Protein dynamics in solution and in a crystalline environment: a molecular dynamics study

The effect of a solvent and a crystalline environment on the dynamics of proteins is investigated by the method of computer simulation. Three 25-ps molecular dynamics simulations at 300 K of the bovine pancreatic trypsin inhibitor (BPTI), consisting of 454 heavy atoms, are compared: one of BPTI in vacuo, one of BPTI in a box with 2647 spherical nonpolar solvent atoms, and one of BPTI surrounded by fixed crystal image atoms. Both average and time-dependent molecular properties are examined to determine the effect of the environment on the behavior of the protein. The dynamics of BPTI in solution or in the crystal environment are found to be very similar to that found in the vacuum calculation. The primary difference in the average properties is that the equilibrium structure in the presence of solvent or the crystal field is significantly closer to the X-ray structure than is the vacuum result; concomitantly, the more realistic environment leads to a number density closer to experiment. The presence of solvent has a negligible effect on the overall magnitude of the positional or dihedral angle fluctuations in the interior of the protein; however, there are changes in the decay times of the fluctuations of interior atoms. For surface residues, both the magnitude and the time course of the motions are significantly altered by the solvent. There tends to be an increase in the displacements of long side chains and the flexible parts of the main chain that protrude into the solvent. Further, these motions tend to have a more diffusive character with longer relaxation times than in vacuo. The crystal environment has a specific effect on a number of side chains which are held in relatively fixed positions through hydrogen-bond and electric interactions with the neighboring protein atoms. Most of the effects of the solution environment seem to be sufficiently nonspecific that it may be possible to model them by applying a mean field and stochastic dynamic methods.     

Molecular dynamics study of the structure and dynamics of a protein molecule in a crystalline ionic environment, Streptomyces griseus protease A

A large-scale molecular dynamics simulation of the behavior of a serine protease (Streptomyces griseus protease A) in a crystalline environment has been performed. All atoms (including hydrogens) of two protein molecules and the surrounding solvent of crystallization, consisting of both water and salt ions, were explicitly represented, and a relatively long range of interactions (up to 15 A) were included. The simulation is the longest so far reported for a protein in such an environment (60 ps). The use of the full crystalline environment allows a direct comparison of the structure and dynamic properties of the protein and surrounding solvent to be made with the experimental X-ray structure. Here we report the comparison of the protein structures and analyze the energetics of the system, including interaction with the aqueous environment. Subsequent papers will deal with other aspects of the simulation. The overall root mean square differences between the time-averaged molecular dynamics structure and that from crystallography, for all well-ordered, non-hydrogen atoms, are 1.67 and 1.25 A for the two molecules taken as the asymmetric unit. An extensive analysis of the conformation of substructural elements and individual residues and their deviation from experiment has revealed a strong influence of the ionic medium on their behavior. Implications of the results for free energy calculations and for future directions are also discussed.     

Differences between the conformations of nitrotyrosyl-248 carboxypeptidase A in the crystalline state and in solution

In solution, nitrocarboxypeptidase A, modified at tyrosyl-248, exhibits a nitrotyrosyl pK apparent of 6.3. In the crystalline state, the pK apparent is about 8.2. This change in ionization is consistent with the hypothesis that crystallization of the enzyme causes a displacement of tyrosine-248 away from the active site zinc ion.     

Molecular dynamics simulations of lignin peroxidase in solution

The dynamical and structural properties of lignin peroxidase and its Trp171Ala mutant have been investigated in aqueous solution using molecular dynamics (MD) simulations. In both cases, the enzyme retained its overall backbone structure and all its noncovalent interactions in the course of the MD simulations. Very interestingly, the analysis of the MD trajectories showed the presence of large fluctuations in correspondence of the residues forming the heme access channel; these movements enlarge the opening and facilitate the access of substrates to the enzyme active site. Moreover, steered molecular dynamics docking simulations have shown that lignin peroxidase natural substrate (veratryl alcohol) can easily approach the heme edge through the access channel.     

