3 Nsec molecular dynamics simulation of the protein ubiquitin and comparison with X-ray crystal and solution NMR structures.

Mainly due to computational limitations, past protein molecular dynamics simulations have rarely been extended to 300 psec; we are not aware of any published results beyond 350 psec. The present work compares a 3000 psec simulation of the protein ubiquitin with the available x-ray crystallographic and solution NMR structures. Aside from experimental structure availability, ubiquitin was studied because of its relatively small size (76 amino acids) and lack of disulfide bridges. An implicit solvent model was used except for explicit treatment of waters of crystallization. We found that the simulated average structure retains most of the character of the starting x-ray crystal structure. In two highly surface accessible regions, the simulation was not in agreement with the x-ray structure. In addition, there are six backbone-backbone hydrogen bonds that are in conflict between the solution NMR and x-ray crystallographic structures; two are bonds that the NMR does not locate, and four are ones that the two methods disagree upon the donor. Concerning these six backbone-backbone hydrogen bonds, the present simulation agrees with the solution NMR structure in five out-of-the six cases, in that if a hydrogen bond is present in the x-ray structure and not in the NMR structure, the bond breaks within 700 psec. Of the two hydrogen bonds that are found in the NMR structure and not in the x-ray structure, one forms at 1400 psec and the other forms rarely. The present results suggest that relatively long molecular dynamics simulations, that use protein x-ray crystal coordinates for the starting structure and a computationally efficient solvent representation, may be used to gain an understanding of conformational and dynamic differences between the solid-crystal and dilute-solution states.     

Structural trees for protein superfamilies

Structural trees for large protein superfamilies, such as β proteins with the aligned β sheet packing, β proteins with the orthogonal packing of α helices, two-layer and three-layer α/β proteins, have been constructed. The structural motifs having unique overall folds and a unique handedness are taken as root structures of the trees. The larger protein structures of each superfamily are obtained by a stepwise addition of α helices and/or β strands to the corresponding root motif, taking into account a restricted set of rules inferred from known principles of the protein structure. Among these rules, prohibition of crossing connections, attention to handedness and compactness, and a requirement for α helices to be packed in α-helical layers and β strands in β layers are the most important. Proteins and domains whose structures can be obtained by stepwise addition of α helices and/or β strands to the same root motif can be grouped into one structural class or a superfamily. Proteins and domains found within branches of a structural tree can be grouped into subclasses or subfamilies. Levels of structural similarity between different proteins can easily be observed by visual inspection. Within one branch, protein structures having a higher position in the tree include the structures located lower. Proteins and domains of different branches have the structure located in the branching point as the common fold. Proteins 28:241–260, 1997. © 1997 Wiley-Liss Inc.     

Molecular dynamics simulation of hydration in myoglobin

This study was carried out to evaluate the stability of the 89 bound water molecules that were observed in the neutron diffraction study of CO myoglobin. The myoglobin structure derived from the neutron analysis was used as the starting point in the molecular dynamics simulation using the software package CHARMM. After solvation of the protein, energy minimization and equilibration of the system, 50 ps of Newtonian dynamics was performed. This data showed that only 4 water molecules are continously bound during the length of this simulation while the other solvent molecules exhibit considerable mobility and are breaking and reforming hydrogen bonds with the protein. At any instant during the simulation, 73 of the hydration sites observed in the neutron structure are occupied by water. © 1995 Wiley-Liss, Inc.     

Molecular dynamics simulation of the RNA complex of a double-stranded RNA-binding domain reveals dynamic features of the intermolecular interface and its hydration

