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The technique of one-step perturbation to explore the relation between particular force-field parameters on the one hand and particular properties of a biomolecular system on the other hand from one or a few molecular dynamics simulations is applied to investigate the dependence of the free enthalpy of dimer formation and of crystal dissolution of a self-complementary fragment (H-CGTACG-NH2) of peptide nucleic acid, PNA, a mimic of DNA. The simulations show that PNA dimer formation in aqueous solution is favored by a decrease in the base charges with respect to values of the GROMOS 45A4 force field, while it is disfavored by a decrease in the backbone charges. In contrast, crystal dissolution of the PNA dimer is favored by a decrease in base charges, while a variation of backbone charges has a minor effect on this free enthalpy change. These opposite effects in a crystalline versus aqueous solution environment can be understood from the different water contents for these systems and have consequences for biomolecular force-field development.

In this study, a hexa-β-peptide whose conformational equilibrium encompasses two different helical folds, a right-handed 2.7(10/12)-helix and a left-handed 3(14)-helix, is simulated using different GROMOS force-field parameter sets. When applying the recently developed GROMOS 54A7 force field, a significant destabilization effect on the 2.7(10/12)-helix of the peptide is observed, and the agreement with the experimental NOE distance bounds is much worse compared with the ones using previous versions of the GROMOS force field. This led us to investigate the free enthalpy difference between the two helices as a function of a variation of different subsets of force-field parameters. Both long time molecular dynamics simulations and one-step perturbation predictions suggest that the disagreement with the experimental NMR data when using the 54A7 force field is caused by the use for β-peptides of the new backbone φ-/ψ-torsional-angle energy terms introduced in this force field which were based on conformational fitting of backbone φ/ψ angles for a large set of proteins. This means that these parameters of backbone φ- and ψ-torsional-angle terms should not be applied to non-α-peptides such as β-peptides. This modified assignment of torsional-angle energy terms and parameters is denoted as 54A7_β. It corrects the wrong description of the conformational ensemble of the hexa-β-peptide obtained using the previous assignment and yields as good agreement with NMR data for other β-peptides that adopt a single helical or a hairpin fold., (Copyright © 2013 Wiley Periodicals, Inc.)

To lessen the problem of insufficient conformational sampling in biomolecular simulations is still a major challenge in computational biochemistry. In this article, an application of the method of enveloping distribution sampling (EDS) is proposed that addresses this challenge and its sampling efficiency is demonstrated in simulations of a hexa-β-peptide whose conformational equilibrium encompasses two different helical folds, i.e., a right-handed 2.7(10∕12)-helix and a left-handed 3(14)-helix, separated by a high energy barrier. Standard MD simulations of this peptide using the GROMOS 53A6 force field did not reach convergence of the free enthalpy difference between the two helices even after 500 ns of simulation time. The use of soft-core non-bonded interactions in the centre of the peptide did enhance the number of transitions between the helices, but at the same time led to neglect of relevant helical configurations. In the simulations of a two-state EDS reference Hamiltonian that envelops both the physical peptide and the soft-core peptide, sampling of the conformational space of the physical peptide ensures that physically relevant conformations can be visited, and sampling of the conformational space of the soft-core peptide helps to enhance the transitions between the two helices. The EDS simulations sampled many more transitions between the two helices and showed much faster convergence of the relative free enthalpy of the two helices compared with the standard MD simulations with only a slightly larger computational effort to determine optimized EDS parameters. Combined with various methods to smoothen the potential energy surface, the proposed EDS application will be a powerful technique to enhance the sampling efficiency in biomolecular simulations.

Nuclear magnetic resonance (NMR) nuclear Overhauser enhancement (NOE) data obtained for a 35-nucleotide RNA segment of a bacterial group II intron indicate a helical hairpin structure in which three parts, a terminal pentaloop, a bulge, and a G-A mismatch, display no Watson-Crick base pairing. The 668 NOE upper distance bounds for atom pairs are insufficient to uniquely determine the conformation of these segments. Therefore, molecular dynamics simulations including time-averaged distance restraints have been used to obtain a conformational ensemble compatible with the observed NMR data. The ensemble shows alternating hydrogen bonding patterns for the mentioned segments. In particular, in the pentaloop and in the bulge, the hydrogen bonding networks correspond to distinct conformational clusters that could not be captured by using conventional single-structure refinement techniques. This implies that, to obtain a realistic picture of the conformational ensemble of such flexible biomolecules, it is necessary to properly account for the conformational variability in the structure refinement of RNA fragments.

NMR experiments have shown that water molecules in the crystal of the protein Crh are still mobile at temperatures well below 273 K. In order to investigate this water anomaly, a molecular dynamics (MD) simulation study of crystalline Crh was carried out to determine the mobility of water in this crystal. The simulations were carried out at three temperatures, 150, 200, and 291 K. Simulations of bulk water at these temperatures were also done to obtain the properties of the simple point charge (SPC) water model used at these temperatures and to allow a comparison of the properties of water in the Crh crystal with those of bulk water at the same temperatures. According to the simulations, water is immobilized at 150 K both in crystal and in bulk water. As expected, at 291 K it diffuses and rotates more slowly in the protein crystal than in bulk water. However, at 200 K, the translational and rotational mobility of the water molecules is larger in the crystal than in bulk water. The enhancement of water mobility in the crystal at 200 K was further investigated by MD simulations in which the backbone or all protein atoms were positionally restrained, and in which additionally the electrostatic protein-water interactions were removed. Of these changes in the environment of the water molecules, rigidifying the protein backbones slightly enhanced water diffusion, while it slowed down rotation. In contrast, removal of electrostatic protein-water interactions did not change water diffusion but enhanced rotational motion significantly. Further investigations are required to delineate particular features of the protein crystal that induce the anomalous behavior of water at 200 K.

In a recent study (Lin et al., Helv. Chim. Acta 2011, 94, 597), the one-step perturbation method was applied to tackle a challenging computational problem, that is, the calculation of the folding free enthalpies ΔGF,U of six hepta-β-peptides with different, Ala, Val, Leu, Ile, Ser, or Thr, side chains in the fifth residue. The ΔGF,U values obtained using one-step perturbation based on a single molecular dynamics simulation of a judiciously chosen reference state with soft-core atoms in the side chain of the fifth residue showed an overall accuracy of about kB T for the four peptides with nonpolar side chains, but twice as large deviations were observed for the peptides with polar side chains. Here, alternative reference-state Hamiltonians that better cover the conformational space relevant to these peptides are investigated, and post simulation rotational sampling of the χ1 and χ2 torsional angles of the fifth residue is carried out to sample different orientations of the side chain. A reference state with rather soft atoms yields accurate ΔGF,U values for the peptides with the Ser and Thr side chains, but it failed to correctly predict the folding free enthalpy for one peptide with a nonpolar side chain, that is, Leu. Based on the results and those of earlier studies, possible ways to improve the accuracy of the efficient one-step perturbation technique to compute free enthalpies of folding are discussed., (Copyright © 2013 Wiley Periodicals, Inc.)

