Structure and energy landscape of a photoswitchable peptide: a replica exchange molecular dynamics study

A replica exchange molecular dynamics (REMD) simulation of a bicyclic azobenzene peptide in explicit dimethyl sulfoxide solution is presented in order to characterize the conformational structures and energy landscape of a photoswitchable peptide. It is shown that an enhanced-sampling technique such as the REMD method is essential to obtain a converged conformational sampling of the peptide at room temperature. This is because conventional MD simulations of less than approximately 100-ns length are either trapped in local minima (at 295 K) or-if run at high temperature-do not resemble the room-temperature REMD results. Calculating various nuclear Overhauser effects (NOEs) and (3)J-couplings, a good overall agreement between the REMD simulations and the NMR experiments of Renner et al. (Biopolymers 2000;54:501-514) is found. In particular, the REMD study confirms the general picture drawn by Renner et al. that the trans-isomer of the azobenzene peptide exhibits a well-defined structure, while the cis-isomer is a conformational heterogeneous system; that is, the trans-isomer occurs in 2 well-defined conformers, while the cis-isomer represents an energetically frustrated system that leads to an ensemble of conformational structures. Employing a principal component analysis of the REMD data, the free energy landscape of the systems is studied at various temperatures. The implications for the folding and unfolding pathways of the system are discussed.     

Picosecond dynamics of bacteriorhodopsin, probed by time-resolved infrared spectroscopy.

The photoinduced reaction cycle of bacteriorhodopsin (BR) has been studied by means of a recently developed picosecond infrared spectroscopic method at ambient temperature. BR - K difference spectra between 1560 and 1700 cm-1 have been recorded at delay times from 100 ps to 14 ns. The spectrum remains unchanged during this period. The negative difference OD band at 1660 cm-1 indicates the peptide backbone responds within 50 ps. A survey in the region of carboxylic side chain absorption around 1740 cm-1 reveals that perturbations of those groups, present in low-temperature FTIR spectra, are not observable within 10 ns, suggesting a slow conformational change.     

Combined use of molecular dynamics simulations and NMR to explore peptide bond isomerization and multiple intramolecular hydrogen-bonding possibilities in a cyclic pentapeptide, cyclo(Gly-Pro-D-Phe-Gly-Val).

The conformational behavior of a model cyclic pentapeptide--cyclo(Gly-L-Pro-D-Phe-Gly-L-Val)--has been explored through the combined use of in vacuo molecular dynamics simulations and a range of nmr experiments (preceding paper). The molecular dynamics analysis suggests that, despite the conformational constraints imposed by formation of the pentapeptide cycle, this pentapeptide undergoes conformational transitions between various hydrogen-bonded conformations, characterized by low energy barriers. An inverse gamma turn with Pro in position i + 1 and a gamma turn with D-Phe in position i + 1 are two alternatives occurring frequently. Like other DLDDL cyclic pentapeptides, cyclo(Gly-Pro-D-Phe-Gly-Val) is also stabilized by an inverse gamma-turn structure with the beta-branched Val residue in position i + 1, and this hydrogen bond is retained in the different conformational families. The gamma-turn around D-Phe3 and the inverse gamma turn around Val5 are consistent with the nmr observations. 3JNH-CH alpha coupling constants of the all-trans forms were calculated from one of the molecular dynamics trajectories and are comparable to nmr experimental data, suggesting that the conformational states visited during the simulation are representative of the conformational distribution in solution. In addition to the equilibrium among various hydrogen-bonded all-trans conformers, the observation in nmr spectra of two sets of resonances for all peptide protons indicated a slow conformational interconversion of the Gly-Pro peptide bond between trans and cis isomers. The activation energy between these two conformers was determined experimentally by magnetization transfer and was calculated by high temperature constrained molecular dynamics simulation. Both methods yield a free energy of activation of ca. 20 kcal/mol. Furthermore, the free energy of activation is dependent on the direction of rotation of the Gly-Pro peptide bond.     

