Parametrization of Backbone Flexibility in a Coarse-Grained Force Field for Proteins (COFFDROP) Derived from All-Atom Explicit-Solvent Molecular Dynamics Simulations of All Possible Two-Residue Peptides.

PubMed

Frembgen-Kesner, Tamara; Andrews, Casey T; Li, Shuxiang; Ngo, Nguyet Anh; Shubert, Scott A; Jain, Aakash; Olayiwola, Oluwatoni J; Weishaar, Mitch R; Elcock, Adrian H

2015-05-12

Recently, we reported the parametrization of a set of coarse-grained (CG) nonbonded potential functions, derived from all-atom explicit-solvent molecular dynamics (MD) simulations of amino acid pairs and designed for use in (implicit-solvent) Brownian dynamics (BD) simulations of proteins; this force field was named COFFDROP (COarse-grained Force Field for Dynamic Representations Of Proteins). Here, we describe the extension of COFFDROP to include bonded backbone terms derived from fitting to results of explicit-solvent MD simulations of all possible two-residue peptides containing the 20 standard amino acids, with histidine modeled in both its protonated and neutral forms. The iterative Boltzmann inversion (IBI) method was used to optimize new CG potential functions for backbone-related terms by attempting to reproduce angle, dihedral, and distance probability distributions generated by the MD simulations. In a simple test of the transferability of the extended force field, the angle, dihedral, and distance probability distributions obtained from BD simulations of 56 three-residue peptides were compared to results from corresponding explicit-solvent MD simulations. In a more challenging test of the COFFDROP force field, it was used to simulate eight intrinsically disordered proteins and was shown to quite accurately reproduce the experimental hydrodynamic radii (Rhydro), provided that the favorable nonbonded interactions of the force field were uniformly scaled downward in magnitude. Overall, the results indicate that the COFFDROP force field is likely to find use in modeling the conformational behavior of intrinsically disordered proteins and multidomain proteins connected by flexible linkers.

On the complexity of Engh and Huber refinement restraints: the angle τ as example

DOE Office of Scientific and Technical Information (OSTI.GOV)

Touw, Wouter G.; Vriend, Gert, E-mail: vriend@cmbi.ru.nl

2010-12-01

The angle τ (backbone N—C{sup α}—C) is the most contested Engh and Huber refinement target parameter. It is shown that this parameter is ‘correct’ as a PDB-wide average, but can be improved by taking into account residue types, secondary structures and many other aspects of our knowledge of the biophysical relations between residue type and protein structure. The Engh and Huber parameters for bond lengths and bond angles have been used uncontested in macromolecular structure refinement from 1991 until very recently, despite critical discussion of their ubiquitous validity by many authors. An extensive analysis of the backbone angle τ (N—C{supmore » α}—C) illustrates that the Engh and Huber parameters can indeed be improved and a recent study [Tronrud et al. (2010 ▶), Acta Cryst. D66, 834–842] confirms these ideas. However, the present study of τ shows that improving the Engh and Huber parameters will be considerably more complex than simply making the parameters a function of the backbone ϕ, ψ angles. Many other aspects, such as the cooperativity of hydrogen bonds, the bending of secondary-structure elements and a series of biophysical aspects of the 20 amino-acid types, will also need to be taken into account. Different sets of Engh and Huber parameters will be needed for conceptually different refinement programs.« less

Determination of Multiple φ-Torsion Angles in Proteins by Selective and Extensive 13C Labeling and Two-Dimensional Solid-State NMR

NASA Astrophysics Data System (ADS)

Hong, Mei

1999-08-01

We describe an approach to efficiently determine the backbone conformation of solid proteins that utilizes selective and extensive 13C labeling in conjunction with two-dimensional magic-angle-spinning NMR. The selective 13C labeling approach aims to reduce line broadening and other multispin complications encountered in solid-state NMR of uniformly labeled proteins while still enhancing the sensitivity of NMR spectra. It is achieved by using specifically labeled glucose or glycerol as the sole carbon source in the protein expression medium. For amino acids synthesized in the linear part of the biosynthetic pathways, [1-13C]glucose preferentially labels the ends of the side chains, while [2-13C]glycerol labels the Cα of these residues. Amino acids produced from the citric-acid cycle are labeled in a more complex manner. Information on the secondary structure of such a labeled protein was obtained by measuring multiple backbone torsion angles φ simultaneously, using an isotropic-anisotropic 2D correlation technique, the HNCH experiment. Initial experiments for resonance assignment of a selectively 13C labeled protein were performed using 15N-13C 2D correlation spectroscopy. From the time dependence of the 15N-13C dipolar coherence transfer, both intraresidue and interresidue connectivities can be observed, thus yielding partial sequential assignment. We demonstrate the selective 13C labeling and these 2D NMR experiments on a 8.5-kDa model protein, ubiquitin. This isotope-edited NMR approach is expected to facilitate the structure determination of proteins in the solid state.