Experimental studies of barley and maize lipid transfer proteins (LTPs) show that the two proteins bind the ligand palmitate in opposite orientations in their internal cavities. Moreover, maize LTP is reported to bind the ligand caprate in the internal cavity in a mixture of two orientations with approximately equal occupancy. Six 30 ns molecular dynamics (MD) simulations of maize and barley LTP with ligands bound in two orientations (modes M and B) have been used to understand the different ligand binding preferences. The simulations show that both maize and barley LTP could bind palmitate in the orientation observed experimentally for maize LTP (mode M), with the predominant interaction being a salt bridge between the ligand carboxylate headgroup and a conserved arginine side chain. However, the simulation of barley LTP with palmitate in the mode B orientation shows the most favorable protein-ligand interaction energy. In contrast, the simulations of maize LTP with palmitate and with caprate in the mode B orientation show no persistent ligand binding, the ligands leaving the cavity during the simulations. Sequence differences between maize and barley LTP in the AB loop region, in residues at the base of the hydrophobic cavity, and in the helix A region are identified as contributing to the different behavior. The simulations reproduce well the experimentally observed binding preferences for palmitate and suggest that the experimental data for maize LTP with caprate reflect ligand mobility in binding mode M rather than the population of binding modes M and B.

Enveloping distribution sampling was used to calculate free-enthalpy changes associated with single amino acid mutations for a pair of proteins, GA95 and GB95, that show 95% sequence identity yet fold into topologically different structures. Of the L → A, I → F, and L → Y mutations at positions 20, 30, and 45, respectively, of the 56-residue sequence, the first and the last contribute the most to the free-enthalpy difference between the native and non-native sequence-structure combinations, in agreement with the experimental findings for this protein pair. The individual free-enthalpy changes are almost sequence-independent in the four-strand/one-helix structure, the stable form of GB95, while in the three-helix bundle structure, the stable form of GA95, an interplay between residues 20 and 45 is observed.

It is often assumed that intramolecular hydrogen-bonding (H-bonding) exerts a significant influence on the conformational properties of aqueous (bio-)polymers. To discuss this statement, one should, however, distinguish between solvent-exposed and buried H-bonds, and between their respective roles in promoting stability (i.e., as a driving force) and specificity (for which the term steering force is introduced here). In this study, the role of solvent-exposed H-bonding in carbohydrates as a driving or steering force is probed using explicit-solvent molecular dynamics simulations with local elevation umbrella sampling in the simple context of cellobiose stereoisomers. More specifically, four β(1→4)-linked D-aldohexopyranose disaccharides are considered, which present a different stereochemisty of the potentially H-bonding groups neighboring the glycosidic linkage. Although the epimerization may largely alter the intramolecular trans-glycosidic H-bonding pattern, it is found to have only very limited influence on the Ramachandran free-energy map of the disaccharide, a loss of intramolecular H-bonding being merely compensated for by an enhancement of the interaction with the solvent molecules. This finding suggests that solvent-exposed trans-glycosidic H-bonding (and in particular the HO'(3)→O5 H-bond) is not the cause of the 21-helical secondary structure characteristic of cellooligosaccharides, but rather the opportunistic consequence of a sterically and stereoelectronically dictated conformational preference. In other words, for these compounds, solvent-exposed H-bonding appears to represent a minor (possibly adverse) conformational driving as well as steering force.

The enzyme chorismate mutase EcCM from Escherichia coli catalyzes one of the few pericyclic reactions in biology, the transformation of chorismate to prephenate. The isochorismate pyruvate lyase PchB from Pseudomonas aeroginosa catalyzes another pericyclic reaction, the isochorismate to salicylate transformation. Interestingly, PchB possesses weak chorismate mutase activity as well thus being able to catalyze two distinct pericyclic reactions in a single active site. EcCM and PchB possess very similar folds, despite their low sequence identity. Using molecular dynamics simulations of four combinations of the two enzymes (EcCM and PchB) with the two substrates (chorismate and isochorismate) we show that the electrostatic field due to EcCM at atoms of chorismate favors the chorismate to prephenate transition and that, analogously, the electrostatic field due to PchB at atoms of isochorismate favors the isochorismate to salicylate transition. The largest differences between EcCM and PchB in electrostatic field strengths at atoms of the substrates are found to be due to residue side chains at distances between 0.6 and 0.8 nm from particular substrate atoms. Both enzymes tend to bring their non-native substrate in the same conformation as their native substrate. EcCM and to a lower extent PchB fail in influencing the forces on and conformations of the substrate such as to favor the other chemical reaction (isochorismate pyruvate lyase activity for EcCM and chorismate mutase activity for PchB). These observations might explain the difficulty of engineering isochorismate pyruvate lyase activity in EcCM by solely mutating active site residues., (© 2013 The Protein Society.)

Various experimental studies of hen egg white lysozyme (HEWL) in water and TFE/water clearly indicate structural differences between the native state and TFE state of HEWL, e.g. the helical content of the protein in the TFE state is much higher than in the native state. However, the available detailed NMR studies were not sufficient to determine fully a structure of HEWL in the TFE state. Different molecular dynamics (MD) simulations, i.e. at room temperature, at increased temperature and using proton-proton distance restraints derived from NMR NOE data, have been used to generate configurational ensembles corresponding to the TFE state of HEWL. The configurational ensemble obtained at room temperature using atom-atom distance restraints measured for HEWL in TFE/water solution satisfies the experimental data and has the lowest protein energy. In this ensemble residues 50-58, which are part of the β-sheet in native HEWL, adopt fluctuating α-helical secondary structure.

The methodology of Enveloping Distribution Sampling (EDS) is extended to probe a single-simulation alternative to the thermodynamic cycle that is standardly used for measuring the effect of a modification of a chemical compound, e.g. from a given species to a chemical derivative for a ligand or solute molecule, on the free-enthalpy change associated with a change in environment, e.g. from the unbound state to the bound state for a protein-ligand system or from one solvent to another one for a solute molecule. This alternative approach relies on the coupled simulation of two systems (computational boxes) 1 and 2, and the method is therefore referred to as twin-system EDS. Systems 1 and 2 account for the two choices of environment. The end states of the alchemical perturbation for the twin-system associate the two alternative forms X and Y of the molecule to systems 1 and 2 or 2 and 1, respectively. In this way, the processes of transforming one molecule into the other are carried out simultaneously in opposite directions in the two environments, leading to a change in free enthalpy that is smaller than for the two individual processes and to an energy-difference distribution that is more symmetric. As an illustration, the method is applied to the calculation of octanol-water partition coefficients for C4 to C8 alkanes, 1-hexanol and 1,2-dimethoxyethane. It is shown in particular that the consideration of the residual hydration of octanol leads to calculated partition coefficients that are in better agreement with reported experimental numbers.