The interaction between double-stranded RNA (dsRNA) and the third double-stranded domain (dsRBD) from Drosophila Staufen protein represents a paradigm to understand how the dsRBD protein family, one of the most common RNA-binding protein units, binds dsRNA. The nuclear magnetic resonance (NMR) structure of this complex and the x-ray structure of another family member revealed the stereochemical basis for recognition, but also raised new questions. Although the crystallographic studies revealed a highly ordered interface containing numerous water-mediated contacts, NMR suggested extensive residual motion at the interface. To address how interfacial motion contributes to molecular recognition in the dsRBD-dsRNA system, we conducted a 2-ns molecular dynamics simulation of the complex derived from Staufen protein and of the separate protein and RNA components. The results support the observation that a high degree of conformational flexibility is retained upon complex formation and that this involves interfacial residues that are critical for dsRBD-dsRNA binding. The structural origin of this residual flexibility is revealed by the analysis of the trajectory of motion. Individual basic side chains switch continuously from one RNA polar group to another with a residence time seldom exceeding 100 ps, while retaining favorable interaction with RNA throughout much of the simulation. Short-lived water molecules mediate some of these interactions for a large fraction of the trajectory studied here. This result indicates that water molecules are not statically associated with the interface, but continuously exchange with the bulk solvent on a 1-10-ps time scale. This work provides new insight into dsRBD-dsRNA recognition and builds upon a growing body of evidence, suggesting that short-lived dynamic interactions play important roles in protein-nucleic acid interactions.     

Structure and dynamics of calcium-activated calmodulin in solution.

Two 4-ns molecular dynamics simulations of calcium loaded calmodulin in solution have been performed, using both standard nonbonded cutoffs and Ewald summation to treat electrostatic interactions. Our simulation results are generally consistent with solution experimental studies of calmodulin structure and dynamics, including NMR, cross-linking, fluorescence and x-ray scattering. The most interesting result of the molecular dynamics simulations is the detection of large-scale structural fluctuations of calmodulin in solution. The globular N- and C-terminal domains tend to move approximately like rigid bodies, with fluctuations of interdomain distances within a 7 A range and of interdomain angles by up to 60 deg. Essential dynamics analysis indicates that the three dominant types of motion involve bending of the central helix in two perpendicular planes and a twist in which the domains rotate in opposite directions around the central helix. In the more realistic Ewald trajectory the protein backbone remains mostly within a 2-3 A root-mean-square distance from the crystal structure, the secondary structure within the domains is conserved and middle part of the central helix becomes disordered. The central helix itself exhibits limited fluctuations, with its bend angle exploring the 0-50 degrees range and the end-to-end distance falling in 39-43 A. The results of the two simulations were similar in many respects. However, the cutoff trajectory exhibited a larger deviation from the crystal, loss of several helical hydrogen bonds in the N-terminal domain and lack of structural disorder in the central helix.     

Refolding simulations of an isolated fragment of barnase into a native-like β hairpin: Evidence for compactness and hydrogen bonding as concurrent stabilizing factors

Experimental evidence and theoretical models both suggest that protein folding is initiated within specific fragments intermittently adopting conformations close to that found in the protein native structure. These folding initiation sites encompassing short portions of the protein are ideally suited for study in isolation by computational methods aimed at peering into the very early events of folding. We have used Molecular Dynamics (MD) technique to investigate the behavior of an isolated protein fragment formed by residues 85 to 102 of barnase that folds into a β hairpin in the protein native structure. Three independent MD simulations of 1.3 to 1.8 ns starting from unfolded conformations of the peptide portrayed with an all-atom model in water were carried out at gradually decreasing temperature. A detailed analysis of the conformational preferences adopted by this peptide in the course of the simulations is presented. Two of the unfolded peptide conformations fold into a hairpin characterized by native and a larger bulk of nonnative interactions. Both refolding simulations substantiate the close relationship between interstrand compactness and hydrogen bonding network involving backbone atoms. Persistent compactness witnessed by side-chain interactions always occurs concomitantly with the formation of backbone hydrogen bonds. No highly populated conformations generated in a third simulation starting from the remotest unfolded conformer relative to the native structure are observed. However, nonnative long-range and medium-range contacts with the aromatic moiety of Trp94 are spotted, which are in fair agreement with a former nuclear magnetic resonance study of a denaturing solution of an isolated barnase fragment encompassing the β hairpin. All this lends reason to believe that the 85–102 barnase fragment is a strong initiation site for folding. Proteins 29:212–227, 1997. © 1997 Wiley-Liss, Inc.     