Parameterization of backbone flexibility in a coarse-grained force field for proteins (COFFDROP) derived from all-atom explicit-solvent molecular dynamics simulations of all possible two-residue peptides

PubMed Central

Frembgen-Kesner, Tamara; Andrews, Casey T.; Li, Shuxiang; Ngo, Nguyet Anh; Shubert, Scott A.; Jain, Aakash; Olayiwola, Oluwatoni; Weishaar, Mitch R.; Elcock, Adrian H.

2015-01-01

Recently, we reported the parameterization of a set of coarse-grained (CG) nonbonded potential functions, derived from all-atom explicit-solvent molecular dynamics (MD) simulations of amino acid pairs, and designed for use in (implicit-solvent) Brownian dynamics (BD) simulations of proteins; this force field was named COFFDROP (COarse-grained Force Field for Dynamic Representations Of Proteins). Here, we describe the extension of COFFDROP to include bonded backbone terms derived from fitting to results of explicit-solvent MD simulations of all possible two-residue peptides containing the 20 standard amino acids, with histidine modeled in both its protonated and neutral forms. The iterative Boltzmann inversion (IBI) method was used to optimize new CG potential functions for backbone-related terms by attempting to reproduce angle, dihedral and distance probability distributions generated by the MD simulations. In a simple test of the transferability of the extended force field, the angle, dihedral and distance probability distributions obtained from BD simulations of 56 three-residue peptides were compared to results from corresponding explicit-solvent MD simulations. In a more challenging test of the COFFDROP force field, it was used to simulate eight intrinsically disordered proteins and was shown to quite accurately reproduce the experimental hydrodynamic radii (Rhydro), provided that the favorable nonbonded interactions of the force field were uniformly scaled downwards in magnitude. Overall, the results indicate that the COFFDROP force field is likely to find use in modeling the conformational behavior of intrinsically disordered proteins and multi-domain proteins connected by flexible linkers. PMID:26574429

Building alternate protein structures using the elastic network model.

PubMed

Yang, Qingyi; Sharp, Kim A

2009-02-15

We describe a method for efficiently generating ensembles of alternate, all-atom protein structures that (a) differ significantly from the starting structure, (b) have good stereochemistry (bonded geometry), and (c) have good steric properties (absence of atomic overlap). The method uses reconstruction from a series of backbone framework structures that are obtained from a modified elastic network model (ENM) by perturbation along low-frequency normal modes. To ensure good quality backbone frameworks, the single force parameter ENM is modified by introducing two more force parameters to characterize the interaction between the consecutive carbon alphas and those within the same secondary structure domain. The relative stiffness of the three parameters is parameterized to reproduce B-factors, while maintaining good bonded geometry. After parameterization, violations of experimental Calpha-Calpha distances and Calpha-Calpha-Calpha pseudo angles along the backbone are reduced to less than 1%. Simultaneously, the average B-factor correlation coefficient improves to R = 0.77. Two applications illustrate the potential of the approach. (1) 102,051 protein backbones spanning a conformational space of 15 A root mean square deviation were generated from 148 nonredundant proteins in the PDB database, and all-atom models with minimal bonded and nonbonded violations were produced from this ensemble of backbone structures using the SCWRL side chain building program. (2) Improved backbone templates for homology modeling. Fifteen query sequences were each modeled on two targets. For each of the 30 target frameworks, dozens of improved templates could be produced In all cases, improved full atom homology models resulted, of which 50% could be identified blind using the D-Fire statistical potential. (c) 2008 Wiley-Liss, Inc.