Atomistic molecular dynamics simulations of peptides or proteins in aqueous solution are still limited to the multi-nanosecond time scale and multi-nanometer range by computational cost. Combining atomic solutes with a supramolecular solvent model in hybrid fine-grained/coarse-grained (FG/CG) simulations allows atomic detail in the region of interest while being computationally more efficient. We used enveloping distribution sampling (EDS) to calculate the free enthalpy differences between different helical conformations, i.e., α-, π-, and 310-helices, of an atomic level FG alanine deca-peptide solvated in a supramolecular CG water solvent. The free enthalpy differences obtained show that by replacing the FG solvent by the CG solvent, the π-helix is destabilized with respect to the α-helix by about 2.5 kJ mol(-1), and the 310-helix is stabilized with respect to the α-helix by about 9 kJ mol(-1). In addition, the dynamics of the peptide becomes faster. By introducing a FG water layer of 0.8 nm around the peptide, both thermodynamic and dynamic properties are recovered, while the hybrid FG/CG simulations are still four times more efficient than the atomistic simulations, even when the cutoff radius for the nonbonded interactions is increased from 1.4 to 2.0 nm. Hence, the hybrid FG/CG model, which yields an appropriate balance between reduced accuracy and enhanced computational speed, is very suitable for molecular dynamics simulation investigations of biomolecules.

Theoretical-computational modeling with an eye to explaining experimental observations in regard to a particular chemical phenomenon or process requires choices concerning essential degrees of freedom and types of interactions and the generation of a Boltzmann ensemble or trajectories of configurations. Depending on the degrees of freedom that are essential to the process of interest, for example, electronic or nuclear versus atomic, molecular or supra-molecular, quantum- or classical-mechanical equations of motion are to be used. In multi-resolution simulation, various levels of resolution, for example, electronic, atomic, supra-atomic or supra-molecular, are combined in one model. This allows an enhancement of the computational efficiency, while maintaining sufficient detail with respect to particular degrees of freedom. The basic challenges and choices with respect to multi-resolution modeling are reviewed and as an illustration the differential catalytic properties of two enzymes with similar folds but different substrates with respect to these substrates are explored using multi-resolution simulation at the electronic, atomic and supra-molecular levels of resolution., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

One-step perturbation is an efficient method to estimate free energy differences in molecular dynamics (MD) simulations, but its accuracy depends critically on the choice of an appropriate, possibly unphysical, reference state that optimizes the sampling of the physical end states. In particular, the perturbation from a polar moiety to a nonpolar one and vice versa in a polar environment such as water poses a challenge which is of importance when estimating free energy differences that involve entropy changes and the hydrophobic effect. In this work, we systematically study the performance of the one-step perturbation method in the calculation of the free enthalpy difference between a polar water solute and a nonpolar "water" solute molecule solvated in a box of 999 polar water molecules. Both these polar and nonpolar physical reference states fail to predict the free enthalpy difference as obtained by thermodynamic integration, but the result is worse using the nonpolar physical reference state, because both a properly sized cavity and a favorable orientation of the polar solute in a polar environment are rarely, if ever, sampled in a simulation of the nonpolar solute in such an environment. Use of nonphysical soft-core reference states helps to sample properly sized cavities, and post-MD simulation rotational and translational sampling of the solute to be perturbed leads to much improved free enthalpy estimates from one-step perturbation., (Copyright © 2012 Wiley Periodicals, Inc.)

Folding free enthalpies of many not too different polypeptides can be efficiently and accurately predicted with the one-step perturbation (OSP) method using only one or a few molecular dynamics (MD) simulations. In this article, we introduce a combination of enveloping distribution sampling (EDS) and the OSP method (EDS-OSP) and apply it to predict the free enthalpy differences between a right-handed 2.710/12-helix and a left-handed 314-helix for 16 β-peptides with slightly different side-chain substitution patterns. An EDS simulation of a designed soft-core reference-state peptide was carried out in which both helices were sampled. Then, the soft-core atoms were perturbed into physical atoms. Thus, free enthalpy differences between the two helices for the 16 β-peptides can be predicted from only one simulation. The results predicted by EDS-OSP and a previous OSP study are very similar, i.e., the deviations between the results of the 16 peptides are mostly within the order of kBT, and the average absolute deviation is 1.2 kJ mol(-1). Together with the EDS parameter update simulation, about 128 ns of MD simulations needed to be carried out using the EDS-OSP method, while 700 ns of MD simulations were required in the previous OSP study where two separate reference-state simulations and an additional long time MD simulation of one of the 16 β-peptides were carried out. Thus, the computational effort was significantly reduced, i.e., by more than a factor of 5, using the EDS-OSP method. Hence, we consider this method an efficient tool to predict conformational free enthalpy differences from MD simulations.

"Seven deadly sins" in modern academic research and publishing can be condensed into a list ranging from poorly described experimental or computational setups to falsification of data. This Essay describes these sins and their ramifications, and serves as a code of best practice for researchers in their quest for scientific truth., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The contemporary proteinogenic repertoire contains 20 amino acids with diverse functional groups and side chain geometries. Primordial proteins, in contrast, were presumably constructed from a subset of these building blocks. Subsequent expansion of the proteinogenic alphabet would have enhanced their capabilities, fostering the metabolic prowess and organismal fitness of early living systems. While the addition of amino acids bearing innovative functional groups directly enhances the chemical repertoire of proteomes, the inclusion of chemically redundant monomers is difficult to rationalize. Here, we studied how a simplified chorismate mutase evolves upon expanding its amino acid alphabet from nine to potentially 20 letters. Continuous evolution provided an enhanced enzyme variant that has only two point mutations, both of which extend the alphabet and jointly improve protein stability by >4 kcal/mol and catalytic activity tenfold. The same, seemingly innocuous substitutions (Ile→Thr, Leu→Val) occurred in several independent evolutionary trajectories. The increase in fitness they confer indicates that building blocks with very similar side chain structures are highly beneficial for fine-tuning protein structure and function., Competing Interests: PM is an inventor of the GM3 culturing device, which is patented under WIPO WO0034433. This patent is licensed to Heurisko USA, of which PM is the president of the board.