Helix bending in alamethicin: molecular dynamics simulations and amide hydrogen exchange in methanol

Molecular dynamics simulations of alamethicin in methanol were carried out with either a regular alpha-helical conformation or the x-ray crystal structure as starting structures. The structures rapidly converged to a well-defined hydrogen-bonding pattern with mixed alpha-helical and 3(10)-helical hydrogen bonds, consistent with NMR structural characterization, and did not unfold throughout the 1-ns simulation, despite some sizable backbone fluctuations involving reversible breaking of helical hydrogen bonds. Bending of the helical structure around residues Aib10-Aib13 was associated with reversible flips of the peptide bonds involving G11 (Aib10-G11 or G11-L12 peptide bonds), yielding discrete structural states in which the Aib10 carbonyl or (rarely) the G11 carbonyl was oriented away from the peptide helix. These peptide bond reversals could be accommodated without greatly perturbing the adjacent helical structure, and intramolecular hydrogen bonding was generally maintained in bent states through the formation of new (non-alpha or 3[10]) hydrogen bonds with good geometries: G11 NH-V9 CO (inverse gamma turn), Aib13 NH-Aib8 CO (pi-helix) and, rarely, L12 NH- Q7 NH (pi-helix). These observations may reconcile potentially conflicting NMR structural information for alamethicin in methanol, in which evidence for conformational flexibility in the peptide sequence before P14 (G11-Aib13) contrasts with the stability of backbone amide NH groups to exchange with solvent. Similar reversible reorientation of the Thr11-Gly12 peptide bond of melittin is also observed in dynamics simulations in methanol (R. B. Sessions, N. Gibbs, and C. E. Dempsey, submitted). This phenomenon may have some role in the orientation of the peptide carbonyl in solvating the channel lumen in membrane ion channel states of these peptides.     

Molecular dynamics of subtilisin Carlsberg in aqueous and nonaqueous solutions

Crystal structures have recently appeared for the enzyme subtilisin Carlsberg in anhydrous acetonitrile and in water. To gain a mechanistic understanding of how the solvent environment affects protein structure and dynamics, we have performed molecular dynamics simulations on subtilisin Carlsberg in water and acetonitrile. We describe a 480 ps simulation of subtilisin in acetonitrile solution and a 450 ps simulation of subtilisin in water. Each simulation employed the all-atom AMBER force field. The calculated rms deviations, from their respective x-ray structures, were similar in each simulation, but ～0.5 ? higher in the acetonitrile simulation. Only in the acetonitrile simulation does one helix undergo a reversible partial unwinding, which lasted for about 100 ps. The other secondary structure elements remain intact or undergo modest fluctuations. In the aqueous simulation, the calculated and experimental temperature factors agree very well. In the acetonitrile simulation, however, the calculated temperature factors are much higher than the experimental values. The larger rms deviation and thermal fluctuations noted in the acetonitrile simulation are consistent with the requirement for protein cross-linking in this crystal and a recent two-dimensional NH-exchange nmr study on horse heart cytochrome c in nonaqueous solution. ? 1996 John Wiley & Sons, Inc.     

Solution structure and backbone dynamics of human Raf-1 kinase inhibitor protein

Human Raf-1 kinase inhibitor protein (hRKIP) is a small multi-functional protein of 187 residues. It contains a conserved pocket, which binds a wide range of ligands from various small molecules to distinct proteins. To provide a structural basis for the ligand diversity of RKIP, we herein determined the solution structure of hRKIP, and analyzed its structural dynamics. In solution, hRKIP mainly comprises two antiparallel β sheets, two α helices and two 3₁₀ helices. NMR dynamic analysis reveals that the overall structure of hRKIP is rigid, but its C-terminal helix which is close to the ligand-binding site is mobile. In addition, residues around the ligand-binding pocket exhibit significant conformational exchange on the μs–ms timescale. Conformational flexibility may allow the ligand-binding pocket and the C-terminal helix to adopt various conformations to interact with different substrates. This work may shed light on the underlying molecular mechanisms of how hRKIP recognizes and binds diverse substrate ligands.     

