trajectory maybe crucial to get correct protonation states. A relationship is found between the arginine group 82, the aspartic group 85 and the glutamate group 204. Glu 204 is protonated in the ground state but the pK(a) value decreases towards deprotonation when the chromophore isomerizes into the cis state.     

Excited-state dynamics of bacteriorhodopsin.

Near infrared emission of bacteriorhodopsin at neutral pH and at room temperature was characterized by a large Stokes shift. This characteristic was lost in an acidic pH (approximately pH 2) where a remarkable enchancement (more than 10 times) in the fluorescence quantum yield accompanied the red shift in the main absorption band. It is suggested from fluorescence polarization measurements that the emission occurs from the first allowed excited state of the retinylidene chromophore, irrespective of pH. We suggest that the large Stokes shift observed at neutral pH is a result of a charge displacement (e.g., proton translocation) that occurs immediately after excitation, and is prevented by protonation (in the ground state) of an amino-acid residue in the protein.     

The stability of transmembrane helices: A molecular dynamics study on the isolated helices of bacteriorhodopsin

Bacteriorhodopsin (bR) continues to be a proven testing ground for the study of integral membrane proteins (IMPs). It is important to study the stability of the individual helices of bR, as they are postulated to exist as independently stable transmembrane helices (TMHs) and also for their utility as templates for modeling other IMPs with the postulated seven-helix bundle topology. Toward this purpose, the seven helices of bR have been studied by molecular dynamics simulation in this study. The suitability of using the backbone-dependent rotamer library of side-chain conformations arrived at from the data base of globular protein structures in the case TMHs has been tested by another set of 7 helix simulations with the side-chain orientations taken from this library. The influence of the residue's net charge on the helix stability was examined by simulating the helices III, IV, and VI (from both of the above sets of helices) with zero net charge on the side chains. The results of these 20 simulations demonstrate in general the stability of the isolated helices of bR in conformity with the two-stage hypothesis of IMP folding. However, the helices I, II, V, and VII are more stable than the other three helices. The helical nature of certain regions of III, IV, and VI are influenced by factors such as the net charge and orientation of several residues. It is seen that the residues Arg, Lys, Asp, and Glu (charged residues), and Ser, Thr, Gly, and Pro, play a crucial role in the stability of the helices of bR. The backbone-dependent rotamer library for the side chains is found to be suitable for the study of TMHs in IMP. © 1996 John Wiley & Sons, Inc.     

Molecular mechanisms of adefovir sensitivity and resistance in HBV polymerase mutants: a molecular dynamics study

Molecular modeling studies of adefovir diphosphate with the wild type and the mutant HBV polymerase-DNA complex demonstrated that the increase in adefovir sensitivity toward HBV polymerase mutants (rtL180M, rtM204V/I, rtL180M-M204V/I) is a result of increased van der Waals interaction and is supplemented by the decreased affinity of natural substrate toward the mutant HBV polymerase. In the case of rtN236T mutant, loss of two hydrogen bonds accompanied by significant decrease in electrostatic interactions is observed, which explains the observed decrease in drug sensitivity and binding affinity of adefovir diphosphate toward the rtN236T mutant HBV polymerase.     

Molecular dynamics study of the M412 intermediate of bacteriorhodopsin.

Molecular dynamics simulations have been carried out to study the M412 intermediate of bacteriorhodopsin's (bR) photocycle. The simulations start from two simulated structures for the L550 intermediate of the photocycle, one involving a 13-cis retinal with strong torsions, the other a 13,14-dicis retinal, from which the M412 intermediate is initiated through proton transfer to Asp-85. The simulations are based on a refined structure of bR568 obtained through all-atom molecular dynamics simulations and placement of 16 waters inside the protein. The structures of the L550 intermediates were obtained through simulated photoisomerization and subsequent molecular dynamics, and simulated annealing. Our simulations reveal that the M412 intermediate actually comprises a series of conformations involving 1) a motion of retinal; 2) protein conformational changes; and 3) diffusion and reconfiguration of water in the space between the retinal Schiff base nitrogen and the Asp-96 side group. (1) turns the retinal Schiff base nitrogen from an early orientation toward Asp-85 to a late orientation toward Asp-96; (2) disconnects the hydrogen bond network between retinal and Asp-85 and tilts the helix F of bR, enlarging bR's cytoplasmic channel; (3) adds two water molecules to the three water molecules existing in the cytoplasmic channel at the bR568 stage and forms a proton conduction pathway. The conformational change (2) of the protein involves a 60 degrees bent of the cytoplasmic side of helix F and is induced through a break of a hydrogen bond between Tyr-185 and a water-side group complex in the counterion region.     