Water molecules in the binding pocket of a protein and their role in ligand binding have increasingly raised interest in recent years. Displacement of such water molecules by ligand atoms can be either favourable or unfavourable for ligand binding depending on the change in free enthalpy. In this study, we investigate the displacement of water molecules by an apolar probe in the binding pocket of two proteins, cyclin-dependent kinase 2 and tRNA-guanine transglycosylase, using the method of enveloping distribution sampling (EDS) to obtain free enthalpy differences. In both cases, a ligand core is placed inside the respective pocket and the remaining water molecules are converted to apolar probes, both individually and in pairs. The free enthalpy difference between a water molecule and a CH(3) group at the same location in the pocket in comparison to their presence in bulk solution calculated from EDS molecular dynamics simulations corresponds to the binding free enthalpy of CH(3) at this location. From the free enthalpy difference and the enthalpy difference, the entropic contribution of the displacement can be obtained too. The overlay of the resulting occupancy volumes of the water molecules with crystal structures of analogous ligands shows qualitative correlation between experimentally measured inhibition constants and the calculated free enthalpy differences. Thus, such an EDS analysis of the water molecules in the binding pocket may give valuable insight for potency optimization in drug design.

The protein chorismate mutase MtCM from Mycobacterium tuberculosis catalyzes one of the few pericyclic reactions known in biology: the transformation of chorismate to prephenate. Chorismate mutases have been widely studied experimentally and computationally to elucidate the transition state of the enzyme catalyzed reaction and the origin of the high catalytic rate. However, studies about substrate entry and product exit to and from the highly occluded active site of the enzyme have to our knowledge not been performed on this enzyme. Crystallographic data suggest a possible substrate entry gate, that involves a slight opening of the enzyme for the substrate to access the active site. Using multiple molecular dynamics simulations, we investigate the natural dynamic process of the product exiting from the binding pocket of MtCM. We identify a dominant exit pathway, which is in agreement with the gate proposed from the available crystallographic data. Helices H2 and H4 move apart from each other which enables the product to exit from the active site. Interestingly, in almost all exit trajectories, two residues arginine 72 and arginine 134, which participate in the burying of the active site, are accompanying the product on its exit journey from the catalytic site., (Copyright © 2012 The Protein Society.)

Molecular dynamics simulations have been performed to investigate the binding of tris(hydroxymethyl)-aminomethane to the surface of the core domain of the mouse cellular tumor antigen p53 employing the GROMOS and 53A6 force field parameter sets. A close investigation of the crystal structure reported by Ho et al. revealed that the protonated form is bound to the protein, i.e. a tris(hydroxymethyl)-methylammonium ion (TRSH). Molecular Dynamics (MD) simulations indicate that the p53 protein gains stability upon binding the ligand. In addition to MD simulations of the p53 protein with and without the TRSH compound, thermodynamic integration was utilised to estimate the free enthalpy of binding of the TRSH-p53 complex, which was estimated to be -49 and -54 kJ mol(-1) utilising the and 53A6 force fields, respectively.

Phosphorylation is an important mechanism regulating protein-protein interactions involving intrinsically disordered protein regions. Stathmin, an archetypical example of an intrinsically disordered protein, is a key regulator of microtubule dynamics in which phosphorylation of 63Ser within the helical nucleation sequence strongly down-regulates the tubulin binding and microtubule destabilizing activities of the protein. Experimental studies on a peptide encompassing the 19-residue helical nucleation sequence of stathmin (residues 55-73) indicate that phosphorylation of 63Ser destabilizes the peptide's secondary structure by disrupting the salt bridges supporting its helical conformation. In order to investigate this hypothesis at atomic resolution, we performed molecular dynamics simulations of nonphosphorylated and phosphorylated stathmin-[55-73] at room temperature and pressure, neutral pH, and explicit solvation using the recently released GROMOS force field 54A7. In the simulations of nonphosphorylated stathmin-[55-73] emerged salt bridges associated with helical configurations. In the simulations of 63Ser phosphorylated stathmin-[55-73] these configurations dispersed and were replaced by a proliferation of salt bridges yielding disordered configurations. The transformation of the salt bridges was accompanied by emergence of numerous interactions between main and side chains, involving notably the oxygen atoms of the phosphorylated 63Ser. The loss of helical structure induced by phosphorylation is reversible, however, as a final simulation showed. The results extend the hypothesis of salt bridge derangement suggested by experimental observations of the stathmin nucleation sequence, providing new insights into regulation of intrinsically disordered protein systems mediated by phosphorylation.

The newly implemented quantum-chemical/molecular-mechanical (QM/MM) functionality of the Groningen molecular simulation (GROMOS) software for (bio)molecular simulation is described. The implementation scheme is based on direct coupling of the GROMOS C++ software to executables of the quantum-chemical program packages MNDO and TURBOMOLE, allowing for an independent further development of these packages. The new functions are validated for different test systems using program and model testing techniques. The effect of truncating the QM/MM electrostatic interactions at various QM/MM cutoff radii is discussed and the application of semiempirical versus density-functional Hamiltonians for a solute molecule in aqueous solution is compared., (Copyright © 2012 Wiley Periodicals, Inc.)

So-called coarse-grained models are a popular type of model for accessing long time scales in simulations of biomolecular processes. Such models are coarse-grained with respect to atomic models. But any modelling of processes or substances involves coarse-graining, i.e. the elimination of non-essential degrees of freedom and interactions from a more fine-grained level of modelling. The basic ingredients of developing coarse-grained models based on the properties of fine-grained models are reviewed, together with the conditions that must be satisfied in order to preserve the correct physical mechanisms in the coarse-graining process. This overview should help the reader to determine how realistic a coarse-grained model of a biomolecular system is, i.e. whether it reflects the underlying physical mechanisms or merely provides a set of pretty pictures of the process or substances of interest.

Considering N-methylacetamide (NMA) as a model compound, new interaction parameters are developed for the amide function in the GROMOS force field that are compatible with the recently derived 53A6(OXY) parameter set for oxygen-containing chemical functions. The resulting set, referred to as 53A6(OXY+A) , represents an improvement over earlier GROMOS force-field versions in the context of the pure-liquid properties of NMA, including the density, heat of vaporization, dielectric permittivity, self-diffusion constant and viscosity, as well as in terms of the Gibbs hydration free energy of this molecule. Assuming that NMA represents an adequate model compound for the backbone of peptides, 53A6(OXY+A) may be expected to also provide an improved description of polypeptide chains. As an initial test, simulations are reported for two β-peptides characterized by very different folding properties in methanol. For these systems, earlier force-field versions provided good agreement with experimental NMR data, and the test shows that the improved description achieved in the context of NMA is not accompanied by any deterioration in the representation of the conformational properties of these peptides., (Copyright © 2012 Wiley Periodicals, Inc.)