The Ribbon of Hydrogen Bonds in the Three-Dimensional Structure of Globular Proteins

We report quantum mechanical computations and experimental evidence which suggest that the backbone conformation of globular proteins depends generally on the conservation of that part of the hydrogen bond network or ribbon which is joined, in general, directly to the backbone and is largely independent of the remainder of this whole network of hydrogen bonds. The familiar hydrogen bonds of the helix and the sheet form about one-half of this ribbon of hydrogen bonds. Both water molecules and hydrogen bonding side chain groups are involved in the formation of the ribbon.This view of the three-dimensional structure of globular proteins in terms of the `molecule' allows us to deal with the non-secondary structure as well as with the familiar secondary structure. It also suggests that the ribbon contains approximately the same number of hydrogen bonds within all three structures – the helix, the sheet and the coil – and that this is the reason for the ease of interconversion of these three structures.The quantum mechanical computations on hydrogen bonding suggest that delocalised water molecules which have substantial mobility are an essential part of the ribbon. This situation arises because the hydrogen bonding groups of the protein molecule are not free to move to optimise the hydrogen bonding geometries as are the oxygen atoms in the waters and ices. Such delocalised water molecules either have high B values or are invisible in the X-ray data and yet are able to form a structure which is as strong as a normal hydrogen bond.The experimental data on the point mutations of the THRI57 residue of the T4 phage lysome provides an initial test of this model. Both the local backbone conformation and the ribbon of hydrogen bonds are conserved throughout all the mutations of residue 157,providing that the delocalised water molecules are accepted as a genuine part of the structure. These mutations include the introduction of hydrocarbon side chains at position 157 when water molecules or other side chain groups take over the formation of the hydrogen bonds.We suggest that, provided steric effects are not important, many point mutations succeed because they leave the ribbon of hydrogen bonds (and so the backbone conformation) largely unchanged.     

Structure and hydration of BamHI DNA recognition site: a molecular dynamics investigation

The results of a 3-ns molecular dynamics simulation of the dodecamer duplex d(TATGGATCCATA)(2) recognized by the BamHI endonuclease are presented here. The DNA has been simulated as a flexible molecule using an AMBER force field and the Ewald summation method, which eliminates the undesired effects of truncation and permits evaluation of the full effects of electrostatic forces. The starting B conformation evolves toward a configuration quite close to that observed through x-ray diffraction in its complex with BamHI. This configuration is fairly stable and the Watson-Crick hydrogen bonds are well maintained over the simulation trajectory. Hydration analysis indicates a preferential hydration for the phosphate rather than for the ester oxygens. Hydration shells in both the major and minor groove were observed. In both grooves the C-G pairs were found to be more hydrated than A-T pairs. The "spine of hydration" in the minor groove was clear. Water residence times are longer in the minor groove than in the major groove, although relatively short in both cases. No special long values are observed for sites where water molecules were observed by x-ray diffraction, indicating that water molecules having a high probability of being located in a specific site are also fast-exchanging.     

Spectroscopy-Based Modelling of the 3D Structure of the β Subunit of the High Affinity IgE Receptor

Abstract

The high affinity IgE receptor, possesses a tetrameric structure. The 243 residue β subunit is a polytopic protein with four hydrophobic membrane-spanning segments, whereas the individual α and γ subunits are bitopic proteins each containing one transmembrane domain in their monomeric form. In the proposed topographical model (Blank et al., 1989), the four trans-membrane α helices of the β subunit are connected by three loop sequences.

To study the individual subunits and intact receptor, this membrane protein was divided into domains such as its loop peptides, cytoplasmic peptides and transmembrane helices according to Blank et al., 1989. The 3D structure of the synthesized loop peptides and cytoplasmic peptides were calculated; CD and/or NMR data were used as appropriate to generate the resultant structures which were then used as data basis for the higher level calculations.

The four individual transmembrane helices of the β subunit were characterised, first of all, by mapping the relative lipophilicity of their surfaces using lipophilic probes. A second procedure, docking of the individual helices in pairs, was used to predict helix–helix interactions.

The data on the relative lipophilicity of the surfaces as well as the surfaces that favoured helix–helix interactions were used in combination with the spectroscopy-based structures of the loops and cytoplasmic domains to calculate via molecular dynamics, the helix arrangement and 3D structure of the β subunit of the high affinity IgE receptor. In the final analysis, the molecular simulations yielded two structures of the β subunit, which should form a basis for the modelling of the whole high affinity IgE receptor.     