Principles of carbopeptoid folding: a molecular dynamics simulation study.

The conformational spaces of five oligomers of tetrahydrofuran-based carbopeptoids in chloroform and dimethyl sulfoxide were investigated through nine molecular dynamics simulations. Prompted by nuclear magnetic resonance experiments that indicated various stable folds for some but not all of these carbopeptoids, their folding behaviour was investigated as a function of stereochemistry, chain length and solvent. The conformational distributions of these molecules were analysed in terms of occurrence of hydrogen bonds, backbone torsional-angle distributions, conformational clustering and solute configurational entropy. While a cis-linkage across the tetrahydrofuran ring favours right-handed helical structures, a trans-linkage results in a larger conformational variability. Intra-solute hydrogen bonding is reduced with increasing chain length and with increasing solvent polarity. Solute configurational entropies confirm the picture obtained: they are smaller for cis- than for trans-linked peptides, for chloroform than for dimethyl sulfoxide as solvent and for shorter peptide chains. The simulations provide an atomic picture of molecular conformational variability that is consistent with the available experimental data.     

Structural stability of disulfide mutants of basic pancreatic trypsin inhibitor: A molecular dynamics study

The structure and folding of basic pancreatic trypsin inhibitor (BPTI) has been studied extensively by experimental means. We report a computer simulation study of the structural stability of various disulfide mutants of BPTI, involving eight 250-psec molecular dynamics simulations of the proteins in water, with and without a phosphate counterion. The presence of the latter alters the relative stability of the single disulfide species [5–55] and [30–51]. This conclusion can explain results of mutational studies and the conservation of residues in homologues of BPTI, and suggests a possible role of ions in stabilizing one intermediate over another in unfolding or folding processes. © 1996 Wiley-Liss, Inc.     

Conformational dynamics of RNA-peptide binding: a molecular dynamics simulation study

Molecular dynamics simulations of the binding of the heterochiral tripeptide KkN to the transactivation responsive (TAR) RNA of HIV-1 is presented, using an all-atom force field with explicit water. To obtain starting structures for the TAR-KkN complex, semirigid docking calculations were performed that employ an NMR structure of free TAR RNA. The molecular dynamics simulations show that the starting structures in which KkN binds to the major groove of TAR (as it is the case for the Tat-TAR complex of HIV-1) are unstable. On the other hand, the minor-groove starting structures are found to lead to several binding modes, which are stabilized by a complex interplay of stacking, hydrogen bonding, and electrostatic interactions. Although the ligand does not occupy the binding position of Tat protein, it is shown to hinder the interhelical motion of free TAR RNA. The latter is presumably necessary to achieve the conformational change of TAR RNA to bind Tat protein. Considering the time evolution of the trajectories, the binding process is found to be ligand-induced and cooperative. That is, the conformational rearrangement only occurs in the presence of the ligand and the concerted motion of the ligand and a large part of the RNA binding site is necessary to achieve the final low-energy binding state.     

