Large‐scale analysis of the dynamics of enzymes

Protein enzymes enable the cell to execute chemical reactions in short time by accelerating the rate of the reactions in a selective manner. The motions or dynamics of the enzymes are essential for their function. Comparison of the dynamics of a set of 1247 nonhomologous enzymes was performed. For each enzyme, the slowest modes of motion are calculated using the Gaussian network model (GNM) and they are globally aligned. Alignment is done using the dynamic programming algorithm of Needleman and Wunsch, commonly used for sequence alignment. Only 96 pairs of proteins were identified to have three similar GNM slow modes with 63 of them having a similar structure. The most frequent slowest mode of motion describes a two domains anticorrelated motion that characterizes at least 23% of the enzymes. Therefore, dynamics uniqueness cannot be accounted for by the slowest mode itself but rather by the combination of several slow modes. Different quaternary structure packing can restrain the motion of enzyme subunits differently and may serve as another mechanism that increases the dynamics uniqueness. Proteins 2013; 81:1910–1918. © 2013 Wiley Periodicals, Inc.     

Predicting the order in which contacts are broken during single molecule protein stretching experiments

We combine two methods to enable the prediction of the order in which contacts are broken under external stretching forces in single molecule experiments. These two methods are Gō-like models and elastic network models. The Gō-like models have shown remarkable success in representing many aspects of protein behavior, including the reproduction of experimental data obtained from atomic force microscopy. The simple elastic network models are often used successfully to predict the fluctuations of residues around their mean positions, comparing favorably with the experimentally measured crystallographic B-factors. The behavior of biomolecules under external forces has been demonstrated to depend principally on their elastic properties and the overall shape of their structure. We have studied in detail the muscle protein titin and green fluorescent protein and tested for ten other proteins. First, we stretch the proteins computationally by performing stochastic dynamics simulations with the Gō-like model. We obtain the force-displacement curves and unfolding scenarios of possible mechanical unfolding. We then use the elastic network model to calculate temperature factors (B-factors) and compare the slowest modes of motion for the stretched proteins and compare them with the predicted order of breaking contacts between residues in the Gō-like model. Our results show that a simple Gaussian network model is able to predict contacts that break in the next time stage of stretching. Additionally, we have found that the contact disruption is strictly correlated with the highest force exerted by the backbone on these residues. Our prediction of bond-breaking agrees well with the unfolding scenario obtained with the Gō-like model. We anticipate that this method will be a useful new tool for interpreting stretching experiments.     

Molecular mechanism of domain swapping in proteins: an analysis of slower motions

Domain swapping is a structural phenomenon that plays an important role in the mechanism of oligomerization of some proteins. The monomer units in the oligomeric structure become entangled with each other. Here we investigate the mechanism of domain swapping in diphtheria toxin and the structural criteria required for it to occur by analyzing the slower modes of motion with elastic network models, Gaussian network model and anisotropic network model. We take diphtheria toxin as a representative of this class of domain-swapped proteins and show that the domain, which is being swapped in the dimeric state, rotates and twists, in going from the "open" to the "closed" state, about a hinge axis that passes through the middle of the loop extending between two domains. A combination of the intra- and intermolecular contacts of the dimer is almost equivalent to that of the monomer, which shows that the relative orientations of the residues in both forms are almost identical. This is also reflected in the calculated B-factors when compared with the experimentally determined B-factors in x-ray crystal structures. The slowest modes of both the monomer and dimer show a common hinge centered on residue 387. The differences in distances between the monomer and the dimer also shows the hinge at nearly the same location (residue 381). Finally, the first three dominant modes of anisotropic network model together shows a twisting motion about the hinge centered on residue 387. We further identify the location of hinges for a set of another 12 domain swapped proteins and give the quantitative measures of the motions of the swapped domains toward their "closed" state, i.e., the overlap and correlation between vectors.     

An analysis of core deformations in protein superfamilies

An analysis is presented on how structural cores modify their shape across homologous proteins, and whether or not a relationship exists between these structural changes and the vibrational normal modes that proteins experience as a result of the topological constraints imposed by the fold. A set of 35 representative, well-populated protein families is studied. The evolutionary directions of deformation are obtained by using multiple structural alignments to superimpose the structures and extract a conserved core, together with principal components analysis to extract the main deformation modes from the three-dimensional superimposition. In parallel, a low-resolution normal mode analysis technique is employed to study the properties of the mechanical core plasticity of these same families. We show that the evolutionary deformations span a low dimensional space of 4-5 dimensions on average. A statistically significant correspondence exists between these principal deformations and the approximately 20 slowest vibrational modes accessible to a particular topology. We conclude that, to a significant extent, the structural response of a protein topology to sequence changes takes place by means of collective deformations along combinations of a small number of low-frequency modes. The findings have implications in structure prediction by homology modeling.     