We briefly review advances in computational actinoid (An) chemistry during the past ten years in regard to two issues: the geometrical and electronic structures, and reactions. The former addresses the An-O, An-C, and M-An (M is a metal atom including An) bonds in the actinoid molecular systems, including actinoid oxo and oxide species, actinoid-carbenoid, dinuclear and diatomic systems, and the latter the hydration and ligand exchange, the disproportionation, the oxidation, the reduction of uranyl, hydroamination, and the photolysis of uranium azide. Concerning their relevance to the electronic structures and reactions of actinoids and their importance in the development of an advanced nuclear fuel cycle, we also mentioned the work on actinoid carbides and nitrides, which have been proposed to be candidates of the next generation of nuclear fuel, and the oxidation of PuO(x), which is important to understand the speciation of actinoids in the environment, followed by a brief discussion on the urgent need for a heavier involvement of computational actinoid chemistry in developing advanced reprocessing protocols of spent nuclear fuel. The paper is concluded with an outlook.

Recently, the method of enveloping distribution sampling (EDS) to efficiently obtain free enthalpy differences between different molecular systems from a single simulation has been generalized to compute free enthalpy differences between different conformations of a system [Z. X. Lin, H. Y. Liu, S. Riniker, and W. F. van Gunsteren, J. Chem. Theory Comput. 7, 3884 (2011)]. However, the efficiency of EDS in this case is hampered if the parts of the conformational space relevant to the two end states or conformations are far apart and the conformational diffusion from one state to the other is slow. This leads to slow convergence of the EDS parameter values and free enthalpy differences. In the present work, we apply the EDS methodology to a challenging case, i.e., to calculate the free enthalpy difference between a right-handed 2.7(10∕12)-helix and a left-handed 3(14)-helix of a hexa-β-peptide in solution from a single simulation. No transition between the two helices was detected in a standard EDS parameter update simulation, thus enhanced sampling techniques had to be applied, which included adiabatic decoupling (AD) of solute and solvent motions in combination with increasing the solute temperature, and lowering the shear viscosity of the solvent. AD was found to be unsuitable to enhance the sampling of the solute conformations in the EDS parameter update simulations. Lowering the solvent shear viscosity turned out to be useful during EDS parameter update simulations, i.e., it did speed up the conformational diffusion of the solute, more transitions between the two helices were observed. This came at the cost of more CPU time spent due to the shorter time step needed for simulations with the lower solvent shear viscosity. Using an improved EDS parameter update scheme, parameter convergence was five-fold enhanced. The resulting free enthalpy difference between the two helices calculated from EDS agrees well with the result obtained through direct counting from a long MD simulation, while the EDS technique significantly enhances the sampling of both helices over non-helical conformations.

Atomistic molecular dynamics simulations of proteins in aqueous solution are still limited to the multinanosecond time scale and multinanometer range by computational cost. Combining atomic solutes with a supra-molecular solvent model in hybrid fine-grained/coarse-grained (FG/CG) simulations allows atomic detail in the region of interest while being computationally more efficient. A recent comparison of the properties of four proteins in CG water versus FG water showed the preservation of the secondary and tertiary structure with a computational speed-up of at least an order of magnitude. However, an increased occurrence of hydrogen bonds between side chains was observed due to a lack of hydrogen-bonding partners in the supra-molecular solvent. Here, the introduction of a FG water layer around the protein to recover the hydrogen-bonding pattern of the atomistic simulations is studied. Three layer thicknesses of 0.2, 0.4, and 0.8 nm are considered. A layer thickness of 0.8 nm is found sufficient to recover the behavior of the proteins in the atomistic simulations, whereas the hybrid simulation is still three times more efficient than the atomistic one and the cutoff radius for nonbonded interactions could be increased from 1.4 to 2.0 nm.

Simulation of the dynamics of a protein in aqueous solution using an atomic model for both the protein and the many water molecules is still computationally extremely demanding considering the time scale of protein motions. The use of supra-atomic or supra-molecular coarse-grained (CG) models may enhance the computational efficiency, but inevitably at the cost of reduced accuracy. Coarse-graining solvent degrees of freedom is likely to yield a favourable balance between reduced accuracy and enhanced computational speed. Here, the use of a supra-molecular coarse-grained water model that largely preserves the thermodynamic and dielectric properties of atomic level fine-grained (FG) water in molecular dynamics simulations of an atomic model for four proteins is investigated. The results of using an FG, a CG, an implicit, or a vacuum solvent environment of the four proteins are compared, and for hen egg-white lysozyme a comparison to NMR data is made. The mixed-grained simulations do not show large differences compared to the FG atomic level simulations, apart from an increased tendency to form hydrogen bonds between long side chains, which is due to the reduced ability of the supra-molecular CG beads that represent five FG water molecules to make solvent-protein hydrogen bonds. But, the mixed-grained simulations are at least an order of magnitude faster than the atomic level ones.

Understanding of the driving forces of protein folding is a complex challenge because different types of interactions play a varying role. To investigate the role of hydrogen bonding involving the backbone, the effect of thio substitutions in a protein, hen egg white lysozyme (HEWL), was investigated through molecular dynamics simulations of native as well as partly (only residues in loops) and fully thionated HEWL using the GROMOS 54A7 force field. The results of the three simulations show that the structural properties of fully thionated HEWL clearly differ from those of the native protein, while for partly thionated HEWL they only changed slightly compared with native HEWL. The analysis of the torsional-angle distributions and hydrogen bonds in the backbone suggests that the α-helical segments of native HEWL tend to show a propensity to convert to 3(10)-helical geometry in fully thionated HEWL. A comparison of the simulated quantities with experimental NMR data such as nuclear overhauser effect (NOE) atom-atom distance bounds and (3)J((H)(N)(H)(α))-couplings measured for native HEWL illustrates that the information content of these quantities with respect to the structural changes induced by thionation of the protein backbone is rather limited., (Copyright © 2012 The Protein Society.)

