Bioinformatics. Reality simulation--observe while it happens.
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Amino Acid Motifs↗
Biomedical subjects
H J Berendsen
Publications and source records attributed to H J Berendsen.
DSC studies of Fusarium solani pisi cutinase: consequences for stability in the presence of surfactants.
The application of cutinase from Fusarium solani pisi as a fat-stain removing ingredient in laundry washing is hampered by its lack of stability in the presence of anionic surfactants. We postulate that the stability of cutinase towards anionics can be improved by mutations increasing its temperature stability. Thermal unfolding as measured with DSC, appears to be irreversible, though the thermograms are more symmetric than predicted by a simple irreversible model. In the presence of taurodeoxycholate (TDOC), the unfolding temperature is lower and the unfolding is reversible. We conclude that an early reversible unfolding intermediate exists in which a number of additional hydrophobic patches are exposed to the solvent, or preferentially are covered with TDOC. Improvement of the stability of cutinase with respect to both surfactants and thermal denaturation, should thus be directed toward the prevention of exposure of hydrophobic patches in the early intermediate.
Algorithms↗
Voltage-dependent insertion of alamethicin at phospholipid/water and octane/water interfaces.
Understanding the binding and insertion of peptides in lipid bilayers is a prerequisite for understanding phenomena such as antimicrobial activity and membrane-protein folding. We describe molecular dynamics simulations of the antimicrobial peptide alamethicin in lipid/water and octane/water environments, taking into account an external electric field to mimic the membrane potential. At cis-positive potentials, alamethicin does not insert into a phospholipid bilayer in 10 ns of simulation, due to the slow dynamics of the peptide and lipids. However, in octane N-terminal insertion occurs at field strengths from 0.33 V/nm and higher, in simulations of up to 100 ns duration. Insertion of alamethicin occurs in two steps, corresponding to desolvation of the Gln7 side chain, and the backbone of Aib10 and Gly11. The proline induced helix kink angle does not change significantly during insertion. Polyalanine and alamethicin form stable helices both when inserted in octane and at the water/octane interface, where they partition in the same location. In water, both polyalanine and alamethicin partially unfold in multiple simulations. We present a detailed analysis of the insertion of alamethicin into the octane slab and the influence of the external field on the peptide structure. Our findings give new insight into the mechanism of channel formation by alamethicin and the structure and dynamics of membrane-associated helices.
Alamethicin↗
Towards the design and computational characterization of a membrane protein.
The design of a transmembrane four-helix bundle is described. We start with an idealized four-helix bundle geometry, then use statistical information to build a plausible transmembrane bundle. Appropriate residues are chosen using database knowledge on the sequences of membrane helices and loops, then the packing of the bundle core is optimized, and favorable side chain rotamers from rotamer libraries are selected. Next, we use explicit physical knowledge from biomolecular simulation force fields and molecular dynamics simulations to test whether the designed structure is physically possible. These procedures test whether the designed protein will indeed be alpha-helical, well packed and stable over a time scale of several nanoseconds in a realistic lipid bilayer environment. We then test a modeling approach that does not include sophisticated database knowledge about proteins, but rather relies on applying our knowledge of the physics that governs protein motions. This independent validation of the design is based on simulated annealing and restrained molecular dynamics simulation in vacuo, comparable to procedures used to refine NMR and X-ray structures.
Computer Simulation↗
Collective protein dynamics in relation to function.
Several techniques for the analysis of the internal motions of proteins are available - separating large collective motions from small, presumably uninteresting motions. Such descriptions are helpful in the characterization of internal motions and provide insight into the energy landscape of proteins. The real challenge, however, is to relate large collective motions to functional properties, such as binding and regulation, or to folding. These issues have been recently addressed in several papers.
Allosteric Regulation↗
Computer simulations of the dynamics of human choriogonadotropin and its alpha subunit.
Human choriogonadotropin (hCG) belongs to a family of heterodimeric glycoprotein hormones involved in reproduction. Over 75 ns of molecular dynamics simulations of this heterodimer and the free alpha subunit were performed and validated by experimental information to arrive at a qualitative dynamical description of these molecules. A number of 5-ns simulations at 400 degrees K describe a sufficiently stable heterodimer structure, whereas the free alpha subunit shows the experimentally observed partial unfolding. From the main collective fluctuations of the free alpha subunit, it can be derived that residues alpha35-55 form a domain that is highly flexible with respect to the other domain, which contains all five disulfide bonds. The apparent loss of secondary structure in the region alpha33-58 may very well be induced by this. Dynamic domains can also be determined from the hCG heterodimer simulations. The most important collective mode of motion shows that the flexibility of the alpha subunit is reduced by concerted rotation with both the long loop and the determinant loop of the beta subunit. The motion of the free alpha subunit does not differ significantly from the motion it has in the hCG heterodimer, but the amplitudes along the most important eigenvectors are larger.