Application of the local regression method interval partial least-squares to the elucidation of protein secondary structure

The infrared amide bands are sensitive to the conformation of the polypeptide backbone of proteins. Since the backbone of proteins folds in complex spatial arrangements, the amide bands of these proteins result from the superimposition of vibration modes corresponding to the different types of structural motifs (alpha helices, beta sheets, etc.). Initially, band deconvolution techniques were applied to determine the secondary structure of proteins, i.e., the abundance of each structural motif in the polypeptide chain was directly related to the area of the suitable deconvolved vibration modes under the amide I band (1700-1600 cm(-1)). Recently, several multivariate regression methods have been used to predict the secondary structure of proteins as an alternative to the previous methods. They are based on establishing a relationship between a matrix of infrared protein spectra and another that includes their secondary structure, expressed as the fractions of the different structural motifs, determined from X-ray analysis. In this study, we investigated the use of the local regression method interval partial least-squares (iPLS) to seek improvements to the full-spectrum PLS and other regression methods. The local character of iPLS avoids the use of spectral regions that can introduce noise or that can be irrelevant for prediction and focuses on finding specific spectral ranges related to each secondary structure motif in all the proteins. This study has been applied to a representative protein data set with infrared spectra covering a large wavenumber range, including amides I-III bands (1700-1200 cm(-1)). iPLS has revealed new structural mode assignments related to less explored amide bands and has offered a satisfactory predictive ability using a small amount of selected specific spectral information.     

Systematic Study of Anharmonic Features in a Principal Component Analysis of Gramicidin A

We use principal component analysis (PCA) to detect functionally interesting collective motions in molecular-dynamics simulations of membrane-bound gramicidin A. We examine the statistical and structural properties of all PCA eigenvectors and eigenvalues for the backbone and side-chain atoms. All eigenvalue spectra show two distinct power-law scaling regimes, quantitatively separating large from small covariance motions. Time trajectories of the largest PCs converge to Gaussian distributions at long timescales, but groups of small-covariance PCs, which are usually ignored as noise, have subdiffusive distributions. These non-Gaussian distributions imply anharmonic motions on the free-energy surface. We characterize the anharmonic components of motion by analyzing the mean-square displacement for all PCs. The subdiffusive components reveal picosecond-scale oscillations in the mean-square displacement at frequencies consistent with infrared measurements. In this regime, the slowest backbone mode exhibits tilting of the peptide planes, which allows carbonyl oxygen atoms to provide surrogate solvation for water and cation transport in the channel lumen. Higher-frequency modes are also apparent, and we describe their vibrational spectra. Our findings expand the utility of PCA for quantifying the essential features of motion on the anharmonic free-energy surface made accessible by atomistic molecular-dynamics simulations.     

More compact protein globules exhibit slower folding rates

We have demonstrated that, among proteins of the same size, alpha/beta proteins have on the average a greater number of contacts per residue due to their more compact (more "spherical") structure, rather than due to tighter packing. We have examined the relationship between the average number of contacts per residue and folding rates in globular proteins according to general protein structural class (all-alpha, all-beta, alpha/beta, alpha+beta). Our analysis demonstrates that alpha/beta proteins have both the greatest number of contacts and the slowest folding rates in comparison to proteins from the other structural classes. Because alpha/beta proteins are also known to be the oldest proteins, it can be suggested that proteins have evolved to pack more quickly and into looser structures.     

Dynamics alignment: comparison of protein dynamics in the SCOP database

A novel methodology for comparison of protein dynamics is presented. Protein dynamics is calculated using the Gaussian network model and the modes of motion are globally aligned using the dynamic programming algorithm of Needleman and Wunsch, commonly used for sequence alignment. The alignment is fast and can be used to analyze large sets of proteins. The methodology is applied to the four major classes of the SCOP database: "all alpha proteins," "all beta proteins," "alpha and beta proteins," and "alpha/beta proteins". We show that different domains may have similar global dynamics. In addition, we report that the dynamics of "all alpha proteins" domains are less specific to structural variations within a given fold or superfamily compared with the other classes. We report that domain pairs with the most similar and the least similar global dynamics tend to be of similar length. The significance of the methodology is that it suggests a new and efficient way of mapping between the global structural features of protein families/subfamilies and their encoded dynamics.     