The use of a supra-molecular coarse-grained (CG) model for liquid water as solvent in molecular dynamics simulations of biomolecules represented at the fine-grained (FG) atomic level of modelling may reduce the computational effort by one or two orders of magnitude. However, even if the pure FG model and the pure CG model represent the properties of the particular substance of interest rather well, their application in a hybrid FG/CG system containing varying ratios of FG versus CG particles is highly non-trivial, because it requires an appropriate balance between FG-FG, FG-CG, and CG-CG energies, and FG and CG entropies. Here, the properties of liquid water are used to calibrate the FG-CG interactions for the simple-point-charge water model at the FG level and a recently proposed supra-molecular water model at the CG level that represents five water molecules by one CG bead containing two interaction sites. Only two parameters are needed to reproduce different thermodynamic and dielectric properties of liquid water at physiological temperature and pressure for various mole fractions of CG water in FG water. The parametrisation strategy for the FG-CG interactions is simple and can be easily transferred to interactions between atomistic biomolecules and CG water.

Implicit solvation is a mean force approach to model solvent forces acting on a solute molecule. It is frequently used in molecular simulations to reduce the computational cost of solvent treatment. In the first instance, the free energy of solvation and the associated solvent-solute forces can be approximated by a function of the solvent-accessible surface area (SASA) of the solute and differentiated by an atom-specific solvation parameter σ(i) (SASA). A procedure for the determination of values for the σ(i) (SASA) parameters through matching of explicit and implicit solvation forces is proposed. Using the results of Molecular Dynamics simulations of 188 topologically diverse protein structures in water and in implicit solvent, values for the σ(i) (SASA) parameters for atom types i of the standard amino acids in the GROMOS force field have been determined. A simplified representation based on groups of atom types σ(g) (SASA) was obtained via partitioning of the atom-type σ(i) (SASA) distributions by dynamic programming. Three groups of atom types with well separated parameter ranges were obtained, and their performance in implicit versus explicit simulations was assessed. The solvent forces are available at http://mathbio.nimr.mrc.ac.uk/wiki/Solvent_Forces.

Deriving structural information about a protein from NMR experimental data is still a non-trivial challenge to computational biochemistry. This is because of the low ratio of the number of independent observables to the number of molecular degrees of freedom, the approximations involved in the different relationships between particular observable quantities and molecular conformation, and the averaged character of the experimental data. For example, protein (3)J-coupling data are seldom used for structure refinement because of the multiple-valuedness and limited accuracy of the Karplus relationship linking a (3)J-coupling to a torsional angle. Moreover, sampling of the large conformational space is still problematic. Using the 99-residue protein plastocyanin as an example we investigated whether use of a thermodynamically calibrated force field, inclusion of solvent degrees of freedom, and application of adaptive local-elevation sampling that accounts for conformational averaging produces a more realistic representation of the ensemble of protein conformations than standard single-structure refinement in a non-explicit solvent using restraints that do not account for averaging and are partly based on non-observed data. Yielding better agreement with observed experimental data, the protein conformational ensemble is less restricted than when using standard single-structure refinement techniques, which are likely to yield a picture of the protein which is too rigid.

Structural knowledge about proteins is mainly derived from values of observables, measurable in NMR spectroscopic or X-ray diffraction experiments, i.e. absorbed or scattered intensities, through theoretically derived relationships between structural quantities such as atom positions or torsional angles on the one hand and observable quantities such as squared structure factor amplitudes, NOE intensities or (3) J-coupling constants on the other. The standardly used relation connecting (3) J-couplings to torsional angles is the Karplus relation, which is used in protein structure refinement as well as in the evaluation of simulated properties of proteins. The accuracy of the simple and generalised Karplus relations is investigated using side-chain structural and (3) J (αβ)-coupling data for three different proteins, Plastocyanin, Lysozyme, and FKBP, for which such data are available. The results show that the widely used Karplus relations are only a rough estimate for the relation between (3) J (αβ)-couplings and the corresponding χ(1)-angle in proteins.

Free energy calculations are described for the small copper-containing redox protein Azurin from Pseudomonas aeruginosa. A thermodynamic cycle connecting the reduced and oxidized states at pH 5 and pH 9 is considered, allowing for an assessment of convergence in terms of hysteresis and cycle closure. Previously published thermodynamic integration (TI) data is compared to Hamiltonian replica exchange TI (RE-TI) simulations using different simulation setups. The effects of varying simulation length, initial structure, position restraints on particular atoms, and the strength of temperature coupling are studied. Although the overall simulation times are too short to observe an experimentally described peptide plane rotation, it is found that RE-TI simulations do stimulate the distribution of conformational changes over the relevant values of the TI coupling parameter λ. This results in significantly improved values for hysteresis and cycle closure when compared to regular TI., (Copyright © 2012 Wiley Periodicals, Inc.)

For most liquids, the static relative dielectric permittivity is a decreasing function of temperature, because enhanced thermal motion reduces the ability of the molecular dipoles to orient under the effect of an external electric field. Monocarboxylic fatty acids ranging from acetic to octanoic acid represent an exception to this general rule. Close to room temperature, their dielectric permittivity increases slightly with increasing temperature. Herein, the causes for this anomaly are investigated based on molecular dynamics simulations of acetic and propionic acids at different temperatures in the interval 283-363 K, using the GROMOS 53A6(OXY) force field. The corresponding methyl esters are also considered for comparison. The dielectric permittivity is calculated using either the box-dipole fluctuation (BDF) or the external electric field (EEF) methods. The normal and anomalous temperature dependences of the permittivity for the esters and acids, respectively, are reproduced. Furthermore, in the EEF approach, the response of the acids to an applied field of increasing strength is found to present two successive linear regimes before reaching saturation. The low-field permittivity ε, comparable to that obtained using the BDF approach, increases with increasing temperature. The higher-field permittivity ε' is slightly larger, and decreases with increasing temperature. Further analyses of the simulations in terms of radial distribution functions, hydrogen-bonded structures, and diffusion properties suggest that increasing the temperature or the applied field strength both promote a relative population shift from cyclic (mainly dimeric) to extended (chain-like) hydrogen-bonded structures. The lower effective dipole moment associated with the former structures compared to the latter ones provides an explanation for the peculiar dielectric properties of the two acids compared to their methyl esters., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The performance of enveloping distribution sampling (EDS) simulations to estimate free enthalpy differences associated with seven alchemical transformations of A-T into G-C base pairs at the netropsin binding site in the minor groove of a 13-base pair DNA duplex in aqueous solution is evaluated. It is demonstrated that sufficient sampling can be achieved with a two-state EDS Hamiltonian even for large perturbations such as the simultaneous transformation of up to three A-T into three G-C base pairs. The two parameters required to define the EDS reference state Hamiltonian are obtained automatically using a modified version of a scheme presented in earlier work. The sensitivity of the configurational sampling to a variation of these parameters is investigated in detail. Although for relatively small perturbations, that is, one base pair, the free enthalpy estimate depends only weakly on the EDS parameters, the sensitivity is stronger for the largest perturbation. Yet, EDS offers various convenient measures to evaluate the degree of sampling and thus the reliability of the free enthalpy estimate and appears to be an efficient alternative to the conventional thermodynamic integration methodology to obtain free energy differences for molecular systems., (Copyright © 2012 Wiley Periodicals, Inc.)

