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α-Helical membrane-active antimicrobial peptides (AMPs) are known to act via a range of mechanisms, including the formation of barrel-stave and toroidal pores and the micellisation of the membrane (carpet mechanism). Different mechanisms imply that the peptides adopt different 3D structures when bound at the water-membrane interface, a highly amphipathic environment. Here, an evolutionary algorithm is used to predict the 3D structure of a range of α-helical membrane-active AMPs at the water-membrane interface by optimising amphipathicity. This amphipathic structure prediction (ASP) is capable of distinguishing between curved and linear peptides solved experimentally, potentially allowing the activity and mechanism of action of different membrane-active AMPs to be predicted. The ASP algorithm is accessible via a web interface at http://atb.uq.edu.au/asp/.

Background and Purpose The spider‐venom peptide PcTx1 is the most potent and selective inhibitor of acid‐sensing ion channel (ASIC) 1a. It has centrally acting analgesic activity and is neuroprotective in rodent models of ischaemic stroke. Understanding the molecular details of the PcTx1 : ASIC1a interaction should facilitate development of therapeutically useful ASIC1a modulators. Previously, we showed that several key pharmacophore residues of PcTx1 reside in a dynamic β‐hairpin loop; conclusions confirmed by recent crystal structures of the complex formed between PcTx1 and chicken ASIC1 (cASIC1). Numerous peptide : channel contacts were observed in these crystal structures, but it remains unclear which of these are functionally important.

Phox-homology (PX) domains target proteins to the organelles of the secretary and endocytic systems by binding to phosphatidylinositol phospholipids (PIPs). Among all the structures of PX domains that have been solved, only three have been solved in a complex with the main physiological ligand: PtdIns3P. In this work, molecular dynamic simulations have been used to explore the structure and dynamics of the p40(phox)-PX domain and the SNX17-PX domain and their interaction with membrane-bound PtdIns3P. In the simulations, both PX domains associated spontaneously with the membrane-bound PtdIns3P and formed stable complexes. The interaction between the p40(phox)-PX domain and PtdIns3P in the membrane was found to be similar to the crystal structure of the p40(phox)-PX-PtdIns3P complex that is available. The interaction between the SNX17-PX domain and PtdIns3P was similar to that observed in the p40(phox)-PX-PtdIns3P complex; however, some residues adopted different orientations. The simulations also showed that nonspecific interactions between the beta 1-beta 2 loop and the membrane play an important role in the interaction of membrane bound PtdIns3P and different PX domains. The behaviour of unbound PtdIns3P within a 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC) membrane environment was also examined and compared to the available experimental data and simulation studies of related molecules. (C) 2014 Wiley Periodicals, Inc.

Phosphorescent emissive materials in organic light-emitting diodes (OLEDs) manufactured using evaporation are usually blended with host materials at a concentration of 3–15 wt% to avoid concentration quenching of the luminescence. Here, experimental measurements of hole mobility and photoluminescence are related to the atomic level morphology of films created using atomistic nonequilibrium molecular dynamics simulations mimicking the evaporation process with similar guest concentrations as those used in operational test devices. For blends of fac-tris[2-phenylpyridinato-C2,N]iridium(III) [Ir(ppy)] in tris(4-carbazoyl-9-ylphenyl)amine (TCTA), it is found that clustering of the Ir(ppy) (surface of the molecules within ≈0.4 nm) in the simulated films is directly relatable to the experimentally-measured hole mobility. Films containing 1–10 wt% of Ir(ppy) in TCTA have a mobility of up to two orders of magnitude lower (≈10 cm V s) than the neat TCTA film, which is consistent with the Ir(ppy) molecules acting as hole traps due to their smaller ionization potential. Comparison of the simulated film morphologies with the measured photoluminescence properties shows that for luminescence quenching to occur, the Ir(ppy) molecules have to have their ligands partially overlapping. Thus, the results show that the effect of guest interactions on charge transport and luminescence are markedly different for OLED light-emitting layers.

