A new amphipathy scale: I. Determination of the scale from molecular dynamics data

Two new amphipathy scales elaborated from molecular dynamics data are presented. Their applications contribute for the identification of the hydrophobic or hydrophilic regions in proteins solely from the primary structure. The new amphipathy coefficients (AC) reflect the side chain/solvent molecules configurational energies. A polar (water) and an apolar solvent, CCl₄, were used resulting in the two AC_water and AC_CCl4 scales. These solvents were chosen to simulate the aqueous phases and the transmembrane ambients of cellular membranes where the membrane proteins act. The new amphipathy scales were compared with some previous scales determined by different methods, which were also compared between them, indicating more than 90% of the correlation coefficients are less than 0.9: the scales are strictly dependent on the methodologies used in their determination. The AC_CCl4 scale is related with the size of side chain amino acids while AC_water is related with the hydrophobicity of side chain amino acids. The quality of the scales was confirmed by an example of application where AC_water was able to identify correctly the transmembrane, hydrophobic regions of a membrane protein. These results also indicate that water is an important factor responsible for the tertiary structure of membrane proteins.     

Recognition of alpha-helix transmembrane domains with an amphipathy scale generated by molecular dynamics using only the primary sequence of proteins

We recently developed an amphipathy scale, elaborated from molecular dynamics data that can be used for the identification of hydrophobic or hydrophilic regions in proteins. This amphipathy scale reflects side chain/water molecule interaction energies. We have now used this amphipathy scale to find candidates for transmembrane segments, by examining a large sample of membrane proteins with alpha-helix segments. The candidates were selected based on an amphipathy coefficient value range and the minimum number of residues in a segment. We compared our results with the transmembrane segments previously identified in the PDB_TM database by the TMDET algorithm. We expected that the hydrophobic segments would be identified using only the primary structures of the proteins and the amphipathy scale. However, some of these hydrophobic segments may pertain to hydrophobic pockets not included in transmembrane regions. We found that our amphipathy scale could identify alpha-helix transmembrane regions with a probability of success of 76% when all segments were included and 90% when all membrane proteins were included.     

A molecular dynamics approach for the generation of complete protein structures from limited coordinate data

Generation of full protein coordinates from limited information, e.g., the Cα coordinates, is an important step in protein homology modeling and structure determination, and molecular dynamics (MD) simulations may prove to be important in this task. We describe a new method, in which the protein backbone is built quickly in a rather crude way and then refined by minimization techniques. Subsequently, the side chains are positioned using extensive MD calculations. The method is tested on two proteins, and results compared to proteins constructed using two other MD-based methods. In the first method, we supplemented an existing backbone building method with a new procedure to add side chains. The second one largely consists of available methodology. The constructed proteins are compared to the corresponding X-ray structures, which became available during this study, and they are in good agreement (backbone RMS values of 0.5–0.7 Å, and all-atom RMS values of 1.5–1.9 Å). This comparative study indicates that extensive MD simulations are able, to some extent, to generate details of the native protein structure, and may contribute to the development of a standardized methodology to predict reliably (parts of) protein structures when only partial coordinate data are available. © 1994 John Wiley & Sons, Inc.     

A new method for the analysis of the dynamics of the molecular genetic control systems. I. Description of the method of generalized threshold models.

In the molecular system of coding polymers and metabolites a control subsystem has been singled out that forms controlling variables showing the action of regulatory molecules and a controlled subsystem where, depending on the values of controlling variables controlled variables are formed, i.e. concentrations of DNA, m-RNA, proteins and metabolites. Relationships have been obtained which enable controlling variables to be found. Equations showing the dynamics of molecular genetic control systems' components have been obtained. A method of generalized threshold models that enables kinetic curves to be obtained by pure mathematical means for macromolecular components (DNA, RNA, proteins) of molecular genetic control systems of varying complexity is suggested.     

On the interpretation of biochemical data by molecular dynamics computer simulation.