Proline-Rich Salivary Proteins Have Extended Conformations

PubMed Central

Boze, Hélène; Marlin, Thérèse; Durand, Dominique; Pérez, Javier; Vernhet, Aude; Canon, Francis; Sarni-Manchado, Pascale; Cheynier, Véronique; Cabane, Bernard

2010-01-01

Abstract Three basic proline-rich salivary proteins have been produced through the recombinant route. IB5 is a small basic proline-rich protein that is involved in the binding of plant tannins in the oral cavity. II-1 is a larger protein with a closely related backbone; it is glycosylated, and it is also able to bind plant tannins. II-1ng has the same polypeptidic backbone as II-1, but it is not glycosylated. Small angle x-ray scattering experiments on dilute solutions of these proteins confirm that they are intrinsically disordered. IB5 and II-1ng can be described through a chain model including a persistence length and cross section. The measured radii of gyration (Rg = 27.9 and 41.0 ± 1 Å respectively) and largest distances (rmax = 110 and 155 ± 10 Å respectively) show that their average conformations are rather extended. The length of the statistical segment (twice the persistence length) is b = 30 Å, which is larger than the usual value (18 Å − 20 Å) for unstructured polypeptide chains. These characteristics are presumably related to the presence of polyproline helices within the polypeptidic backbones. For both proteins, the radius of gyration of the chain cross-section is Rc = 2.7 ± 0.2Å. The glycosylated protein II-1 has similar conformations but the presence of large polyoside sidegroups yields the structure of a branched macromolecule with the same hydrophobic backbone and hydrophilic branches. It is proposed that the unusually extended conformations of these proteins in solution facilitate the capture of plant tannins in the oral cavity. PMID:20643086

DNA minor groove electrostatic potential: influence of sequence-specific transitions of the torsion angle gamma and deoxyribose conformations.

PubMed

Zhitnikova, M Y; Shestopalova, A V

2017-11-01

The structural adjustments of the sugar-phosphate DNA backbone (switching of the γ angle (O5'-C5'-C4'-C3') from canonical to alternative conformations and/or C2'-endo → C3'-endo transition of deoxyribose) lead to the sequence-specific changes in accessible surface area of both polar and non-polar atoms of the grooves and the polar/hydrophobic profile of the latter ones. The distribution of the minor groove electrostatic potential is likely to be changing as a result of such conformational rearrangements in sugar-phosphate DNA backbone. Our analysis of the crystal structures of the short free DNA fragments and calculation of their electrostatic potentials allowed us to determine: (1) the number of classical and alternative γ angle conformations in the free B-DNA; (2) changes in the minor groove electrostatic potential, depending on the conformation of the sugar-phosphate DNA backbone; (3) the effect of the DNA sequence on the minor groove electrostatic potential. We have demonstrated that the structural adjustments of the DNA double helix (the conformations of the sugar-phosphate backbone and the minor groove dimensions) induce changes in the distribution of the minor groove electrostatic potential and are sequence-specific. Therefore, these features of the minor groove sizes and distribution of minor groove electrostatic potential can be used as a signal for recognition of the target DNA sequence by protein in the implementation of the indirect readout mechanism.

A general method for the derivation of the functional forms of the effective energy terms in coarse-grained energy functions of polymers. I. Backbone potentials of coarse-grained polypeptide chains

NASA Astrophysics Data System (ADS)

Sieradzan, Adam K.; Makowski, Mariusz; Augustynowicz, Antoni; Liwo, Adam

2017-03-01

A general and systematic method for the derivation of the functional expressions for the effective energy terms in coarse-grained force fields of polymer chains is proposed. The method is based on the expansion of the potential of mean force of the system studied in the cluster-cumulant series and expanding the all-atom energy in the Taylor series in the squares of interatomic distances about the squares of the distances between coarse-grained centers, to obtain approximate analytical expressions for the cluster cumulants. The primary degrees of freedom to average about are the angles for collective rotation of the atoms contained in the coarse-grained interaction sites about the respective virtual-bond axes. The approach has been applied to the revision of the virtual-bond-angle, virtual-bond-torsional, and backbone-local-and-electrostatic correlation potentials for the UNited RESidue (UNRES) model of polypeptide chains, demonstrating the strong dependence of the torsional and correlation potentials on virtual-bond angles, not considered in the current UNRES. The theoretical considerations are illustrated with the potentials calculated from the ab initio potential-energy surface of terminally blocked alanine by numerical integration and with the statistical potentials derived from known protein structures. The revised torsional potentials correctly indicate that virtual-bond angles close to 90° result in the preference for the turn and helical structures, while large virtual-bond angles result in the preference for polyproline II and extended backbone geometry. The revised correlation potentials correctly reproduce the preference for the formation of β-sheet structures for large values of virtual-bond angles and for the formation of α-helical structures for virtual-bond angles close to 90°.

Correlation between protein secondary structure, backbone bond angles, and side-chain orientations.