The antibiotic vancomycin targets lipid II, blocking cell wall synthesis in Gram-positive bacteria. Despite extensive study, questions remain regarding how it recognizes its primary ligand and what is the most biologically relevant form of vancomycin. In this study, molecular dynamics simulation techniques have been used to examine the process of ligand binding and dimerization of vancomycin. Starting from one or more vancomycin monomers in solution, together with different peptide ligands derived from lipid II, the simulations predict the structures of the ligated monomeric and dimeric complexes to within 0.1 nm rmsd of the structures determined experimentally. The simulations reproduce the conformation transitions observed by NMR and suggest that proposed differences between the crystal structure and the solution structure are an artifact of the way the NMR data has been interpreted in terms of a structural model. The spontaneous formation of both back-to-back and face-to-face dimers was observed in the simulations. This has allowed a detailed analysis of the origin of the cooperatively between ligand binding and dimerization and suggests that the formation of face-to-face dimers could be functionally significant. The work also highlights the possible role of structural water in stabilizing the vancomycin ligand complex and its role in the manifestation of vancomycin resistance.

The efficiency of using a variant of Hamiltonian replica-exchange molecular dynamics (Chaperone H-replica-exchange molecular dynamics [CH-REMD]) for the refinement of protein structural models generated de novo is investigated. In CH-REMD, the interaction between the protein and its environment, specifically, the electrostatic interaction between the protein and the solvating water, is varied leading to cycles of partial unfolding and refolding mimicking some aspects of folding chaperones. In 10 of the 15 cases examined, the CH-REMD approach sampled structures in which the root-mean-square deviation (RMSD) of secondary structure elements (SSE-RMSD) with respect to the experimental structure was more than 1.0 A lower than the initial de novo model. In 14 of the 15 cases, the improvement was more than 0.5 A. The ability of three different statistical potentials to identify near-native conformations was also examined. Little correlation between the SSE-RMSD of the sampled structures with respect to the experimental structure and any of the scoring functions tested was found. The most effective scoring function tested was the DFIRE potential. Using the DFIRE potential, the SSE-RMSD of the best scoring structures was on average 0.3 A lower than the initial model. Overall the work demonstrates that targeted enhanced-sampling techniques such as CH-REMD can lead to the systematic refinement of protein structural models generated de novo but that improved potentials for the identification of near-native structures are still needed.

The possibility of estimating equilibrium free-energy profiles from multiple non-equilibrium simulations using the fluctuation-dissipation theory or the relation proposed by Jarzynski has attracted much attention. Although the Jarzynski estimator has poor convergence properties for simulations far from equilibrium, corrections have been derived for cases in which the work is Gaussian distributed. Here, we examine the utility of corrections proposed by Gore and collaborators using a simple dissipative system as a test case. The system consists of a single methane-like particle in explicit water. The Jarzynski equality is used to estimate the change in free energy associated with pulling the methane particle a distance of 3.9 nm at rates ranging from similar to 0.1 to 100 ms(-1). It is shown that although the corrections proposed by Gore and collaborators have excellent numerical performance, the profiles still converge slowly. Even when the corrections are applied in an ideal case where the work distribution is necessarily Gaussian, performing simulations under quasi-equilibrium conditions is still most efficient. Furthermore, it is shown that even for a single methane molecule in water, pulling rates as low as 1 ms(-1) can be problematic. The implications of this finding for studies in which small molecules or even large biomolecules are pulled through inhomogeneous environments at similar pulling rates are discussed.

Atomistic molecular dynamics simulations have been used to investigate the conformational changes associated with the binding of human growth hormone (hGH) to the extracellular domains (ECD) of the human growth hormone receptor (hGHR), thereby shedding light on the mechanism of activation. It is shown that the removal of hGH from the hormone-bound receptor complex results in a counter-clockwise rotation of the twosubunits relative to each other by 30°–64° (average 45° ± 14°), in close agreement in terms of both the magnitude and direction of the rotation with that proposed based on mutagenesis experiments. In addition to providing evidence to support a rotational activation mechanism, the simulations have enabled the nature of the interaction interfaces in both the cytokine-bound and unliganded hGHR states to be analyzed in detail. Proteins 2010. © 2009 Wiley-Liss, Inc.