Molecular dynamics simulations of calcium-free calmodulin in solution

A 4-ns molecular dynamics simulation of calcium-free calmodulin in solution has been performed, using Ewald summation to treat electrostatic interactions. Our simulation results were mostly consistent with solution experimental studies, including NMR, fluorescence and x-ray scattering. The secondary structures within the N- and C-terminal domains were conserved in the simulation, with trajectory structures similar to the NMR-derived model structure 1CFD. However, the relative orientations of the domains, for which there are no NMR restraints, differed in details between the simulation and the 1CFD model. The most interesting information provided by the simulations is that the dynamics of calcium-free calmodulin in solution is dominated by slow rigid body reorientations of the domains. The interdomain distance fluctuated between 29 and 39 A, and interdomain orientation angle, defined as the pseudo-dihedral formed by the four calcium binding sites, varied between -2 degrees and 108 degrees. Similarly, the domain linker region also exhibited significant fluctuations, with its length varying in the 34-45 A range and its bend angle in the 10-100 degrees range. The simulations are in accord with fluorescence results suggesting that calcium-free calmodulin is more compact and more flexible than the calcium activated form. Surprisingly, quite similar solvent accessibilities of the hydrophobic patches were seen in the calcium-free trajectory described in this work and previously generated calcium-loaded calmodulin simulations. Thus, our simulations suggest a reexamination of the standard model of the structural change of calmodulin upon calcium binding, involving exposure of the hydrophobic patches to solvent.     

Non-B DNA conformations analysis through molecular dynamics simulations

BackgroundNon-B DNA conformations are molecular structures that do not follow the canonical DNA double helix. Mutagenetic instability in nuclear and mitochondrial DNA (mtDNA) genomes has been associated with simple non-B DNA conformations, as hairpins or more complex structures, as G-quadruplexes. One of these structures is Structure A, a cloverleaf-like non-B conformation predicted for a 93-nt (nucleotide) stretch of the mtDNA control region 5′-peripheral domain. Structure A is embedded in a hot spot for the 3′ end of human mtDNA deletions revealing its importance in influencing the mutational instability of the mtDNA genome.MethodsTo better characterize Structure A, we predicted its 3D conformation using state-of-art methods and algorithms. The methodologic workflow consisted in the prediction of non-B conformations using molecular dynamics simulations. The conservation scores of alignments of the Structure A region in humans, primates, and mammals, was also calculated.ResultsOur results show that these computational methods are able to measure the stability of non-B conformations by using the level of base pairing during molecular dynamics. Structure A showed high stability and low flexibility correlated with high conservation scores in mammalian, more specifically in primate lineages.ConclusionsWe showed that 3D non-B conformations can be predicted and characterized by our methodology. This allowed the in-depth analysis of the structure A, and the main results showed the structure remains stable during the simulations.General significanceThe fine-scale atomic molecular determination of this type of non-B conformation opens the way to perform computational molecular studies that can show their involvement in mtDNA cellular mechanisms.     

A QM/MM analysis of the conformations of crystalline sucrose moieties

Both ab initio quantum mechanics (QM) and molecular mechanics (MM) were used to produce a hybrid energy surface for sucrose that simultaneously provides low energies for conformations that are observed in crystal structures and high energies for most unobserved structures. HF/6-31G* QM energies were calculated for an analogue based on tetrahydropyran (THP) and tetrahydrofuran (THF). Remaining contributions to the potential energy of sucrose were calculated with MM. To do this, the MM surface for the analogue was subtracted from the MM surface for the disaccharide, and the QM surface for the analogue was added. Prediction of the distribution of observable geometries was enhanced by reducing the strength of the hydrogen bonding. Reduced hydrogen-bonding strength is probably useful because many crystalline sucrose moieties do not have intramolecular hydrogen bonds between the fructose and glucose residues. Therefore, hydrogen bonding does not play a large role in determining the molecular conformation. On the hybrid energy surface that was constructed with a dielectric constant of 3.5, the average potential energy of 23 sucrose moieties from crystal structures is 1.16 kcal/mol, and the population of observed structures drops off exponentially as the energy increases.     

Melanostatin conformations in solution.

The conformations of melanostatin have been studied experimentally using CD spectroscopy and via calculations. In aqueous solution and 2,2,2-trifluoroethanol (TFE) there is no evidence that monomers of the tripeptide exist in an ordered (β-bend) structure. In water and TFE solutions (3–6 × 10^?4M) the neutral molecules aggregate very slowly, taking about 3 days to attain equilibrium at room temperature. At equivalent concentrations in TFE, although not in water, the cationic molecules also slowly aggregate, although to a lesser extent. Calculations using rotational isomeric state theory give the most probable unperturbed end-to-end distance of the molecule at 9.3 ± 0.1 Å and indicate that a vast majority of the molecules exist in some extended conformation, end-to-end distance ≥6 Å. Only 0.4% of the molecules are calculated to have O…?H separations compatible with a β-bend structure. An intramolecular hydrogen bond must have an energy at least 2 kcal/mol lower than that of an intermolecular hydrogen bond to solvent if a β-bend is to be experimentally observable.     

Two conformations of crystalline adenylate kinase.