Carbohydrates↗
A kinetic model for the internal motions of proteins: diffusion between multiple harmonic wells.
The dynamics of collective protein motions derived from Molecular Dynamics simulations have been studied for two small model proteins: initiation factor I and the B1 domain of Protein G. First, we compared the structural fluctuations, obtained by local harmonic approximations in different energy minima, with the ones revealed by large scale molecular dynamics (MD) simulations. It was found that a limited set of harmonic wells can be used to approximate the configurational fluctuations of these proteins, although any single harmonic approximation cannot properly describe their dynamics. Subsequently, the kinetics of the main (essential) collective protein motions were characterized. A dual-diffusion behavior was observed in which a fast type of diffusion switches to a much slower type in a typical time of about 1-3 ps. From these results, the large backbone conformational fluctuations of a protein may be considered as "hopping" between multiple harmonic wells on a basically flat free energy surface.
Kinetics↗
Conformational changes in the chaperonin GroEL: new insights into the allosteric mechanism.
Conformational changes are known to play a crucial role in the function of the bacterial GroE chaperonin system. Here, results are presented from an essential dynamics analysis of known experimental structures and from computer simulations of GroEL using the CONCOORD method. The results indicate a possible direct form of inter-ring communication associated with internal fluctuations in the nucleotide-binding domains upon nucleotide and GroES binding that are involved in the allosteric mechanism of GroEL. At the level of conformational transitions in entire GroEL rings, nucleotide-induced structural changes were found to be distinct and in principle uncoupled from changes occurring upon GroES binding. However, a coupling is found between nucleotide-induced conformational changes and GroES-mediated transitions, but only in simulations of GroEL double rings, and not in simulations of single rings. This provides another explanation for the fact that GroEL functions a double ring system.
Allosteric Regulation↗
The mechanism of channel formation by alamethicin as viewed by molecular dynamics simulations.
Alamethicin is a 20-residue channel-forming peptide that forms a stable amphipathic alpha-helix in membrane and membrane-mimetic environments. This helix contains a kink induced by a central Gly-X-X-Pro sequence motif. Alamethicin channels are activated by a cis positive transbilayer voltage. Channel activation is suggested to correspond to voltage-induced insertion of alamethicin helices in the bilayer. Alamethicin forms multi-conductance channels in lipid bilayers. These channels are formed by parallel bundles of transmembrane helices surrounding a central pore. A change in the number of helices per bundle switches the single channel conductance level. Molecular dynamics simulations of alamethicin in a number of different environments have been used to explore its channel-forming properties. These simulations include: (i) alamethicin in solution in water and in methanol; (ii) a single alamethicin helix at the surface of a phosphatidylcholine bilayer; (iii) single alamethicin helices spanning a phosphatidylcholine bilayer; and (iv) channels formed by bundles of 5, 6, 7 or 8 alamethicin helices spanning a phosphatidylcholine bilayer. The total simulation time is c. 30 ns. Thus, these simulations provide a set of dynamic snapshots of a possible mechanism of channel formation by this peptide.
Alamethicin↗
Alamethicin helices in a bilayer and in solution: molecular dynamics simulations.
Alamethicin is an alpha-helical channel-forming peptide, which inserts into lipid bilayers in a voltage-dependent, asymmetrical fashion. Nanosecond molecular dynamics simulations have been used to compare alamethicin conformation and dynamics in three different environments: 1) in water; 2) in methanol; and 3) inserted into a lipid (palmitoyl-oleoyl-phosphatidylcholine) bilayer to form a transmembrane helix. In the bilayer and in methanol, there was little change (Calpha RMSD approximately 0.2 nm over 2 ns and 1 ns) from the initial helical conformation of the peptide. In water there were substantial changes (Calpha RMSD approximately 0.4 nm over 1 ns), especially in the C-terminal segment of the peptide, which lost its alpha-helical conformation. In the bilayer and in methanol, the alamethicin molecule underwent hinge-bending motion about its central Gly-X-X-Pro sequence motif. Analysis of H-bonding interactions revealed that the polar C-terminal side chains of alamethicin provided an "anchor" to the bilayer/water interface via formation of multiple H-bonds that persisted throughout the simulation. This explains why the preferred mode of helix insertion into the bilayer is N-terminal, which is believed to underlie the asymmetry of voltage activation of alamethicin channels.