A new force field for the simulation of dipalmitoylphosphatidylcholine (DPPC) in the liquid-crystalline, fluid phase at zero surface tension is presented. The structure of the bilayer with the area per lipid (0.629 nm(2); experiment 0.629-0.64 nm(2)), the volume per lipid (1.226 nm(3); experiment 1.229-1.232 nm(3)), and the ordering of the palmitoyl chains (order parameters) are all in very good agreement with experiment. Experimental electron density profiles are well reproduced in particular with regard to the penetration of water into the bilayer. The force field was further validated by simulating the spontaneous assembly of DPPC into a bilayer in water. Notably, the timescale on which membrane sealing was observed using this model appears closer to the timescales for membrane resealing suggested by electroporation experiments than previous simulations using existing models.

When estimating binding affinities of a ligand, which can exists in multiple forms, for a target molecule, one must consider all possible competing equilibria. Here, a method is presented that estimates the contribution of the protonation equilibria of a ligand in solution to the measured or calculated binding affinity. The method yields a correction to binding constants that are based on the total concentration of inhibitor (the sum of all ionized forms of the inhibitor in solution) to account for the complexed form of the inhibitor only. The method is applied to the calculation of the difference in binding affinity of two inhibitors, 2-phosphoglycolate (PGA) and its phoshonate analog 3-phosphonopropionate (3PP), for the glycolytic enzyme triosephosphate isomerase. Both inhibitors have three titrating sites and exist in solution as a mixture of different forms. In this case the form that actually binds to the enzyme is present at relative low concentrations. The contributions of the alternative forms to the difference in binding energies is estimated by means of molecular dynamics simulations and corrections. The inhibitors undergo a pKa shift upon binding that is estimated by ab initio calculations. An interesting finding is that the affinity difference of the two inhibitors is not due to different interactions in the active site of the enzyme, but rather due to the difference in the solvation properties of the inhibitors. Protein 2009. © 2008 Wiley-Liss, Inc.

A series of molecular dynamics simulations in explicit solvent were started from nine structural models of the transition state of the SH3 domain of alpha-spectrin, which were generated by Lindorff Larsen et al. (Nat Struct Mol Biol 2004;11:443-449) using molecular dynamics simulations in which experimental (Phi-values were incorporated as restraints. Two of the nine models were simulated 10 times for 200 ns and the remaining models simulated two times for 200 us. Complete folding was observed in one case, while in the other simulations partial folding or unfolding even ts were observed, which were characterized by a regularization of elements of secondary structure. These results are consistent with recent experimental evidence that the folding of SH3 domains involves low populated intermediate states. (C) 2007 Wiley-Liss, Inc.

The Death Receptor 5 (DR5), a member of tumor necrosis factor receptor (TNFR) superfamily of receptors, triggers apoptosis (programmed cell death) when stimulated by its tridentate ligand TRAIL. Until recently it was generally assumed that the activation of DR5 resulted from the recruitment of three independent receptor units, leading to the trimerization of intracellular domains. However, there is mounting evidence to suggest that, in the absence of ligand, such cytokine receptors primarily reside as preformed complexes. In this work, molecular dynamics simulations of the TRALL-DR5 complex, the unbound receptor trimer and individual receptor monomers are compared to gain insight in the mechanism of activation. The results suggest that, in the absence of TRAIL, DR5 has a strong propensity to self-associate and that this is primarily mediated through interactions of the membrane proximal domains. The association of the free receptors leads to a loss of the threefold symmetry found within the receptor-ligand complex. The simulations suggest that the primary role of TRAIL is to induce threefold-symmetry within the DR5 complex and to constrain the receptor to a specific conformation. The implications of this in terms of the mechanism by which the receptor switches from an inactive to an active state are discussed.