Conformational sampling using high-temperature molecular dynamics

High-temperature molecular dynamics as a method for conformational search was explored on the antigen combining site of McPC 603, a phosphorylcholine binding immunoglobulin. Simulations at temperatures of 500, 800, and 1500 K were run for 111.5, 101.7, and 76.3 ps, respectively. The effectiveness of the search was assessed using a variety of methods. For the shorter hypervariable loops, molecular dynamics explored an appreciable fraction of the conformational space as evidenced by a comparison to a simple theoretical model of the size of the conformational space. However, for the longer loops and the antigen combining site as a whole, the simulation times were too short for a complete search. The simulations at 500 and 800 K both generated conformations that minimized to energies 200 kcal/mole lower than the crystal structure. However, the 1500 K simulation produced higher energy structures, even after minimization; in addition, this highest temperature run had many cis-trans peptide isomerizations. This suggests that 1500 K is too high a temperature for unconstrained conformational sampling. Comparison of the results of high temperature molecular dynamics with a direct conformational search method, [R. E. Bruccoleri & M. Karplus (1987) Biopolymers 26, 137-168]. showed that the two methods did not overlap much in conformational space. Simple geometric measures of the conformational space indicated that the direct method covered more space than molecular dynamics at the lower temperature, but not at 1500 K. The results suggest that high-temperature molecular dynamics can aid in conformational searches.     

Structure and dynamics of interfacial water in an Lalpha phase lipid bilayer from molecular dynamics simulations

Based on molecular dynamics simulations, an analysis of structure and dynamics is performed on interfacial water at a liquid crystalline dipalmitoylphosphatidycholine/water system. Water properties relevant for understanding NMR relaxation are emphasized. The first and second rank orientational order parameters of the water O-H bonds were calculated, where the second rank order parameter is in agreement with experimental determined quadrupolar splittings. Also, two different interfacial water regions (bound water regions) are revealed with respect to different signs of the second rank order parameter. The water reorientation correlation function reveals a mixture of fast and slow decaying parts. The fast (ps) part of the correlation function is due to local anisotropic water reorientation whereas the much slower part is due to more complicated processes including lateral diffusion along the interface and chemical exchange between free and bound water molecules. The 100-ns-long molecular dynamics simulation at constant pressure (1 atm) and at a temperature of 50 degrees C of 64 lipid molecules and 64 x 23 water molecules lack a slow water reorientation correlation component in the ns time scale. The (2)H(2)O powder spectrum of the dipalmitoylphosphatidycholine/water system is narrow and consequently, the NMR relaxation time T(2) is too short compared to experimental results.     

Accessible conformations of melanin-concentrating hormone: a molecular dynamics approach

Molecular dynamics simulations have been used to search for the accessible conformations of the melanin-concentrating hormone (MCH). The studies have been performed on native MCH and two of its peptide fragments, a cyclic MCH(5-14) fragment and a linear MCH(5-14) fragment. An analysis of the molecular dynamics trajectories of the three peptides indicates that two regions of the peptide have characteristic conformational properties that may be important for the biological activity. One is a region around Gly8, which is conformationally mobile, and the other is around Pro13, which shows unusual rigidity. The molecular dynamics simulation results are discussed in terms of backbone structural features like beta turns, side-chain interactions, and orientations of the disulfide bridge. The results of this analysis are used to suggest new analogues that will modify the conformational features of the peptide and further define the conformational requirements for activity. Finally, the results are related to nmr studies of the peptide and reveal agreements between the experimental nuclear Overhauser effect constraints and some of the accessible conformations obtained from the simulation.     

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.     

Molecular dynamics of myoglobin at 298 degrees K. Results from a 300-ps computer simulation.

We have carried out a very long (300 ps) molecular dynamics simulation of the protein myoglobin. This trajectory is approximately three times longer than the longest previous molecular dynamics simulation of a protein, and ten times longer than protein simulations of comparable size (1,423 atoms in our model). Here we report results from this long simulation concerning the average structure, the mean square fluctuations of atoms about the average structure, and the nuclear magnetic resonance order parameters for various groups in myoglobin. The results demonstrate that the average coordinates change very slowly during the simulation. The relative atomic mobilities are well described by the simulation. For both the mean square atomic fluctuations and the order parameters, however, there are significant quantitative differences when values calculated using shorter portions of the trajectory are compared with results obtained for the entire 300-ps simulation. The implications of this result for obtaining converged properties from protein molecular dynamics simulations for comparison with experiment are discussed.     

Molecular dynamics simulation of dark-adapted rhodopsin and free opsin

Molecular dynamics simulation was carried out for the rhodopsin protein to investigate its conformational changes in respect to inclusion of 11-cis retinal chromophore. Molecular dynamics calculations were performed within the time frame 3000 ps. Totally, 3 X 10(6) configurations ofrhodopsin and free opsin were analyzed and compared. It has been shown that the 11-cis retinal rearrangement (adaptation) in opsin strongly affects the surrounding amino acid residues of protein binding pocket and the protein cytoplasmic region. The extracellular part, however, shows comparatively little changes. On basis of the simulation results obtained we propose a molecular mechanism for the rhodopsin protein function as a G-protein-coupled receptor in the state of darkness. We discuss the role of the retinal chromophore as a ligand-antagonist stabilizing the inactive conformation of rhodopsin.     