Vasopressin conformational fluctuations: a molecular dynamics study

Molecular dynamics simulations have been used to study the conformational fluctuations of the oligopeptide hormone vasopressin. Starting coordinates for these simulations were built upon the crystal structure of pressinoic acid, the cyclic ring moiety of vasopressin, recently determined by x-ray diffraction. Coordinates for the additional tripeptide “tail” of vasopressin were selected by arbitrary positioning of this segment using interactive computer graphics. Two such starting configurations were minimized to relax strains, and long dynamics simulations (20 and 40 ps) in vacuo were then conducted following extensive heating and equilibration sequences (36 ps). In these studies, vasopressin was found to undergo few substantial conformational changes at 300 K on the time scale simulated, in contrast to the results of a shorter previous simulation, but comparable structural transitions were observed during the equilibration periods. The pressinoic acid structure was found to be a reasonably stable possible conformation for vasopressin in vacuum on this time scale.     

Cholesterol-sphingomyelin interactions: a molecular dynamics simulation study

Stearoylsphingomyelin (SSM) bilayers containing 0, 22, and 50 mol % cholesterol (Chol) and a pentadecanoyl-stearoylphosphatidylcholine (15SPC) bilayer containing 22 mol % Chol were molecular dynamics simulated at two temperatures (37 degrees C and 60 degrees C). 15SPC is the best PC equivalent of SSM. The Chol effect on the SSM bilayer differs significantly from that on the 15SPC bilayer. At the same temperature and Chol content, H-bonding of Chol with SSM is more extensive than with 15SPC. SSM-Chol H-bonding anchors the OH group of Chol in the lower regions of the SSM-Chol bilayer interface. Such a location strengthens the influence of Chol on the SSM chains. In effect, the phase of the SSM-Chol bilayer containing 22 mol % Chol at 37 degrees C is shifted from the gel to the liquid-ordered phase, and the bilayer displays similar properties below and above the main phase-transition temperature for a pure SSM bilayer of approximately 45 degrees C. In contrast, due to a higher location, Chol is not able to change the phase of the 15SPC-Chol bilayer, which at 37 degrees C remains in the gel phase. Chol affects both the core and interface of the SSM bilayer. With increasing Chol content, the order of SSM chains and hydration of SSM headgroups increase, whereas polar interactions between lipids decrease.     

Electrooptical measurements on purple membrane containing bacteriorhodopsin mutants.

Electrooptical measurements on purple membrane containing the wild-type and 10 different bacteriorhodopsin mutants have shown that the direction of the permanent electric dipole moment of all these membranes reverses at different pH values in the range 3.2-6.4. The induced dipole moment and the retinal angle exhibit an increased value at these pHs. The results demonstrate that the bacteriorhodopsin protein makes an important contribution to the electrooptical properties of the purple membrane.     

Conformational study of valinomycin: a molecular dynamics approach

Valinomycin is a highly flexible cyclic dodecadepsipeptide that transports ions across membranes. Such a flexibility in the conformation is required for its biological function since it has to encounter a variety of environments and liganding state. Exploration of conformational space of this molecule is therefore important and is one of the objectives of the present study that has been carried out by means of high temperature Molecular Dynamics. Further, the stability of the known bracelet-like structure of the uncomplexed valinomycin and the inherent flexibility around this structure has been investigated. The uncomplexed form of valinomycin has been simulated at 75-100 K for 1 ns in order to elucidate the average conformational properties. An alanine-analog of valinomycin has been simulated under identical conditions in order to evaluate the effect of sidechain on the conformational properties, The studies confirm the effect of sidechain on conformational equilibrium.     

FTIR difference spectroscopy of bacteriorhodopsin: Toward a molecular model

Bacteriorhodopsin (bR) is a light-driven proton pump whose function includes two key membrane-based processes, active transport and energy transduction. Despite extensive research on bR and other membrane proteins, these processes are not fully understood on the molecular level. In the past ten years, the introduction of Fourier transform infrared (FTIR) difference spectroscopy along with related techniques including time-resolved FTIR difference spectroscopy, polarized FTIR, and attenuated total reflection FTIR has provided a new approach for studying these processes. A key step has been the utilization of site-directed mutagenesis to assign bands in the FTIR difference spectrum to the vibrations of individual amino acid residues. On this basis, detailed information has been obtained about structural changes involving the retinylidene chromophore and protein during the bR photocycle. This includes a determination of the protonation state of the four membrane-embedded Asp residues, identification of specific structurally active amino acid residues, and the detection of protein secondary structural changes. This information is being used to develop an increasingly detailed picture of the bR proton pump mechanism.     