Optimal relationship between average conformational entropy and average energy of interactions between residues for fast protein folding

Based on the known experimental data and using the theoretical modeling of protein folding, we demonstrate that there exists an optimal relationship between the average conformational entropy and the average energy of contacts per residue, that is an entropy capacity, for fast protein folding. Statistical analysis of conformational entropy and the number of contacts per residue for 5829 protein structures from four general structural classes (all-alpha, all-beta, +/-/beta, alpha+beta) demonstrates that each class of proteins has its own class-specific average number of contacts and average conformational entropy per residue. These class-specific features determine the folding rates: a proteins are the fastest folding proteins, then follow beta and alpha+beta proteins, and finally alpha/beta proteins are the slowest ones.     

Blockade of alpha 5 beta 1 integrins reverses the inhibitory effect of tenascin on chemotaxis of human monocytes and polymorphonuclear leukocytes through three-dimensional gels of extracellular matrix proteins

Tenascin is an extracellular matrix protein found in adults in T cell-dependent areas of lymphoid tissues, sites of inflammation, and tumors. We report here that it inhibited chemotaxis of chemoattractant-stimulated human monocytes and chemoattractant-stimulated polymorphonuclear leukocytes (PMN) through three-dimensional gels composed of collagen I or Matrigel, and chemotaxis of leukotriene B4-stimulated PMN through fibrin gels. The inhibitory effect of tenascin on monocyte or PMN chemotaxis through these matrices was reversed by Abs directed against alpha5beta1 integrins or by a peptide (GRGDSP) that binds to beta1 integrins. Tenascin did not affect leukotriene B4- or fMLP-stimulated expression of beta1 or beta2 integrins, but did exert a small inhibitory effect on PMN adhesion and closeness of apposition to fibrin(ogen)-containing surfaces. Thus, alpha5beta1 integrins mediate the inhibitory effect of tenascin on monocyte and PMN chemotaxis, without promoting close apposition between these leukocytes and surfaces coated with tenascin alone or with tenascin bound to other matrix proteins. This contrasts with the role played by alpha5beta1 integrins in promoting close apposition between fMLP-stimulated PMN and fibrin containing surfaces, thereby inhibiting chemotaxis of fMLP-stimulated PMN through fibrin gels. Thus, chemoattractants and matrix proteins regulate chemotaxis of phagocytic leukocytes by at least two different mechanisms: one in which specific chemoattractants promote very tight adhesion of leukocytes to specific matrix proteins and another in which specific matrix proteins signal cessation of migration without markedly affecting strength of leukocyte adhesion.     

Entropy capacity determines protein folding

Search and study of the general principles that govern kinetics and thermodynamics of protein folding generate a new insight into the factors controlling this process. Here, based on the known experimental data and using theoretical modeling of protein folding, we demonstrate that there exists an optimal relationship between the average conformational entropy and the average energy of contacts per residue-that is, an entropy capacity-for fast protein folding. Statistical analysis of conformational entropy and number of contacts per residue for 5829 protein structures from four general structural classes (all-alpha, all-beta, alpha/beta, alpha+beta) demonstrates that each class of proteins has its own class-specific average number of contacts (class alpha/beta has the largest number of contacts) and average conformational entropy per residue (class all-alpha has the largest number of rotatable angles phi, psi, and chi per residue). These class-specific features determine the folding rates: alpha proteins are the fastest folding proteins, then follow beta and alpha+beta proteins, and finally alpha/beta proteins are the slowest ones. Our result is in agreement with the experimental folding rates for 60 proteins. This suggests that structural and sequence properties are important determinants of protein folding rates.     

Loop motions of triosephosphate isomerase observed with elastic networks

The internal dynamics of triosephosphate isomerase have been investigated with elastic networks, with and without a substrate bound. The slowest modes of motion involve large domain motions but also a loop motion that conforms to the changes observed between the crystal structures and . Our computations confirm that the different motions of this loop are important in several of the computed slowest modes. We have shown that elastic network computations on this protein system can combine atoms for the functional parts of the structure with coarse-grained (cg) representations of the remainder of the structure in several different ways. Similar loop motions are seen with elastic network models for atomistic and mixed cg models. The loop motions are reproduced with an overlap of 0.75-0.79 by combining the four slowest modes of motion for the free and complex forms of the enzyme.     