The application of computer simulation to molecular systems of biochemical interest is reviewed. It is shown that computer simulation is a tool complementary to experimental methods, which can be used to access atomic details inaccessible to experimental probes. Examples are given in which computer simulation augments the experimental information by providing an atomic picture of high resolution with respect to space, energy or time. The usefulness of a computer simulation largely depends on its quality. The most important factors that limit the accuracy of simulated results are discussed. The accuracy of different simulation studies can differ by orders of magnitude. The accuracy will depend on the type of biomolecular system and process studied. It will also depend on the choice of force field, the simulation set-up and the protocol that is used. A list of quality-determining factors is given, which may be useful when interpreting simulation studies appearing in the literature.     

A new gating site in human aquaporin-4: Insights from molecular dynamics simulations

Aquaporin-4 (AQP4) is the predominant water channel in different organs and tissues. An alteration of its physiological functioning is responsible for several disorders of water regulation and, thus, is considered an attractive target with a promising therapeutic and diagnostic potential. Molecular dynamics (MD) simulations performed on the AQP4 tetramer embedded in a bilayer of lipid molecules allowed us to analyze the role of spontaneous fluctuations occurring inside the pore. Following the approach by Hashido et al. [Hashido M, Kidera A, Ikeguchi M (2007) Biophys J 93: 373–385], our analysis on 200 ns trajectory discloses three domains inside the pore as key elements for water permeation. Herein, we describe the gating mechanism associated with the well-known selectivity filter on the extracellular side of the pore and the crucial regulation ensured by the NPA motifs (asparagine, proline, alanine). Notably, on the cytoplasmic side, we find a putative gate formed by two residues, namely, a cysteine belonging to the loop D (C178) and a histidine from loop B (H95). We observed that the spontaneous reorientation of the imidazole ring of H95 acts as a molecular switch enabling H-bond interaction with C178. The occurrence of such local interaction seems to be responsible for the narrowing of the pore and thus of a remarkable decrease in water flux rate. Our results are in agreement with recent experimental observations and may represent a promising starting point to pave the way for the discovery of chemical modulators of AQP4 water permeability.     

The mechanism of proton translocation in respiratory complex I from molecular dynamics

Abstract

Respiratory complex I, the biggest enzyme of respiratory chain, plays a key role in energy production by the mitochondrial respiratory chain and has been implicated in many human neurodegenerative diseases. Recently, the crystal structure of respiratory complex I is reported. We perform 50?ns molecular dynamics simulations on the membrane domain of respiratory complex I under two hypothetical states (oxidized state and reduced state). We find that the density of water molecules in the trans-membrane domain under reduced state is bigger than that under oxidized state. The connecting elements (helix HL and β-hairpins-helix element) fluctuate stronger under reduced state than that under oxidized state, causing more internal water molecules and facilitating the proton conduction. The conformational changes of helix HL and the crucial charged residue Glu in TM5 play key roles in the mechanism of proton translocation. Our results illustrate the dynamic behavior and the potential mechanism of respiratory complex I, which provides the structural basis for drug design of respiratory complex I.     

Peptide amphipathy: a new strategy in design of potential insecticides

A 30-residue peptide [YAA(KALA)6LAA] with an amphipathic helix repeat unit of Lys-Ala-Leu-Ala (KALA) was synthesized as both the L- and the D-isomer. The peptide was shown to form alpha-helices and lyse lipid vesicles in a pH dependent fashion. The calculated helical amphipathic moment is +1.19 kcal/residue and the mean residue hydrophobicity is +0.4 kcal/residue. The formation of alpha-helices as the pH is increased is similar to poly-lysine, yielding a pK 10.2. Though not toxic when fed to insects, KALA killed Spodoptera frugiperda cells at low doses and Manduca sexta larvae when injected.     

Determination of protein structures from nuclear magnetic resonance data using a restrained molecular dynamics approach: the lac repressor DNA binding domain

A procedure is described to determine from NMR data the three-dimensional structure of biomolecules. This procedure combines model building with a restrained Molecular Dynamics algorithm, in which distance information from NOEs is incorporated in the form of pseudo potentials. The method has been applied to the N-terminal DNA-binding domain or "headpiece" (amino acids 1-51) of the lac repressor from E. coli, for which no crystal structure is available. The spatial structure of the headpiece is discussed in terms of known physical and biochemical data and of its DNA binding properties.     