Pig muscle adenylate kinase (EC2.7.4.3) can exist in three crystal forms, which are interconvertible. For crystal form A the enzyme structure is known in atomic detail. We report the X-ray diffraction analysis of crystal form B at 4.7 Å resolution and a comparison with the A form. During the transition from A to B the packing arrangement of the molecules changes slightly. Moreover, the individual molecule undergoes an appreciable conformational change: by displacing a chain segment of seven residues and two adjacent α-helices a hydrophobic pocket is opened deep in the cleft near the centre of the molecule. Concomitantly the β-pleated sheet is enlarged by about four hydrogen bonds in the B form. Several lines of evidence indicate that the observed conformational change is an intrinsic property of the molecule and is not induced by crystal packing forces.     

Molecular dynamics simulations of a calmodulin-peptide complex in solution

We have performed an 4-ns MD simulation of calmodulin complexed with a target peptide in explicit water, under realistic conditions of constant temperature and pressure, in the presence of a physiological concentration of counterions and using Ewald summation to avoid truncation of long-range electrostatic forces. During the simulation the system tended to perform small fluctuations around a structure similar to, but somewhat looser than the starting crystal structure. The calmodulin-peptide complex was quite rigid and did not exhibit any large amplitude domain motions such as previously seen in apo- and calcium-bound calmodulin. We analyzed the calmodulin-peptide interactions by calculating buried surface areas, CHARMM interaction energies and continuum model interaction free energies. In the trajectory, the protein surface area buried by contact with the peptide is 1373 A(2) approximately evenly divided between the calmodulin N-terminal, C-terminal and central linker regions. A majority of this buried surface, 803 A(2), comes from nonpolar residues, in contrast to the protein as a whole, for which the surface is made up of mostly polar and charged groups. Our continuum calculations indicate that the largest favorable contribution to peptide binding comes from burial of molecular surface upon complex formation. Electrostatic contributions are favorable but smaller in the trajectory structures, and actually unfavorable for binding in the crystal structure. Since nonpolar groups make up most of buried surface of the protein, our calculations suggest that the hydrophobic effect is the main driving force for binding the helical peptide to calmodulin, consistent with thermodynamic analysis of experimental data. Besides the burial of nonpolar surface area, secondary contributions to peptide binding come from burial of polar surface and electrostatic interactions. In the nonpolar interactions a crucial role is played by the nine methionines of calmodulin. In the electrostatic interactions the negatively charged protein residues and positively charged peptide residues play a dominant role.     

Virtual and solution conformations of oligosaccharides

The possibility that observed nuclear Overhauser enhancements and bulk longitudinal relaxation times, parameters measured by 1H NMR and often employed in determining the preferred solution conformation of biologically important molecules, are the result of averaging over many conformational states is quantitatively evaluated. Of particular interest was to ascertain whether certain 1H NMR determined conformations are "virtual" in nature; i.e., the fraction of the population of molecules actually found at any time within the subset of conformational space defined as the "solution conformation" is vanishingly small. A statistical mechanics approach was utilized to calculate an ensemble average relaxation matrix from which (NOE)'s and (T1)'s are calculated. Model glycosidic linkages in four oligosaccharides were studied. The solution conformation at any glycosidic linkage is properly represented by a normalized, Boltzmann distribution of conformers generated from an appropriate potential energy surface. The nature of the resultant population distributions is such that 50% of the molecular population is found within 1% of available microstates, while 99% of the molecular population occupies about 10% of the ensemble microstates, a number roughly equal to that sterically allowed. From this analysis we conclude that in many cases quantitative interpretation of NMR relaxation data, which attempts to define a single set of allowable torsion angle values consistent with the observed data, will lead to solution conformations that are either virtual or reflect torsion angle values possessed by a minority of the molecular population. On the other hand, calculation of ensemble average NMR relaxation data yields values in agreement with experimental results. Observed values of NMR relaxation data are the result of the complex interdependence of the population distribution and NOE (or T1) surfaces in conformational space. In conformational analyses, NMR data can therefore be used to test different population distributions calculated from empirical potential energy functions.     