Solution conformation of a cyclic neurokinin antagonist: A nmr and molecular dynamics study

The solution structure of a hexapeptide, cyclo(Gln-Trp-Phe-Gly-Leu-Met), which is a selective NK-2 antagonist, has been studied by a combination of two-dimensional nmr and molecular dynamics (MD) techniques. The simulation based on nmr and MD data resulted in the convergence to a family of structures. Free molecular dynamics for 50 ps in the presence of DMSO solvent molecules shows that the structure is energetically stable. One intramolecular hydrogen bond between the amide proton of Gin and the carbonyl oxygen of Gly was revealed. This result is consistent with the results from the measurement of the temperature coefficient of the amide protons. The extent of intermolecular hydrogen bonding between the amide protons of the peptide and DMSO was also revealed by the free MD simulation. The resulting structure of the cyclic peptide contains a variation type I′ β-turn in the Gly-Leu-Met-Gln segment. Comparison of the structure of this peptide with that of other NK-2 antagonist cyclic hexapeptides was made, and the activity of cyclic antagonists appears to be inversely related to the conformational rigidity of the cyclic peptides. © 1994 John Wiley & Sons, Inc.     

Molecular dynamics decomposition of temperature-dependent elastic neutron scattering by a protein solution

Molecular dynamics simulations are performed of bovine pancreatic trypsin inhibitor in a cryosolution over a range of temperatures from 80 to 300 K and the origins identified of elastic dynamic neutron scattering from the solution. The elastic scattering and mean-square displacement calculated from the molecular dynamics trajectories are in reasonable agreement with experiments on a larger protein in the same solvent. The solvent and protein contributions to the scattering from the simulation model are determined. At lower temperatures (< approximately 200 K) or on shorter timescales ( approximately 10 ps) the scattering contributions are proportional to the isotopic nuclear scattering cross-sections of each component. However, for T > 200 K marked deviations from these cross-sections are seen due to differences in the dynamics of the components of the solution. Rapid activation of solvent diffusion leads to the variation with temperature of the total elastic intensity being determined largely by that of the solvent. At higher temperatures (>240 K) and longer times ( approximately 100 ps) the protein makes the only significant contribution to the scattering, the solvent scattering having moved out of the accessible time-space window. Decomposition of the protein mean-square displacement shows that the observed dynamical transition in the solution at 200-220 K involves activation of both internal motions and external whole-molecule rotational and translational diffusion. The proportion that the external dynamics contributes to the protein mean-square displacement increases to approximately 30 and 60% at 300 K on the 10- and 100-ps timescales, respectively.     

Conformation of a neurokinin antagonist in solution. 2D NMR and restrained molecular dynamics study.

The hexapeptide [cyclo(Leu1 psi(CH2NH2)Leu2-Gln3-Trp4-Phe5-Gly6)]+1 is a potent antagonist of neurokinin A activity in tissues of hamster urinary bladder. The solution conformation of this cyclic hexapeptide has been characterized by the combined use of two dimensional nuclear magnetic resonance spectroscopy and restrained molecular dynamics. The proton spectrum of the peptide was fully assigned by the sequential assignment procedure. Interproton distances were derived from crosspeak volumes in two dimensional Nuclear Overhauser Effect spectra, and dihedral angles were calculated from appropriate coupling constants. Temperature coefficients of the amide protons were determined. Restrained molecular dynamics simulations were carried out using the backbone interproton distances as constraints. During 210 ps of restrained molecular dynamics the peptide interconverted among three closely related families of conformations. These interconversions occurred at picosecond timescales under the simulation conditions.     

Myoglobin-CO conformational substate dynamics: 2D vibrational echoes and MD simulations

Two-dimensional (2D) infrared vibrational echoes were performed on horse heart carbonmonoxymyoglobin (MbCO) in water over a range of temperatures. The A(1) and A(3) conformational substates of MbCO are found to have different dephasing rates with different temperature dependences. A frequency-frequency correlation function derived from molecular dynamics simulations on MbCO at 298 K is used to calculate the vibrational echo decay. The calculated decay shows substantial agreement with the experimentally measured decays. The 2D vibrational echo probes protein dynamics and provides an observable that can be used to test structural assignments for the MbCO conformational substates.     