Hydrophobins are small ( approximately 100 aa) proteins that have an important role in the growth and development of mycelial fungi. They are surface active and, after secretion by the fungi, self-assemble into amphipathic membranes at hydrophobic/hydrophilic interfaces, reversing the hydrophobicity of the surface. In this study, molecular dynamics simulation techniques have been used to model the process by which a specific class I hydrophobin, SC3, binds to a range of hydrophobic/hydrophilic interfaces. The structure of SC3 used in this investigation was modeled based on the crystal structure of the class II hydrophobin HFBII using the assumption that the disulfide pairings of the eight conserved cysteine residues are maintained. The proposed model for SC3 in aqueous solution is compact and globular containing primarily beta-strand and coil structures. The behavior of this model of SC3 was investigated at an air/water, an oil/water, and a hydrophobic solid/water interface. It was found that SC3 preferentially binds to the interfaces via the loop region between the third and fourth cysteine residues and that binding is associated with an increase in alpha-helix formation in qualitative agreement with experiment. Based on a combination of the available experiment data and the current simulation studies, we propose a possible model for SC3 self-assembly on a hydrophobic solid/water interface.

The effect of the box shape on the dynamic behavior of proteins simulated under periodic boundary conditions is evaluated. In particular, the influence of simulation boxes defined by the near-densest lattice packing (NDLP) in conjunction with rotational constraints is compared to that of standard box types without these constraints. Three different proteins of varying size, shape, and secondary structure content were examined in the study. The statistical significance of differences in RMSD, radius of gyration, solvent-accessible surface, number of hydrogen bonds, and secondary structure content between proteins, box types, and the application or not of rotational constraints has been assessed. Furthermore, the differences in the collective modes for each protein between different boxes and the application or not of rotational constraints have been examined. In total 105 simulations were performed, and the results compared using a three-way multivariate analysis of variance (MANOVA) for properties derived from the trajectories and a three-way univariate analysis of variance (ANOVA) for collective modes. It is shown that application of roto-translational constraints does not have a statistically significant effect on the results obtained from the different simulations. However, the choice of simulation box was found to have a small (5-10%), but statistically significant effect on the behavior of two of the three proteins included in the study. (c) 2005 Wiley Periodicals, Inc.

Bacterial chaperonin, GroEL, together with its co-chaperonin, GroES, facilitates the folding of a variety of polypeptides. Experiments suggest that GroEL stimulates protein folding by multiple cycles of binding and release. Misfolded proteins first bind to an exposed hydrophobic surface on GroEL. GroES then encapsulates the substrate and triggers its release into the central cavity of the GroEL/ES complex for folding. In this work, we investigate the possibility to facilitate protein folding in molecular dynamics simulations by mimicking the effects of GroEL/ES namely, repeated binding and release, together with spatial confinement. During the binding stage, the (metastable) partially folded proteins are allowed to attach spontaneously to a hydrophobic surface within the simulation box. This destabilizes the structures, which are then transferred into a spatially confined cavity for folding. The approach has been tested by attempting to refine protein structural models generated using the ROSETTA procedure for ab initio structure prediction. Dramatic improvements in regard to the deviation of protein models from the corresponding experimental structures were observed. The results suggest that the primary effects of the GroEL/ES system can be mimicked in a simple coarse-grained manner and be used to facilitate protein folding in molecular dynamics simulations. Furthermore, the results Sur port the assumption that the spatial confinement in GroEL/ES assists the folding of encapsulated proteins.

HIV-1 protease is most active under weakly acidic conditions (pH 3.5-6.5), when the catalytic Asp25 and Asp25' residues share 1 proton. At neutral pH, this proton is lost and the stability of the structure is reduced. Here we present an investigation of the effect of pH on the dynamics of HIV-1 protease using MD simulation techniques. MD simulations of the solvated HIV-1 protease with the Asp25/25' residues monoprotonated and deprotonated have been performed. In addition we investigated the effect of the inclusion of Na+ and Cl- ions to mimic physiological salt conditions. The simulations of the monoprotonated form and deprotonated form including Na+ show very similar behavior. In both cases the protein remained stable in the compact, "self-blocked" conformation in which the active site is blocked by the tips of the flaps. In the deprotonated system a Na+ ion binds tightly to the catalytic dyad shielding the repulsion between the COO- groups. Ab initio calculations also suggest the geometry of the active site with the Na+ bound closely resembles that of the monoprotonated case. In the simulations of the deprotonated form (without Na+ ions), a water molecule bound between the Asp25 Asp25' side-chains. This disrupted the dimerization interface and eventually led to a fully open conformation. (C) 2004 Wiley-Liss, Inc.