Alamethicin↗
An alamethicin channel in a lipid bilayer: molecular dynamics simulations.
We present the results of 2-ns molecular dynamics (MD) simulations of a hexameric bundle of Alm helices in a 1-palmitoyl-2-oleoylphosphatidylcholine bilayer. These simulations explore the dynamic properties of a model of a helix bundle channel in a complete phospholipid bilayer in an aqueous environment. We explore the stability and conformational dynamics of the bundle in a phospholipid bilayer. We also investigate the effect on bundle stability of the ionization state of the ring of Glu18 side chains. If all of the Glu18 side chains are ionised, the bundle is unstable; if none of the Glu18 side chains are ionized, the bundle is stable. pKA calculations suggest that either zero or one ionized Glu18 is present at neutral pH, correlating with the stable form of the helix bundle. The structural and dynamic properties of water in this model channel were examined. As in earlier in vacuo simulations (Breed et al., 1996 .Biophys. J. 70:1643-1661), the dipole moments of water molecules within the pore were aligned antiparallel to the helix dipoles. This contributes to the stability of the helix bundle.
Alamethicin↗
Surface binding of alamethicin stabilizes its helical structure: molecular dynamics simulations.
Alamethicin is an amphipathic alpha-helical peptide that forms ion channels. An early event in channel formation is believed to be the binding of alamethicin to the surface of a lipid bilayer. Molecular dynamics simulations are used to compare the structural and dynamic properties of alamethicin in water and alamethicin bound to the surface of a phosphatidylcholine bilayer. The bilayer surface simulation corresponded to a loosely bound alamethicin molecule that interacted with lipid headgroups but did not penetrate the hydrophobic core of the bilayer. Both simulations started with the peptide molecule in an alpha-helical conformation and lasted 2 ns. In water, the helix started to unfold after approximately 300 ps and by the end of the simulation only the N-terminal region of the peptide remained alpha-helical and the molecule had collapsed into a more compact form. At the surface of the bilayer, loss of helicity was restricted to the C-terminal third of the molecule and the rod-shaped structure of the peptide was retained. In the surface simulation about 10% of the peptide/water H-bonds were replaced by peptide/lipid H-bonds. These simulations suggest that some degree of stabilization of an amphipathic alpha-helix occurs at a bilayer surface even without interactions between hydrophobic side chains and the acyl chain core of the bilayer.
Alamethicin↗
A molecular dynamics study of the 41-56 beta-hairpin from B1 domain of protein G.
The structural and dynamical behavior of the 41-56 beta-hairpin from the protein G B1 domain (GB1) has been studied at different temperatures using molecular dynamics (MD) simulations in an aqueous environment. The purpose of these simulations is to establish the stability of this hairpin in view of its possible role as a nucleation site for protein folding. The conformation of the peptide in the crystallographic structure of the protein GB1 (native conformation) was lost in all simulations. The new equilibrium conformations are stable for several nanoseconds at 300K (>10 ns), 350 K (>6.5 ns), and even at 450 K (up to 2.5 ns). The new structures have very similar hairpin-like conformations with properties in agreement with available experimental nuclear Overhauser effect (NOE) data. The stability of the structure in the hydrophobic core region during the simulations is consistent with the experimental data and provides further evidence for the role played by hydrophobic interactions in hairpin structures. Essential dynamics analysis shows that the dynamics of the peptide at different temperatures spans basically the same essential subspace. The main equilibrium motions in this subspace involve large fluctuations of the residues in the turn and ends regions. Of the six interchain hydrogen bonds, the inner four remain stable during the simulations. The space spanned by the first two eigenvectors, as sampled at 450 K, includes almost all of the 47 different hairpin structures found in the database. Finally, analysis of the hydration of the 300 K average conformations shows that the hydration sites observed in the native conformation are still well hydrated in the equilibrium MD ensemble.
Amino Acid Sequence↗
Early steps in the unfolding of thermolysin-like proteases.