Conformation and fluctuations of free stefin B: a molecular dynamics study.

Molecular dynamics study was performed on the cysteine proteinase inhibitor stefin B. Structure of inhibitor from the complex with papain was used as a starting point. Amino terminal "trunk" of the inhibitor which lies extended along the cleft of the enzyme in the complex, folded onto the body of inhibitor during MD simulation, thereby reducing the total and particularly hydrophobic surface exposed to the solvent. This effect counterbalances hydrophobic contribution of the "trunk" and explains why its deletion in stefin B and related inhibitors doesn't reduce the dissociation constant. The rest of stefin B conformation is conserved together with main chain hydrogen bonds. Fluctuations of C alpha atoms resembles crystallographic B factors with exception of residues in contact with enzyme.     

Photoinduced conformational dynamics of a photoswitchable peptide: a nonequilibrium molecular dynamics simulation study

Employing nonequilibrium molecular dynamics simulations, a comprehensive computational study of the photoinduced conformational dynamics of a photoswitchable bicyclic azobenzene octapeptide is presented. The calculation of time-dependent probability distributions along various global and local reaction coordinates reveals that the conformational rearrangement of the peptide is rather complex and occurs on at least four timescales: 1) After photoexcitation, the azobenzene unit of the molecule undergoes nonadiabatic photoisomerization within 0.2 ps. 2) On the picosecond timescale, the cooling (13 ps) and the stretching (14 ps) of the photoexcited peptide is observed. 3) Most reaction coordinates exhibit a 50-100 ps component reflecting a fast conformational rearrangement. 4) The 500-1000 ps component observed in the simulation accounts for the slow diffusion-controlled conformational equilibration of the system. The simulation of the photoinduced molecular processes is in remarkable agreement with time-resolved optical and infrared experiments, although the calculated cooling as well as the initial conformational rearrangements of the peptide appear to be somewhat too slow. Based on an ab initio parameterized vibrational Hamiltonian, the time-dependent amide I frequency shift is calculated. Both intramolecular and solvent-induced contributions to the frequency shift were found to change by < or = 2 cm(-1), in reasonable agreement with experiment. The potential of transient infrared spectra to characterize the conformational dynamics of peptides is discussed in some detail.     

DNA and its counterions: a molecular dynamics study

The behaviour of mobile counterions, Na⁺ and K⁺, was analysed around a B-DNA double helix with the sequence CCATGCGCTGAC in aqueous solution during two 50 ns long molecular dynamics trajectories. The movement of both monovalent ions remains diffusive in the presence of DNA. Ions sample the complete space available during the simulation time, although individual ions sample only about one-third of the simulation box. Ions preferentially sample electronegative sites around DNA, but direct binding to DNA bases remains a rather rare event, with highest site occupancy values of <13%. The location of direct binding sites depends greatly on the nature of the counterion. While Na⁺ binding in both grooves is strongly sequence-dependent with the preferred binding site in the minor groove, K⁺ mainly visits the major groove and binds close to the centre of the oligomer. The electrostatic potential of an average DNA structure therefore cannot account for the ability of a site to bind a given cation; other factors must also play a role. An extensive analysis of the influence of counterions on DNA conformation showed no evidence of minor groove narrowing upon ion binding. A significant difference between the conformations of the double helix in the different simulations can be attributed to extensive α/γ transitions in the phosphate backbone during the simulation with Na⁺. These transitions, with lifetimes over tens of nanoseconds, however, appear to be correlated with ion binding to phosphates. The ion-specific conformational properties of DNA, hitherto largely overlooked, may play an important role in DNA recognition and binding.