How orientational order governs collectivity of folded proteins

The past decade has witnessed the development and success of coarse‐grained network models of proteins for predicting many equilibrium properties related to collective modes of motion. Curiously, the results are usually robust toward the different cutoff distances used for constructing the residue networks from the knowledge of the experimental coordinates. In this study, we present a systematical study of network construction and their effect on the predicted properties. Probing bond orientational order around each residue, we propose a natural partitioning of the interactions into an essential and a residual set. In this picture, the robustness originates from the way with which new contacts are added, so that an unusual local orientational order builds up. These residual interactions have a vanishingly small effect on the force vectors on each residue. The stability of the overall force balance then translates into the Hessian as small shifts in the slow modes of motion and an invariance of the corresponding eigenvectors. We introduce a rescaled version of the Hessian matrix and point out a link between the matrix Frobenius norm based on spectral stability arguments and orientational local order. A recipe for the optimal choice of partitioning the interactions into essential and residual components is prescribed. Implications for the study of biologically relevant properties of proteins are discussed with specific examples. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

The cAMP-Epac-Rap1 pathway regulates cell spreading and cell adhesion to laminin-5 through the alpha3beta1 integrin but not the alpha6beta4 integrin

Laminin-5 is an important constituent of the basal lamina. The receptors for laminin-5, the integrins alpha3beta1 and alpha6beta4, have been associated with epithelial wound migration and carcinoma invasion. The signal transduction mechanisms that regulate these integrins are not well understood. We report here that the small GTPase Rap1 regulates the adhesion of a number of cell lines to various extracellular matrix proteins including laminin-5. cAMP also mediates cell adhesion and spreading on laminin-5, a process that is independent of protein kinase A but rather dependent on Epac1, a cAMP-dependent exchange factor for Rap. Interestingly, although both alpha3beta1 and alpha6beta4 mediate adhesion to laminin-5, only alpha3beta1-dependent adhesion is dependent on Rap1. These results provide evidence for a function of the cAMP-Epac-Rap1 pathway in cell adhesion and spreading on different extracellular matrix proteins. They also define different roles for the laminin-binding integrins in regulated cell adhesion and subsequent cell spreading.     

Dynamics of firefly luciferase inhibition by general anesthetics: Gaussian and anisotropic network analyses

The new crystal structures of the product-bound firefly luciferase combined with the previously determined substrate-free structures allow for a detailed analysis of the dynamics basis for the luciferase enzymatic activities. Using the Gaussian network model and the anisotropic network model, we show here that the superposition of the three slowest anisotropic network model modes, consisting of the bending, rotating, and rocking motions of the C-domain, accounts for large rearrangement of domains from the substrate-free (open) to product-bound (closed) conformation and thus constitutes a critical component of the enzyme's functions. The analysis also offers a unique platform to reexamine the molecular mechanism of the anesthetic inhibition of the firefly luciferase. Through perturbing the protein backbone network by introducing additional nodes to represent anesthetics, we found that the presence of two representative anesthetics, halothane and n-decanol, in different regions of luciferase had distinctively different effects on the protein's global motion. Only at the interface of the C- and N-domains did the anesthetics cause the most profound reduction in the overall flexibility of the C-domain and the concomitant increase in the flexibility of the loop, where the substitution of a conserved lysine residue was found experimentally to lead to >2-3 orders of magnitude reduction in activity. These anesthetic-induced dynamics changes can alter the normal function of the protein, appearing as an epiphenomenon of an "inhibition". The implication of the study is that a leading element for general anesthetic action on proteins is to disrupt the modes of motion essential to protein functions.     

Flexibility of alpha-helices: results of a statistical analysis of database protein structures

Alpha-helices stand out as common and relatively invariant secondary structural elements of proteins. However, alpha-helices are not rigid bodies and their deformations can be significant in protein function (e.g. coiled coils). To quantify the flexibility of alpha-helices we have performed a structural principal-component analysis of helices of different lengths from a representative set of protein folds in the Protein Data Bank. We find three dominant modes of flexibility: two degenerate bend modes and one twist mode. The data are consistent with independent Gaussian distributions for each mode. The mode eigenvalues, which measure flexibility, follow simple scaling forms as a function of helix length. The dominant bend and twist modes and their harmonics are reproduced by a simple spring model, which incorporates hydrogen-bonding and excluded volume. As an application, we examine the amount of bend and twist in helices making up all coiled-coil proteins in SCOP. Incorporation of alpha-helix flexibility into structure refinement and design is discussed.     