Several series of site-directed mutations in thermolysin-like proteases are presented that show remarkable nonadditivity in their effect on thermal stability. A simple model is proposed that relates this nonadditivity to the occurrence of independent partial unfolding processes that occur in parallel at elevated temperatures. To prove this model, a thermolysin-like protease was designed in which two mutations located approximately 35 A apart in the structure individually exert small stabilizing effects of 2.3 and 4. 1 degreesC, respectively, but when combined stabilize the protease by 14.6 degreesC. This overadditivity, which follows directly from the model, confirms that unfolding of this engineered protease starts in parallel at two different regions of the protein.
Bacillus↗
Lipid properties and the orientation of aromatic residues in OmpF, influenza M2, and alamethicin systems: molecular dynamics simulations.
Molecular dynamics simulations allow a direct study of the structure and dynamics of membrane proteins and lipids. We describe the behavior of aromatic residues and lipid properties in POPE and POPC bilayer models with the Escherichia coli OmpF trimer, single alamethicin and Influenza M2 helices, 4-helix M2 bundles, and two alamethicin 6-helix channel models. The total simulation time is over 24 ns, of systems containing solvent, protein, and between 104 and 318 lipids. Various types of adjustment between lipids and proteins occur, depending on the size of the protein and the degree of hydrophobic mismatch between lipid and protein. Single helices cause little measurable effect on nearby lipids whereas the 4-helix bundles, 6-helix channel models, and OmpF cause a significant lowering of order parameters in nearby lipid chains, an increased difference between odd and even chain dihedrals in the magnitude of the trans dihedral fractions and dihedral transition rates, and in most cases a decreased gauche population and a decrease in bilayer thickness. An increased tilt of the lipid chains near the proteins can account for most of the observed decrease in order parameters. The orientation of tryptophans and tyrosines on the outside of the proteins is determined by packing at the protein exterior and non-specific hydrogen bonding with lipids and solvent. The tyrosines in the broad bands that delimit the hydrophobic exterior of OmpF show little change in orientation over one nanosecond. Their rings are oriented predominantly perpendicular to the bilayer plane, with the hydroxyl group pointing toward the lipid-water interface. Phenylalanines in OmpF, alamethicin, and Influenza M2 are more mobile and assume a variety of orientations.
Alamethicin↗
Identification of functional and unfolding motions of cutinase as obtained from molecular dynamics computer simulations.
The implementation of cutinase from Fusarium solani pisi as a fat-stain removing ingredient in laundry washing is hampered by its unfolding in the presence of anionic surfactants. In this work we present molecular dynamics (MD) computer simulations on cutinase and analysis procedures to distinguish the movements related to its functional behavior (e.g., substrate binding) from those related to the unfolding of the enzyme. Two kinds of MD-simulations were performed: a simulation mimicking the thermal motion at room temperature, and several simulations in which unfolding is induced either by high temperature or by using a modified water-protein interaction potential. Essential dynamics analyses (A. Amadei et al., Proteins 17:412-425, 1993) on the simulations identify distinct regions in the molecular structure of cutinase in which the motions occur for function and initial unfolding. The unfolding in various simulations starts in a similar way, suggesting that mutations in the regions involved might stabilize the enzyme without affecting its functionality.
Carboxylic Ester Hydrolases↗
A glimpse of the Holy Grail?
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Computer Simulation↗
Domain motions in bacteriophage T4 lysozyme: a comparison between molecular dynamics and crystallographic data.
A comparison of a series of extended molecular dynamics (MD) simulations of bacteriophage T4 lysozyme in solvent with X-ray data is presented. Essential dynamics analyses were used to derive collective fluctuations from both the simulated trajectories and a distribution of crystallographic conformations. In both cases the main collective fluctuations describe domain motions. The protein consists of an N- and C-terminal domain connected by a long helix. The analysis of the distribution of crystallographic conformations reveals that the N-terminal helix rotates together with either of these two domains. The main domain fluctuation describes a closure mode of the two domains in which the N-terminal helix rotates concertedly with the C-terminal domain, while the domain fluctuation with second largest amplitude corresponds to a twisting mode of the two domains, with the N-terminal helix rotating concertedly with the N-terminal domain. For the closure mode, the difference in hinge-bending angle between the most open and most closed X-ray structure along this mode is 49 degrees. In the MD simulation that shows the largest fluctuation along this mode, a rotation of 45 degrees was observed. Although the twisting mode has much less freedom than the closure mode in the distribution of crystallographic conformations, experimental results suggest that it might be functionally important. Interestingly, the twisting mode is sampled more extensively in all MD simulations than it is in the distribution of X-ray conformations.
Bacteriophage T4↗