The incorporation of explicit ions to mimic the effect of ionic strength or to neutralize the overall charge on a system in free energy calculations using molecular dynamics simulations is investigated. The difference in the free energy of hydration between two triosephosphate isomerase inhibitors calculated at five different ion concentrations is used as an example. We show that the free energy difference can be highly sensitive to the presence of explicit ions even in cases where the mutation itself does not involve a change in the overall charge. The effect is most significant if the molecule carries a net charge close to the site mutated. Furthermore, it is shown that the introduction of a small number of ions can lead to very severe sampling problems suggesting that in practical calculations convergence can best be achieved by incorporating either no counterions or by simulating at high ionic strength to ensure sufficient sampling of the ion distribution. (C) 2004 Wiley Periodicals, Inc.

Molecular dynamics simulation techniques together with time-dependent density functional theory calculations have been used to investigate the effect of photon absorption by a 4-hydroxy-cinnamic acid chromophore on the structural properties of the photoactive yellow protein (PYP) from Ectothiorodospira halophila. The calculations suggest that the protein not only modifies the absorption spectrum of the chromophore but also regulates the subsequent isomerization of the chromophore by stabilizing the isomerization transition state. Although signaling from PYP is thought to involve partial unfolding of the protein, the mechanical effects accompanying isomerization do not appear to directly destabilize the protein.

The ability of the GROMOS96 force field to reproduce partition constants between water and two less polar solvents (cyclohexane and chloroform) for analogs of 18 of the 20 naturally occurring amino acids has been investigated. The estimations of the solvation free energies in water, in cyclohexane solution, and chloroform solution are based on thermodynamic integration free energy calculations using molecular dynamics simulations. The calculations show that while the force field reproduces the experimental solvation free energies of nonpolar analogs with reasonable accuracy the solvation free energies of polar analogs in water are systematically overestimated (too positive). The dependence of the calculated free energies on the atomic partial charges was also studied. (C) 2002 Wiley Periodicals, Inc.

The dynamics of the three-stranded beta-sheet peptide Betanova has been studied at four different temperatures (280, 300, 350, and 450 K by molecular dynamics simulation techniques, in explicit water. Two 20-ns simulations at 280 K indicate that the peptide remains very flexible under "folding" conditions sampling a range of conformations that together satisfy the nuclear magnetic resonance (NMR)-derived experimental constraints. Two simulations at 300 K (above the experimental folding temperature) of 20 ns each show partial formation of "native"-like structure, which also satisfies most of the NOE constraints at 280 K. At higher temperature, the presence of compact states, in which a series of hydrophobic contacts remain present, are observed. This is consistent with experimental observations regarding the role of hydrophobic contacts in determining the peptide's stability and in initiating the formation of turns and loops. A set of different structures is shown to satisfy NMR-derived distance restraints and a possible mechanism for the folding of the peptide into the NMR-determined structure is proposed.

We present entropy estimates based on molecular dynamics simulations of models of the molten globule state of the protein alpha-lactalbumin at low pH. The entropy calculations use the covariance matrix of atom-positional fluctuations and yield the complete configurational entropy. The configurational entropy of the entire protein and of each of its side chains is calculated. Exposed side chains show a larger entropy compared to buried side chains. A comparison to data from rotamer counting is made and significant differences are found.