Molecular dynamics simulation of carboxy and deoxy human cytoglobin in solution

Cytoglobin (Cgb), the fourth member of the vertebrate heme globin family, is widely expressed in mammalian tissues, and reversibly binds to CO, O₂ and other small ligands. The diverse functions of Cgb may include ligand transport, redox reactions and enzymatic catalysis. Recent studies indicate that Cgb is a potential gene medicine for fibrosis and cancer therapy. In the present work, molecular dynamics (MD) simulations were performed to investigate the functionally related structural properties and dynamic characteristics in carboxy and deoxy human Cgb. The simulation results showed that the loop regions and internal cavities were significantly affected through the binding of an exogenous ligand. The AB, GH and EF loops were found to undergo significant rearrangement and this led to distinct cavity adjustments in Xe2, Xe4 and the distal pocket. In addition, solvent accessibility and torsion angle analyses revealed an interactive distal network comprised of His⁸¹(E7), Leu⁴⁶(B10) and Arg⁸⁴(E10). The MD study of carboxy and deoxy human Cgb revealed that CO-ligated Cgb modulates the protein conformation primarily by loop and cavity rearrangements rather than the heme sliding mechanism found in neuroglobin (Ngb). The significant differences between Cgb and Ngb in the loop and cavity properties are presumably linked to their various biological functions.     

Molecular dynamics simulation of the calmodulin-trifluoperazine complex in aqueous solution

Trifluoperazine (TFP) has been widely studied in relation to its mode of binding and its inactivation of calmodulin (CaM). Most studies in solution have indicated that CaM has two high-affinity binding sites for TFP. The crystal structure of the 1:4 CaM-TFP complex (CaM-4TFP) shows that three TFP molecules bind to the C-domain of CaM, and that one TFP molecule binds to the N-domain. In contrast, the crystal structure of the 1:1 CaM-TFP complex (CaM-1TFP) shows that one TFP molecule binds to the C-domain. It has been thought that the binding of one TFP molecule to the C-domain is followed by binding to the N-domain. The crystal structure of the 1:2 CaM-TFP complex (CaM-2TFP), moreover, has recently been determined, showing that two TFP molecules bind to the C-domain. In order to determine the structure of the CaM-TFP complex and to clarify the interaction between CaM and TFP in solution, we performed a molecular dynamics simulation of the CaM-TFP complex in aqueous solution starting from the CaM-4TFP crystal structure. The obtained solution structure is very similar to the CaM-2TFP crystal structure. The computer simulation showed that the binding ability of the secondary binding site of the C-domain is higher than that of the primary binding site of the N-domain.     

Molecular dynamics simulations of polypeptide conformations in water: A comparison of alpha, beta, and poly(pro)II conformations.

A significant fraction of the so-called "random coil" residues in globular proteins exists in the left-handed poly(Pro)II conformation. In order to compare the behavior of this secondary structure with that of the other regular secondary structures, molecular dynamics simulations, with the GROMOS suite of programs, of an alanine octapeptide in water, in alpha-helix, beta-strand, and left-handed poly(Pro)II conformations, have been performed. Our results indicate a limited flexibility for the alpha-helix conformation and a relatively larger flexibility for the beta-strand and poly(Pro)II conformations. The behavior of oligopeptides with a starting configuration of beta-strand and poly(Pro)II conformations, both lacking interchain hydrogen bonds, were similar. The (phi, psi) angles reflect a continuum of structures including both beta and P(II) conformations, but with a preference for local P(II) regions. Differences in the network of water molecules involved in hydrogen bonding with the backbone of the polypeptide were observed in local regions of beta and P(II) conformations. Such water bridges help stabilize the P(II) conformation relative to the beta conformation. Proteins 1999;36:400-406.     

DNA fine structure and dynamics in crystals and in solution: the impact of BI/BII backbone conformations

Sugar-phosphate backbone conformations are an important structural element for a complete understanding of specific recognition in nucleic acid-protein interactions. They can be involved both in early stages of target discrimination and in structural adaptation upon binding. In the first part of this study, we have analyzed high-resolution structures of double-stranded B-DNA either isolated or bound to proteins, and explored the impact of both the standard BI and the unusual BII phosphate backbone conformations on neighboring sugar puckers and on selected helical parameters. Correlations are found to be similar for free and bound DNA, and in both categories, the possible facing backbone conformations (BI.BI, BI.BII, and BII.BII) define well-characterized substates in the B-DNA conformational space. Notably, BII.BII steps are characterized by specific, and sequence-independent, structural effects involving reduced standard deviations for almost all conformational parameters. In the second part of this work, we analyze four 10 ns molecular dynamics simulations in explicit solvent on the DNA targets of NF-kappaB and bovine papillomavirus E2 proteins, highlighting the multiplicity of backbone dynamical behavior. These results show sequence effects on the percentages of BI and BII conformers, the preferential state of facing backbones, the occurrence of coupled transitions. The backbone states can consequently be seen as a mechanism for transmitting information from the bases to the phosphate groups and thus for modulating the overall structural properties of the target DNA.