Patterns,structures, and amino acid frequencies in structural building blocks,a protein secondary structure classification scheme

To study local structures in proteins, we previously developed an autoassociative artificial neural network (autoANN) and clustering tool to discover intrinsic features of macromolecular structures. The hidden unit activations computed by the trained autoANN are a convenient low-dimensional encoding of the local protein backbone structure. Clustering these activation vectors results in a unique classification of protein local structural features called Structural Building Blocks (SBBs). Here we describe application of this method to a larger database of proteins, verification of the applicability of this method to structure classification, and subsequent analysis of amino acid frequencies and several commonly occurring patterns of SBBs. The SBB classification method has several interesting properties: 1) it identifies the regular secondary structures, α helix and β strand; 2) it consistently identifies other local structure features (e.g., helix caps and strand caps); 3) strong amino acid preferences are revealed at some positions in some SBBs; and 4) distinct patterns of SBBs occur in the “random coil” regions of proteins. Analysis of these patterns identifies interesting structural motifs in the protein backbone structure, indicating that SBBs can be used as “building blocks” in the analysis of protein structure. This type of pattern analysis should increase our understanding of the relationship between protein sequence and local structure, especially in the prediction of protein structures. © 1997 Wiley-Liss, Inc.     

β Sheet to α helix transition in the binding subunit of cholera toxin

Circular dichroism spectra have been measured for the binding subunit of cholera toxin in water and in the presence of dodecyl sulfate. In water the protein has an appreciable amount of β structure and almost no α helix. In the presence of dodecyl sulfate the spectrum undergoes drastic change over a time period of approximately four hours, and at equilibrium resembles that expected for a chain with an appreciable amount of α helix but no β structure. The change in helicity is in good agreement with that expected from our formulation of the configuration partition function for the binding subunit. The conformational change may have an important relationship to the means by which the binding subunit permits penetration of the active subunit into the cell.     

The presence of non‐native helical structure in the unfolding of a beta‐sheet protein MPT63

MPT63, a major secreted protein from Mycobacterium tuberculosis, has been shown to have immunogenic properties and has been implicated in virulence. MPT63 is a β‐sandwich protein containing 11 β strands and a very short stretch of 3₁₀ helix. The detailed experimental and computational study reported here investigates the equilibrium unfolding transition of MPT63. It is shown that in spite of being a complete β‐sheet protein, MPT63 has a strong propensity toward helix structures in its early intermediates. Far UV‐CD and FTIR spectra clearly suggest that the low‐pH intermediate of MTP63 has enhanced helical content, while fluorescence correlation spectroscopy suggests a significant contraction. Molecular dynamics simulation complements the experimental results indicating that the unfolded state of MPT63 traverses through intermediate forms with increased helical characteristics. It is found that this early intermediate contains exposed hydrophobic surface, and is aggregation prone. Although MPT63 is a complete β‐sheet protein in its native form, the present findings suggest that the secondary structure preferences of the local interactions in early folding pathway may not always follow the native conformation. Furthermore, the Gly25Ala mutant supports the proposed hypothesis by increasing the non‐native helical propensity of the protein structure.     

The structural properties of magainin in water, TFE/water, and aqueous urea solutions: molecular dynamics simulations

Here, the MD simulations and comparative structural analysis of Magainin in water, TFE/water, and 2M, 4M, and BM urea solutions is reported. For MAG-TFE/water and MAG-2M urea the largely alpha helical conformation of the peptide is maintained throughout the 9-ns simulation. While in water, 4M urea, and 8M urea, the helix length decreases and at the same time helix radius increases. This suggests a more destabilized magainin secondary structure. Our simulation data reveals that the stabilizing effect of TFE is induced by preferential accumulation of TFE molecules around the alpha helical peptide. These results indicate that an aqueous urea solution solvates the surface of polypeptide chain more favorably than pure water. Urea molecules interact more favorably with nonpolar groups of the peptide in comparison with water, and the presence of urea improves the interactions of water molecules with the hydrophilic groups of the peptide. At 8M urea, there are more direct interactions between the urea and solute, and the helix is destabilized. At 2M urea, the interaction of urea molecules and nonpolar residues are weak, therefore, the presence of urea molecules decreases the interactions of water molecules with hydrophilic groups. Urea could not deteriorate the peptide secondary structure with time from an initial helix structure.     