To evaluate the ability of molecular dynamics (MD) simulations using atomic force-fields to correctly predict stable folded conformations of a peptide in solution, we show results from MD simulations of the reversible folding of an octapeptide rich in alpha-aminoisobutyric acid (2-amino-2-methyl-propanoic acid, Aib) solvated in di-methyl-sulfoxide (DMSO). This solvent generally prevents the formation of secondary structure, whereas Aib-rich peptides show a high propensity to form secondary structural elements, in particular 3(10)- and alpha-helical structures. Aib is, moreover, achiral, so that Aib-rich peptides can form left- or right-handed helices depending on the overall composition of the peptide, the temperature, and the solvation conditions. This makes the system an interesting case to study the ensembles of peptide conformations as a function of temperature by MD simulation. Simulations involving the folding and unfolding of the peptide were performed starting from two initial structures, a right-handed alpha-helical structure and an extended structure, at three temperatures, 298 K, 340 K, and 380 K, and the results are compared with experimental nuclear magnetic resonance (NMR) data measured at 298 K and 340 K. The simulations generally reproduce the available experimental nuclear Overhauser effect (NOE) data, even when a wide range of conformations is sampled at each temperature. The importance of adequate statistical sampling in order to reliably interpret the experimental data is discussed.

The configurational entropy of a beta -heptapeptide in solution at four different temperatures is calculated, The contributions of the backbone and of the side-chain atoms to the total peptide entropy are analyzed separately and the effective contribution to the entropy arising from correlations between these terms determined. The correlation between the backbone and side-chain atoms amounts to about 17% and is rather insensitive to the temperature. The correlation of motion within the backbone and within side-chains is much larger and decreases with temperature. As the peptide reversibly folds at higher temperatures, its change in entropy and enthalpy upon folding is analyzed. The change in entropy and enthalpy upon folding of the peptide alone cannot account for the observed change in free energy on folding of the peptide in solution. Enthalpic and entropic contributions of the solvent thus also play a key role. Proteins 2001; 43:45-56. (C) 2001 Wiley Liss, Inc.

The effect of motional averaging when relating structural properties inferred from nuclear magnetic resonance (NMR) experiments to molecular dynamics simulations of peptides is considered. In particular, the effect of changing populations of conformations, the extent of sampling, and the sampling frequency on the estimation of nuclear Overhauser effect (NOE) inter-proton distances, vicinal (3)J-coupling constants, and chemical shifts are investigated. The analysis is based on 50-ns simulations of a beta-heptapeptide in methanol at 298 K, 340 K, 350 K, and 360 K. This peptide undergoes reversible folding and samples a significant proportion of the available conformational space during the simulations, with at 298 K being predominantly folded and at 360 K being predominantly unfolded. The work highlights the fact that when motional averaging is included, NMR data has only limited capacity to distinguish between a single fully folded peptide conformation and various mixtures of folded and unfolded conformations. Proteins 1999;36:542-555.

The thermodynamics of folding and unfolding of a beta-heptapeptide in methanol solution has been studied at four different temperatures, 298 K, 340 K, 350 K, and 360 K, by molecular dynamics simulation. At each of these temperatures, the 50-ns simulations were sufficient to generate an equilibrium distribution between a relatively small number of conformations (approximately 10(2)), showing that, even above the melting temperature (approximately 340 K), the peptide does not randomly sample conformational space. The free energy of folding and the free energy difference between pairs of conformations have been calculated from their relative populations. The experimentally determined folded conformation at 298 K, a left-handed 3(1)-helix, is at each of the four temperatures the predominant conformation, with its probability and average lifetime decreasing with increasing temperature. The most common intermediates of folding and unfolding are also the same at the four temperatures. Paths and rates of interconversion between different conformations have been determined. It has been found that folding can occur through multiple pathways, not necessarily downhill in free energy, although the final step involves a reduced number of intermediates.

Die akkurate Reproduktion des Mechanismus der reversiblen Peptidfaltung in Losung sowie von Konformationsunterschieden als Funktion der Aminosaurensequenz ermoglichen Molekuldynamik-Simulationen bei atomarer Auflosung. So wurde fur das Hepta-β-peptid 1 (Molekulmodell siehe Bild) eine linksgangige 31-Helixstruktur und fur das Hexa-β-peptid 2 eine rechtsgangige Helix als Faltungsmuster vorhergesagt, was durch Vergleich mit den NMR-spektroskopisch bestimmten Strukturen bestatigt wurde.