The time- and length-scale accessible to molecular dynamics simulations of biomolecular systems using atomic-level (AL) models is most limited by the calculation of the solvent-solvent interactions, which comprise the majority of the interactions and yet are seldom of specific interest. Coarse-graining (CG), in which multiple solvent molecules are subsumed into a single bead, provides a means of overcoming this limitation without resorting to implicit solvation models, which basically misrepresent the hydrophobic effect. Most existing CG models, however, do not explicitly include electrostatic interactions, and thus fail to reproduce important properties of the solvent such as dielectric screening. Moreover, CG models for one type of solvent molecule are seldom compatible with those for other solvents. Here, we develop polarizable CG models for the solvents dimethyl sulfoxide, chloroform, and methanol that are compatible with an existing CG model for water. The inclusion of polarizability greatly improves the reproduction of thermodynamic data measured experimentally and calculated from AL simulations for both the pure liquids and binary mixtures.

During the past years, the use of combined quantum-classical, QM/MM, methods for the study of complex biomolecular processes, such as enzymatic reactions and photocycles, has increased considerably. The quality of the results obtained from QM/MM calculations is largely dependent on five aspects to be considered when setting up a molecular model: the QM Hamiltonian, the MM Hamiltonian or force field, the boundary and coupling between the QM and MM regions, the size of the QM region and the boundary condition for the MM region. In this study, we systematically investigate the influence of a variation of the molecular mechanics force field and the size of the QM region in QM/MM MD simulations on properties of the photoactive part of the blue light photoreceptor protein AppA. For comparison, we additionally performed classical MD simulations and studied the effect of a variation of the type of spatial boundary condition. The classical boundary conditions and the force field used in a QM/MM MD simulation are shown to have non-neglegible effects upon the structural and energetic properties of the protein which makes it advisable to minimize computational artifacts in QM/MM MD simulations by application of periodic boundary conditions and a thermodynamically calibrated force field. A comparison of the structural and energetic properties of MD simulations starting from two alternative, different X-ray structures for the blue light utilizing flavin protein in its dark state indicates a slight preference of the two force fields used for the so-called Anderson structure over the Jung structure., (Copyright © 2011 Wiley Periodicals, Inc.)

Since the most recent description of the functionalities of the GROMOS software for biomolecular simulation in 2005 many new functions have been implemented. In this article, the new functionalities that involve modified forces in a molecular dynamics (MD) simulation are described: the treatment of electronic polarizability, an implicit surface area and internal volume solvation term to calculate interatomic forces, functions for the GROMOS coarse-grained supramolecular force field, a multiplicative switching function for nonbonded interactions, adiabatic decoupling of a number of degrees of freedom with temperature or force scaling to enhance sampling, and nonequilibrium MD to calculate the dielectric permittivity or viscosity. Examples that illustrate the use of these functionalities are given., (Copyright © 2011 Wiley Periodicals, Inc.)

Connecting experimental observables with the underlying conformational ensemble is a long-standing problem in the structure determination of biomolecules. The simulations described in this article attempt to resolve a seeming discrepancy between the conformational features derived from measured NOE intensities, (3)J-coupling constants, and circular dichroism (CD) spectra for two β-peptides differing in a linker between two side-chains. Although both peptides are very similar in terms of the r(-6) averaged distances between atom pairs involved in the observed NOEs, the molecular dynamics trajectories suggest why the CD spectra show a greater 3(14)-helical propensity for the linked, cyclic peptide than for the linear one, whereas slightly more NMR NOE peaks are observed and assigned for the latter. The nine 100 ns unrestrained simulations show better agreement with the observed experimental data than the single conformations derived from the published NMR structures by additional energy minimization with the GROMOS force field. They show why the seemingly contradictory quantities obtained by NMR and CD spectroscopy can arise from a single conformational ensemble., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Prediction and understanding of the folding and stability of the 3D structure of proteins is still a challenge. The different atomic interactions, such as non polar contacts and hydrogen bonding, are known but their exact relative weights and roles when contributing to protein folding and stability are not identified. Initiated by a previous molecular dynamics simulation of fully ester-linked hen egg white lysozyme (HEWL), which showed a more compact fold of the ester-linked molecule compared to the native one, three variants of this protein are analyzed in the present study. These are 129-residue native HEWL, partly ester-linked HEWL, in which only 34 peptide linkages that are not involved in the helical or β-strand parts of native HEWL were replaced by ester linkages, and fully (126 residues) ester-linked HEWL. Native and partly ester-linked HEWL showed comparable behaviour, whereas fully ester-linked HEWL could not maintain the native secondary structure of HEWL in the simulation and adopted a more compact fold. The conformational changes were analyzed by comparing simulation averaged values of quantities that can be measured by NMR, such as (1)H-(15)N backbone order parameters, residual dipolar couplings, proton-proton NOE distances and (3)J-couplings with the corresponding values derived from experimental NMR data for native HEWL. The information content of the latter appeared to be insufficient to detect the local conformational rearrangements upon esterification of the loop regions of the protein. For fully ester-linked HEWL, a significantly reduced agreement was observed. Upon esterification, the backbone-side chain and side chain-side chain hydrogen-bonding pattern of HEWL changes to maintain its compactness and thus the structural stability of the ester-linked lysozymes., (© 2011 The Authors Journal compilation © 2011 FEBS.)

The remarkable recent creation of two proteins that fold into two completely different and stable structures, exhibit different functions, yet differ by only a few amino acids poses a conundrum to those hoping to understand how sequence encodes structure. Here, computer modeling uniquely allows the characterization of not only the native structure of each minimally different sequence but also systems in which each sequence was modeled onto the fold of the alternate sequence. The reasons for the different structural preferences of two pairs of highly similar sequences are explored by a combination of structure analyses, comparison of potential energies calculated from energy-minimized single structures and trajectories produced from molecular dynamics simulations, and application of a novel method for calculating free energy differences. The sensitivity of such analyses to the choice of force field is also explored. Many of the hypotheses proposed on the basis of the nuclear magnetic resonance model structures of the proteins with 95% identical sequences are supported. However, each level of analysis provides different predictions regarding which sequence-structure combination should be most favored, highlighting the fact that protein structure and stability result from a complex combination of interdependent factors.