Myosin-mediated cytoskeleton contraction and Rho GTPases regulate laminin-5 matrix assembly

Laminin-5 is a major structural element of epithelial tissue basement membranes. In the matrix of cultured epithelial cells, laminin-5 is arranged into intricate patterns. Here we tested a hypothesis that myosin II-mediated actin contraction is necessary for the proper assembly of a laminin-5 matrix by cultured SCC12 epithelial cells. To do so, the cells were treated with ML-7, a myosin II light chain kinase inhibitor, or Y-27632, an inhibitor of Rho-kinase (ROCK), both of which block actomyosin contraction. Under these conditions, laminin-5 shows an aberrant localization in dense patches at the cell periphery. Since ROCK activity is regulated by the small GTPase Rho, this suggests that members of the Rho family of GTPases may also be important for laminin-5 matrix assembly by SCC12 cells. We confirmed this hypothesis since SCC12 cells expressing mutant proteins that inhibit RhoA, Rac, and Cdc42 assemble the same aberrant laminin-5 protein arrays as drug-treated cells. We have also evaluated the organization of the laminin-5 receptors alpha3beta1 and alpha6beta4 integrin and hemidesmosome proteins in ML-7- and Y-27632-treated cells or in cells in which RhoA, Rac, and Cdc42 activity were inhibited. In all instances, alpha3beta1 and alpha6beta4 integrin heterodimers, as well as hemidesmosome proteins, localize precisely with laminin-5 in the matrix of the cells. In summary, our results provide evidence that myosin II-mediated actin contraction and the activity of Rho GTPases are necessary for the proper organization of a laminin-5 matrix and localization of hemidesmosome protein arrays in epithelial cells.     

The utrophin actin-binding domain binds F-actin in two different modes: implications for the spectrin superfamily of proteins

Utrophin, like its homologue dystrophin, forms a link between the actin cytoskeleton and the extracellular matrix. We have used a new method of image analysis to reconstruct actin filaments decorated with the actin-binding domain of utrophin, which contains two calponin homology domains. We find two different modes of binding, with either one or two calponin-homology (CH) domains bound per actin subunit, and these modes are also distinguishable by their very different effects on F-actin rigidity. Both modes involve an extended conformation of the CH domains, as predicted by a previous crystal structure. The separation of these two modes has been largely dependent upon the use of our new approach to reconstruction of helical filaments. When existing information about tropomyosin, myosin, actin-depolymerizing factor, and nebulin is considered, these results suggest that many actin-binding proteins may have multiple binding sites on F-actin. The cell may use the modular CH domains found in the spectrin superfamily of actin-binding proteins to bind actin in manifold ways, allowing for complexity to arise from the interactions of a relatively few simple modules with actin.     

Analysis of domain movements in glutamine-binding protein with simple models

In this work, the mechanism of domain movements of glutamine-binding protein (GlnBP), especially the influence of the ligand on GlnBP dynamic behavior is investigated with the aid of a Gaussian network model (GNM) and an anisotropy elastic network model. The results show that the "open-closed" transition mainly appears as the large movement of the small domain, especially the top region including two alpha-helices and two beta-strands. The slowest mode of each three forms of GlnBP--ligand-free open, ligand-bound closed, and ligand-free closed GlnBP--shows that the open-closed motion of the two domains has a common hinge axis centered on Lys-87 and Gln-183. Accompanying the conformational transition, the residues within both large and small domains move in a highly coupled way. The peaks of the fast modes correspond to residues that were thought, in the GNM, to be important for the stability of the protein, and these residues may be involved in the interactions with the membrane-bound components. With the contacts between the large domain and the small domain increasing, the ability of the "open-closed" motion is decreased. All the results agree well with those of molecular dynamics simulations, and it is thought that the open-closed conformation transition is the nature of the topology structure of GlnBP. Also, the influence of the ligand on GlnBP is studied with a modified GNM method. The results obtained show that the ligand does not influence the closed-to-open transition tendency.     

Proteomic analysis of the acid-soluble organic matrix of the chicken calcified eggshell layer

The major difference between inorganic minerals and biominerals is the presence of an organic matrix consisting of proteins, glycoproteins, proteoglycans, and polysaccharides, which is synthesized by specialized cells under genetic control before or during mineralization. The organic matrix is thought to play a major role in the assembly of the biomineral and determination of its mechanical properties. The recent elucidation of the chicken genome provided an opportunity to explore the matrix proteome of a biomineral using up-to-date MS-based technology. We identified 520 proteins in this matrix including the ten matrix proteins already known before. The identified proteins were divided into three abundance groups using the exponentially modified protein abundance index described recently which was roughly calibrated with the few known data on protein yield derived from Edman sequence analysis. A small group of 32 highly abundant proteins contained the presently known eggshell-specific proteins and all of the other known eggshell matrix constituents identified before with much less sensitive conventional methods. The present study, which is the first comprehensive proteomic study of a vertebrate biomineral, is intended as a starting point for the detailed molecular characterization of eggshell matrix proteins, their interactions in the matrix network and functional studies.