Mol. dynamics simulation studies on the folding of beta-peptides H-beta3-HVal-beta3-HAla-beta3-HLeu-(S,S)-beta3-HAla(alphaMe)-beta3-HVal-beta3-HAla-beta3-HLeu-OH and H-beta2-HVal-beta3-HAla-beta2-HLeu-beta3-HVal-beta2-HAla-beta3-HLeu-OH were carried out. Despite the small differences in sequence between the two peptides studied, the simulations correctly predict a left-handed 31-helical fold for the beta-heptapeptide and a right-handed helical fold for the beta-hexapeptide.

Modeling the mechanisms of protofibril twisting: Molecular-dynamics simulations of simian viral peptide aggregates show that β sheets of 10 to 30 chains form left-handed helical ribbons with saddlelike curvature (see picture). These structures are highly dynamic, with oscillations around an average twist angle of 9-10°, and a pitch of 15-20 nm, depending on β-sheet length. The peptides studied are key to viral entry into host cells.

Four methods are compared to drive the unfolding of a protein: (1) high tem- perature (T-run), (2) high pressure (P-run), (3) by imposing a gradual increase in the mean ra- dius of the protein using a penalty function added to the physical interaction function (F- run, radial force driven unfolding), and (4) by weak coupling of the difference between the temperature of the radially outward moving at- oms and the radially inward moving atoms to an external temperature bath (K-run, kinetic energy driven unfolding). The characteristic features of the four unfolding pathways are an- alyzed in order to detect distortions due to the size or the type of the applied perturbation, as well as the features that are common to all of them. Hen egg white lysozyme is used as a test system. The simulations are analyzed and com- pared to experimental data like 'H-NMR amide proton exchange-folding competition, heat ca- pacity, and compressibility measurements. 0 1995 Wiley-Liss, Inc.

The continuous advance of experimental techniques is steadily increasing our knowledge of biochemical systems and processes. A detailed picture of many biomolecular processes is emerging due to the possibility of measuring atomic properties of biological macromolecules, such as proteins. X-ray diffraction has provided a three-dimensional picture of biomolecular assemblies of ever increasing size, ranging from small proteins to a complete macromolecular reaction centre. Other experimental techniques, like nuclear magnetic resonance (NMR) and other spectroscopic methods, yield less complete information at the atomic level. However, the development of multi-dimensional NMR has made it possible to determine the spatial structure of small proteins, albeit at low resolution. The advantage of spectroscopic measuring techniques over X-ray diffraction methods is that the former can be used to obtain information on the dynamics of specific atoms or groups of atoms in the biomolecule, while the latter only yield an indication of the mobility of atoms, not of the time scale of their motion. The other major step forward is the advent of the possibility to change the amino acid composition of a protein at will. Instead of studying the proteins with which nature has provided us, we may now make specific mutations and study their effect on protein properties.

Methods to compute free energy differences between different states of a molecular system are reviewed with the aim of identifying their basic ingredients and their utility when applied in practice to biomolecular systems. A free energy calculation is comprised of three basic components: (i) a suitable model or Hamiltonian, (ii) a sampling protocol with which one can generate a representative ensemble of molecular configurations, and (iii) an estimator of the free energy difference itself. Alternative sampling protocols can be distinguished according to whether one or more states are to be sampled. In cases where only a single state is considered, six alternative techniques could be distinguished: (i) changing the dynamics, (ii) deforming the energy surface, (iii) extending the dimensionality, (iv) perturbing the forces, (v) reducing the number of degrees of freedom, and (vi) multi-copy approaches. In cases where multiple states are to be sampled, the three primary techniques are staging, importance sampling, and adiabatic decoupling. Estimators of the free energy can be classified as global methods that either count the number of times a given state is sampled or use energy differences. Or, they can be classified as local methods that either make use of the force or are based on transition probabilities. Finally, this overview of the available techniques and how they can be best used in a practical context is aimed at helping the reader choose the most appropriate combination of approaches for the biomolecular system, Hamiltonian and free energy difference of interest. © 2009 Wiley Periodicals, Inc.