Enveloping distribution sampling (EDS) is a powerful method to compute relative free energies from simulation. So far, the EDS method has only been applied to alchemical free energy differences, i.e., between different Hamiltonians defining different systems, and not yet to obtain free energy differences between different conformations or conformational states of a system. In this article, we extend the EDS formalism such that it can be applied to compute free energy differences of different conformations and apply it to compute the relative free enthalpy ΔG of 310-, α-, and π-helices of an alanine deca-peptide in explicit water solvent. The resulting ΔG values are compared to those obtained by standard thermodynamic integration (TI) and from so-called end-state simulations. A TI simulation requires the definition of a λ-dependent pathway which in the present case is based on hydrogen bonds of the different helical conformations. The values of ⟨(∂VTI)/(∂λ)⟩λ show a sharp change for a particular range of λ values, which is indicative of an energy barrier along the pathway, which lowers the accuracy of the resulting ΔG value. In contrast, in a two-state EDS simulation, an unphysical reference-state Hamiltonian which connects the parts of conformational space that are relevant to the different end states is constructed automatically; that is, no pathway needs to be defined. In the simulation using this reference state, both helices were sampled, and many transitions between them occurred, thus ensuring the accuracy of the resulting free enthalpy difference. According to the EDS simulations, the free enthalpy differences of the π-helix and the 310-helix versus the α-helix are 5 kJ mol(-1) and 47 kJ mol(-1), respectively, for an alanine deca-peptide in explicit SPC water solvent using the GROMOS 53A6 force field. The EDS method, which is a particular form of umbrella sampling, is thus applicable to compute free energy differences between conformational states as well as between systems and has definite advantages over the traditional TI and umbrella sampling methods to compute relative free energies.

The modulation of the properties of lipid membranes by polyhydroxylated cosolutes such as sugars is a phenomenon of considerable biological, technological and medicinal relevance. A few years ago, we proposed the sugar-like mechanism--binding driven by the release of water molecules--as an attempt to rationalize the preferential affinity of carbohydrate molecules compared to water molecules for the surface of lipid bilayers, which is presumably related to the bioprotective action of these compounds. The goal herein is to gain a better understanding of the driving force underlying this mechanism, in terms of specific interactions or effects, as well as in terms of the energy-entropy partitioning. This is done in the simplest possible context of an apolar rigid-wall model representing the membrane, and mixtures of closely related and possibly artificial species in solution, namely monomers or dimers of Lennard-Jones particles, water with physical or reduced charges, and hydroxymethyl groups. The results indicate that although the sugar-like mechanism seems phenomenologically reasonable, the main driving force underlying this mechanism is not the entropy gain upon releasing water molecules into the bulk, as originally suggested, but rather the hydrophobic effect. Note that the latter effect is a generic concept and may in principle involve both a solvent release and an interaction component, depending on the solute considered., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The calculation of the relative free energies of ligand-protein binding, of solvation for different compounds, and of different conformational states of a polypeptide is of considerable interest in the design or selection of potential enzyme inhibitors. Since such processes in aqueous solution generally comprise energetic and entropic contributions from many molecular configurations, adequate sampling of the relevant parts of configurational space is required and can be achieved through molecular dynamics simulations. Various techniques to obtain converged ensemble averages and their implementation in the GROMOS software for biomolecular simulation are discussed, and examples of their application to biomolecules in aqueous solution are given., (© 2011 American Chemical Society)

The ability to design well-folding β-peptides with a specific biological activity requires detailed insight into the relationship between the β-amino acid sequence and the dominant three-dimensional structure of such a peptide. To this end, secondary structure preferences of two sets of 16 β-peptides were investigated by means of one-step perturbation using molecular dynamics (MD) simulations. For each set of peptides, two reference-state simulations and one perturbed-state simulation were carried out to predict the secondary structure preferences for the other 15 peptides. The results show that the substitution of a methyl group in the third or fourth residue stabilizes the left-handed 3(14)-helix over the right-handed 2.7(10/12)-helix for the set of hexapeptides A; for the set of heptapeptides B, having methyl substitutions at both β- and α-carbon positions of the fourth or fifth residue stabilizes the left-handed 3(14)-helix over the right-handed 2.5(12)-helix. Not only the side-chain substitution pattern but also the side-chain composition affects the relative stability of different secondary structures. The approach described here may be of use in peptide design with an eye to obtaining peptides with particular folds and biological activities.

For the understanding of cellular processes the molecular structure of biomolecules has to be accurately determined. Initial models can be significantly improved by structure refinement techniques. Here, we present the refinement methods and analysis techniques implemented in the GROMOS software for biomolecular simulation. The methodology and some implementation details of the computation of NMR NOE data, (3)J-couplings and residual dipolar couplings, X-ray scattering intensities from crystals and solutions and neutron scattering intensities used in GROMOS is described and refinement strategies and concepts are discussed using example applications. The GROMOS software allows structure refinement combining different types of experimental data with different types of restraining functions, while using a variety of methods to enhance conformational searching and sampling and the thermodynamically calibrated GROMOS force field for biomolecular simulation., (© Springer Science+Business Media B.V. 2011)

GROMOS++ is a set of C++ programs for pre- and postprocessing of molecular dynamics simulation trajectories and as such is part of the GROningen MOlecular Simulation software for (bio)molecular simulation. It contains more than 70 programs that can be used to prepare data for the production of molecular simulation trajectories and to analyze these. These programs are reviewed and the various structural, dynamic, and thermodynamic quantities that can be analyzed using time series, correlation functions, and distributions are described together with technical aspects of their implementation in GROMOS. A few examples of the use of GROMOS++ for the analysis of MD trajectories are given. A full list of all GROMOS++ programs, together with an indication of their capabilities, is given in the Appendix .

A method for conformational Boltzmann sampling of loops in proteins in aqueous solution is presented that is based on adiabatic decoupling molecular dynamics (MD) simulation with temperature or force scaling. To illustrate the enhanced sampling, the loop from residues 33 to 43 in the bovine protein ribonuclease A is adiabatically decoupled from the rest of the protein and the solvent with a mass scaling factor s(m) =1000 and the sampling is enhanced with a scaling of the temperature using s(T) =2 or of the force using s(V) =0.667. Over 5 ns of simulation the secondary structure of the protein remains unaltered while a combined dihedral-angle conformational cluster analysis shows an increase of conformations outside the first most populated cluster of loop conformations for adiabatic decoupling MD with temperature scaling using s(T) =2 or force scaling using s(V) =0.667 compared to the standard MD simulation. The atom-positional root-mean-square fluctuations of the C(α) atoms of the loop show an increase in the movement of the loop as well, indicating that adiabatic decoupling MD with upscaling of the temperature or downscaling of the force is a promising method for conformational Boltzmann sampling., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)