Structural classification of αββ and ββα supersecondary structure units in proteins

We present a fully automatic structural classification of supersecondary structure units, consisting of two hydrogen-bonded β strands, preceded or followed by an α helix. The classification is performed on the spatial arrangement of the secondary structure elements, irrespective of the length and conformation of the intervening loops. The similarity of the arrangements is estimated by a structure alignment procedure that uses as similarity measure the root mean square deviation of superimposed backbone atoms. Applied to a set of 141 well-resolved nonhomologous protein structures, the classification yields 11 families of recurrent arrangements. In addition, fragments that are structurally intermediate between the families are found; they reveal the continuity of the classification. The analysis of the families shows that the α helix and β hairpin axes can adopt virtually all relative orientations, with, however, some preferable orientations; moreover, according to the orientation, preferences in the left/right handedness of the α–β connection are observed. These preferences can be explained by favorable side by side packing of the α helix and the β hairpin, local interactions in the region of the α–β connection or stabilizing environments in the parent protein. Furthermore, fold recognition procedures and structure prediction algorithms coupled to database-derived potentials suggest that the preferable nature of these arrangements does not imply their intrinsic stability. They usually accommodate a large number of sequences, of which only a subset is predicted to stabilize the motif. The motifs predicted as stable could correspond to nuclei formed at the very beginning of the folding process. Proteins 30:193–212, 1998. © 1998 Wiley-Liss, Inc.     

Beyond average protein secondary structure content prediction using FTIR spectroscopy

This paper demonstrates that secondary structure information beyond purely protein secondary structure content can be predicted from FTIR (Fourier transform infrared spectroscopy) spectra of proteins with a high degree of accuracy. Both neural networks and adaptive neuro-fuzzy inference systems (ANFISs) were employed to predict helix/sheet segment information. The best results were achieved using ANFISs with fuzzy subtractive clustering based on normalised, compressed amide I data with an average SEP (standard error of prediction, root mean of squared errors) of 1.51. Predictions for average helix/sheet length based merely on the amide I band maximum position in combination with the full-width at half-height resulted in a comparable average SEP of 1.62. This suggests the importance of information on the position and width of the amide I band maximum for the prediction of helix/sheet segment information. Finally, the most promising pattern recognition approaches found in this study were applied to a protein with an as yet unknown x-ray structure: native a1-antichymotrypsin (a1-ACT).     

Crystal structure of cat muscle pyruvate kinase at a resolution of 2.6 A

The structure of pyruvate kinase (EC 2.7.1.40) has been determined from a 2.6 Å resolution electron density map. This map shows more detail than the previous 3.1 Å map (Stammers &; Muirhead, 1977) and has enabled a detailed chain folding to be established for two out of the three domains which make up each of the four identical subunits. A provisional chain folding has been established for the third domain. The results have been briefly reported in a previous paper (Levine et al., 1978). Details of the structure determination and a further discussion of the results are presented in this paper.Domain A (the three domains of pyruvate kinase are referred to as A, B and C) can be described in terms of a cylindrical eight-stranded parallel β sheet and an outer coaxial cylinder of eight α helices. The α helices connect adjacent strands of the β sheet. Domain B is made up of a closed anti-parallel β sheet structure. Domain C is a five-stranded β sheet of which the fourth strand is anti-parallel and the rest parallel. These strands are also interconnected by α helices.Domain A can be dissected into eight consecutive β strand—α helix units starting from the N-terminus. The arrangement of these relative to each other can be most simply described by relating them to eight planes, each at 40 ° to the cylinder axis and symmetrically placed around the cylinder. When unit 2 is aligned with one of these planes then units 1, 3, 4, 5 and 8 are also closely aligned with a plane. This analysis is also applied to triosephosphate isomerase and a strikingly similar arrangement is found. A detailed comparison of the two structures is presented. Although the lack of a chemical sequence makes it difficult to identify the amino acid residues of pyruvate kinase, side-chains are clearly visible in the map and this information is correlated with the results of previous 6 Å substrate soaking experiments and with the structure of triosephosphate isomerase. The similarities and differences are discussed in terms of similarities and differences in the reactions catalysed and also of different subunit packing.