A new Sparassis species from Spain described using morphological and molecular data

Sparassis miniensis, collected in Pinus pinaster forests in Galicia (northwest Iberian Peninsula) is described as a new species, based on morphological and molecular data. Sparassis miniensis is morphologically distinct from all other species in the genus Sparassis based on scattered flabellae, which are strongly laciniated, azonate, and arise from an orange to rose-purplish base. The sporadic presence of clamp connections is restricted to subhymenial hyphae. Molecular data from LSU-rDNA, ITS and partial gene coding RNA polymerase subunit II (rpb2) suggest a close relationship between the new species S. miniensis and S. brevipes, another European species producing large fruiting bodies but with entire flabellae and no clamp connections.     

Modeling of the structure of bacteriorhodopsin. A molecular dynamics study.

Secondary structure predictions for membrane proteins are relatively reliable and permit the construction of model structures that may serve as initial conformations for molecular dynamics simulations. This might provide a scheme to predict the three-dimensional structures of membrane proteins. The feasibility of such an approach is tested for bacteriorhodopsin. We were not able to fully predict the kidney-shaped structure of bacteriorhodopsin. However, features compatible with this structure developed in a simulation starting from a circular arrangement of the seven predicted helices. When instead we started from the kidney shape, assigning the seven predicted helices in different ways to those on the structure, we could distinguish between the different assignments on the basis of energy and tilt of the helices. In this way we could select the correct assignment from a few others. For the correct assignment, the helices spontaneously adopted a tilt that agrees remarkably well with the experimental model structure derived by others. The root-mean-square deviation between our best molecular dynamics structure and the experimental model structure is 3.8 A, caused mainly by deviations in the internal degrees of freedom of the helices.     

A new species of Cudonia based on morphological and molecular data

A discomycete collected in western Sichuan, China, is morphologically intermediate between Cudonia and Spathularia. The fungus has a bright yellow capitate ascigerous head, a white, ridged stalk, and a well-developed membrane covering the whole ascoma. The asci, ascospores, and paraphyses are similar to those of Cudonia and Spathularia. Based on morphology and DNA sequence analysis, a new species, Cudonia sichuanensis, is reported. Cudonia and Spathularia are closely related to members of Rhytismataceae, as has been suggested previously. The similarity of ascoma and ascospore development between these two genera and Lophodermium (Rhytismataceae) is discussed.     

A new cuticle scale hydrolysing protease from Beauveria brongniartii

From a screening for the production of new proteases specific for cuticle scales, Beauveria brongniartii was selected producing an alkaline Ca⁺⁺ dependent protease. The purified had a molecular weight of 27 kDa and a pI value of 8.0. Substrate specificities of model substrates (wool with partially removed cuticles treated with SDS) were analyzed by protein release, dissolved organic carbon (DOC) and nitrogen analysis. The C/N ratio of released material turned out to be a good parameter to determine the site of action of proteases on fibres. Compared to other enzymes, the fungal protease preferentially hydrolyzed cuticle scales and has thus a potential for anti-shrinking pre-treatment of wool fabrics.     

Determination of the local structure of the protein insectotoxin I5A from the scorpion Buthus eupeus from 1H-NMR spectroscopy data

The local structure (torsion angles phi, psi and chi 1 of amino acid residues) of insectotoxin I5A (35 residues) of scorpion Buthus eupeus has been determined from cross-peak integral intensities in two-dimensional nuclear Overhauser enhancement (NOESY) spectra and spin coupling constants of vicinal H--NC alpha--H and H--C alpha C beta--H protons. The local structure determination was carried out by fitting complete relaxation matrix of peptide unit protons (protons of a given residue and NH proton of the next residue in the amino acid sequence) with experimental NOESY cross-peak intensities. The obtained intervals of backbone torsional angles phi and psi consistent with NMR data were determined for all but Gly residues. The predominant C alpha--C beta rotamer of the side chain has been unambiguously determined for 42% of the insectotoxin amino acid residues whereas for another 46% residues experimental data are fitted equally well with two rotamers. Stereospecific assignments were obtained for 38% of beta-methylene groups. The determined torsional angles phi, psi and chi 1 correspond to the sterically allowed conformations of the amino acid residues and agree with the insectotoxin secondary structure established earlier by 1H NMR spectroscopy.     