Synthesis and application of an azobenzene amino acid as a light-switchable turn element in polypeptides

The synthesis of an azobenzene amino acid (aa) for use as a photo-inducible conformational switch in polypeptides is described. The compound can be easily incorporated into an aa sequence by solid-phase peptide synthesis using standard 9-fluorenylmethoxycarbonyl methods. A reversible conformational change of the peptide backbone is induced by switching between the cis and trans configurations of the azobenzene moiety by irradiation with light of suitable wavelength. Thermal cis --> trans isomerization of this azobenzene aa is slow, enabling detailed structural investigations of the modified peptides, e.g., using NMR techniques. The total time for the synthesis of the photoswitch is typically 4 d, with an overall yield of 40-50%.     

Multiple loop conformations of peptides predicted by molecular dynamics simulations are compatible with nuclear magnetic resonance

The affinity and selectivity of protein-protein interactions can be fine-tuned by varying the size, flexibility, and amino acid composition of involved surface loops. As a model for such surface loops, we study the conformational landscape of an octapeptide, whose flexibility is chemically steered by a covalent ring closure integrating an azobenzene dye into and by a disulfide bridge additionally constraining the peptide backbone. Because the covalently integrated azobenzene dyes can be switched by light between a bent cis state and an elongated trans state, six cyclic peptide models of strongly different flexibilities are obtained. The conformational states of these peptide models are sampled by NMR and by unconstrained molecular dynamics (MD) simulations. Prototypical conformations and the free-energy landscapes in the high-dimensional space spanned by the phi/psi angles at the peptide backbone are obtained by clustering techniques from the MD trajectories. Multiple open-loop conformations are shown to be predicted by MD particularly in the very flexible cases and are shown to comply with the NMR data despite the fact that such open-loop conformations are missing in the refined NMR structures.     

Molecular dynamics simulation of melittin in a dimyristoylphosphatidylcholine bilayer membrane.

Molecular dynamics trajectories of melittin in an explicit dimyristoyl phosphatidylcholine (DMPC) bilayer are generated to study the details of lipid-protein interactions at the microscopic level. Melittin, a small amphipathic peptide found in bee venom, is known to have a pronounced effect on the lysis of membranes. The peptide is initially set parallel to the membrane-solution interfacial region in an alpha-helical conformation with unprotonated N-terminus. Solid-state nuclear magnetic resonance (NMR) and polarized attenuated total internal reflectance Fourier transform infrared (PATIR-FTIR) properties of melittin are calculated from the trajectory to characterize the orientation of the peptide relative to the bilayer. The residue Lys7 located in the hydrophobic moiety of the helix and residues Lys23, Arg24, Gln25, and Gln26 at the C-terminus hydrophilic form hydrogen bonds with water molecules and with the ester carbonyl groups of the lipids, suggesting their important contribution to the stability of the helix in the bilayer. Lipid acyl chains are closely packed around melittin, contributing to the stable association with the membrane. Calculated density profiles and order parameters of the lipid acyl chains averaged over the molecular dynamics trajectory indicate that melittin has effects on both layers of the membrane. The presence of melittin in the upper layer causes a local thinning of the bilayer that favors the penetration of water through the lower layer. The energetic factors involved in the association of melittin at the membrane surface are characterized using an implicit mean-field model in which the membrane and the surrounding solvent are represented as structureless continuum dielectric material. The results obtained by solving the Poisson-Bolztmann equation numerically are in qualitative agreement with the detailed dynamics. The influence of the protonation state of the N-terminus of melittin is examined. After 600 ps, the N-terminus of melittin is protonated and the trajectory is continued for 400 ps, which leads to an important penetration of water molecules into the bilayer. These observations provide insights into how melittin interacts with membranes and the mechanism by which it enhances their lysis.     

Locally accessible conformations of proteins: multiple molecular dynamics simulations of crambin.

Multiple molecular dynamics (MD) simulations of crambin with different initial atomic velocities are used to sample conformations in the vicinity of the native structure. Individual trajectories of length up to 5 ns sample only a fraction of the conformational distribution generated by ten independent 120 ps trajectories at 300 K. The backbone atom conformational space distribution is analyzed using principal components analysis (PCA). Four different major conformational regions are found. In general, a trajectory samples only one region and few transitions between the regions are observed. Consequently, the averages of structural and dynamic properties over the ten trajectories differ significantly from those obtained from individual trajectories. The nature of the conformational sampling has important consequences for the utilization of MD simulations for a wide range of problems, such as comparisons with X-ray or NMR data. The overall average structure is significantly closer to the X-ray structure than any of the individual trajectory average structures. The high frequency (less than 10 ps) atomic fluctuations from the ten trajectories tend to be similar, but the lower frequency (100 ps) motions are different. To improve conformational sampling in molecular dynamics simulations of proteins, as in nucleic acids, multiple trajectories with different initial conditions should be used rather than a single long trajectory.