Large‐scale comparison of protein essential dynamics from molecular dynamics simulations and coarse‐grained normal mode analyses

A large‐scale comparison of essential dynamics (ED) modes from molecular dynamic simulations and normal modes from coarse‐grained normal mode methods (CGNM) was performed on a dataset of 335 proteins. As CGNM methods, the elastic network model (ENM) and the rigid cluster normal mode analysis (RCNMA) were used. Low‐frequency normal modes from ENM correlate very well with ED modes in terms of directions of motions and relative amplitudes of motions. Notably, a similar performance was found if normal modes from RCNMA were used, despite a higher level of coarse graining. On average, the space spanned by the first quarter of ENM modes describes 84% of the space spanned by the five ED modes. Furthermore, no prominent differences for ED and CGNM modes among different protein structure classes (CATH classification) were found. This demonstrates the general potential of CGNM approaches for describing intrinsic motions of proteins with little computational cost. For selected cases, CGNM modes were found to be more robust among proteins that have the same topology or are of the same homologous superfamily than ED modes. In view of recent evidence regarding evolutionary conservation of vibrational dynamics, this suggests that ED modes, in some cases, might not be representative of the underlying dynamics that are characteristic of a whole family, probably due to insufficient sampling of some of the family members by MD. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Determination of membrane-insertion free energies by molecular dynamics simulations

The accurate prediction of membrane-insertion probability for arbitrary protein sequences is a critical challenge to identifying membrane proteins and determining their folded structures. Although algorithms based on sequence statistics have had moderate success, a complete understanding of the energetic factors that drive the insertion of membrane proteins is essential to thoroughly meeting this challenge. In the last few years, numerous attempts to define a free-energy scale for amino-acid insertion have been made, yet disagreement between most experimental and theoretical scales persists. However, for a recently resolved water-to-bilayer scale, it is found that molecular dynamics simulations that carefully mimic the conditions of the experiment can reproduce experimental free energies, even when using the same force field as previous computational studies that were cited as evidence of this disagreement. Therefore, it is suggested that experimental and simulation-based scales can both be accurate and that discrepancies stem from disparities in the microscopic processes being considered rather than methodological errors. Furthermore, these disparities make the development of a single universally applicable membrane-insertion free energy scale difficult.     

Structure and dynamics of the antifungal molecules Syringotoxin-B and Syringopeptin-25A from molecular dynamics simulation

The phytopathogen Pseudomonas syringae pv. syringae produces toxic cyclic lipodepsipeptides (CLPs): nona-peptides and syringopeptins. All CLPs inhibit the growth of many fungal species, including human pathogens, although different fungi display different degrees of sensitivity. The best studied CLPs are Syringomycin-E (SR-E), Syringotoxin-B (ST-B) and Syringopeptin-25A (SP-25A). Their biological activity is affected by membrane composition and their structural differences. We previously (Matyus et al. in Eur Biophys J 35:459–467, 2006) reported the molecular features and structural preferences of SR-E in water and octane environments. Here we investigate in atomic detail the molecular features of the two other main CLP components, ST-B and SP-25A, in water and octane by 200 ns molecular dynamics simulations (MD), using distance restraints derived from NMR NOE data (Ballio et al. in Eur J Biochem 234:747–758, 1995). We have obtained three-dimensional models of ST-B and SP-25A CLPs in different environments. These models can now be used as a basis to investigate the interactions of ST-B and SP-25A with lipid membranes an important further step towards a better understanding of the antifungal and antibacterial activity of these peptides. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.