PubMed

Lundgren, Martin; Niemi, Antti J

2012-08-01

We investigate the fine structure of the sp3 hybridized covalent bond geometry that governs the tetrahedral architecture around the central C(α) carbon of a protein backbone, and for this we develop new visualization techniques to analyze high-resolution x-ray structures in the Protein Data Bank. We observe that there is a correlation between the deformations of the ideal tetrahedral symmetry and the local secondary structure of the protein. We propose a universal coarse-grained energy function to describe the ensuing side-chain geometry in terms of the C(β) carbon orientations. The energy function can model the side-chain geometry with a subatomic precision. As an example we construct the C(α)-C(β) structure of HP35 chicken villin headpiece. We obtain a configuration that deviates less than 0.4 Å in root-mean-square distance from the experimental x-ray structure.

Multistep modeling of protein structure: application towards refinement of tyr-tRNA synthetase

NASA Technical Reports Server (NTRS)

Srinivasan, S.; Shibata, M.; Roychoudhury, M.; Rein, R.

1987-01-01

The scope of multistep modeling (MSM) is expanding by adding a least-squares minimization step in the procedure to fit backbone reconstruction consistent with a set of C-alpha coordinates. The analytical solution of Phi and Psi angles, that fits a C-alpha x-ray coordinate is used for tyr-tRNA synthetase. Phi and Psi angles for the region where the above mentioned method fails, are obtained by minimizing the difference in C-alpha distances between the computed model and the crystal structure in a least-squares sense. We present a stepwise application of this part of MSM to the determination of the complete backbone geometry of the 321 N terminal residues of tyrosine tRNA synthetase to a root mean square deviation of 0.47 angstroms from the crystallographic C-alpha coordinates.

A new way to see RNA

PubMed Central

Keating, Kevin S.; Humphris, Elisabeth L.; Pyle, Anna Marie

2015-01-01

Unlike proteins, the RNA backbone has numerous degrees of freedom (eight, if one counts the sugar pucker), making RNA modeling, structure building and prediction a multidimensional problem of exceptionally high complexity. And yet RNA tertiary structures are not infinite in their structural morphology; rather, they are built from a limited set of discrete units. In order to reduce the dimensionality of the RNA backbone in a physically reasonable way, a shorthand notation was created that reduced the RNA backbone torsion angles to two (η and θ, analogous to ϕ and ψ in proteins). When these torsion angles are calculated for nucleotides in a crystallographic database and plotted against one another, one obtains a plot analogous to a Ramachandran plot (the η/θ plot), with highly populated and unpopulated regions. Nucleotides that occupy proximal positions on the plot have identical structures and are found in the same units of tertiary structure. In this review, we describe the statistical validation of the η/θ formalism and the exploration of features within the η/θ plot. We also describe the application of the η/θ formalism in RNA motif discovery, structural comparison, RNA structure building and tertiary structure prediction. More than a tool, however, the η/θ formalism has provided new insights into RNA structure itself, revealing its fundamental components and the factors underlying RNA architectural form. PMID:21729350

Deep learning methods for protein torsion angle prediction.

PubMed

Li, Haiou; Hou, Jie; Adhikari, Badri; Lyu, Qiang; Cheng, Jianlin

2017-09-18

Deep learning is one of the most powerful machine learning methods that has achieved the state-of-the-art performance in many domains. Since deep learning was introduced to the field of bioinformatics in 2012, it has achieved success in a number of areas such as protein residue-residue contact prediction, secondary structure prediction, and fold recognition. In this work, we developed deep learning methods to improve the prediction of torsion (dihedral) angles of proteins. We design four different deep learning architectures to predict protein torsion angles. The architectures including deep neural network (DNN) and deep restricted Boltzmann machine (DRBN), deep recurrent neural network (DRNN) and deep recurrent restricted Boltzmann machine (DReRBM) since the protein torsion angle prediction is a sequence related problem. In addition to existing protein features, two new features (predicted residue contact number and the error distribution of torsion angles extracted from sequence fragments) are used as input to each of the four deep learning architectures to predict phi and psi angles of protein backbone. The mean absolute error (MAE) of phi and psi angles predicted by DRNN, DReRBM, DRBM and DNN is about 20-21° and 29-30° on an independent dataset. The MAE of phi angle is comparable to the existing methods, but the MAE of psi angle is 29°, 2° lower than the existing methods. On the latest CASP12 targets, our methods also achieved the performance better than or comparable to a state-of-the art method. Our experiment demonstrates that deep learning is a valuable method for predicting protein torsion angles. The deep recurrent network architecture performs slightly better than deep feed-forward architecture, and the predicted residue contact number and the error distribution of torsion angles extracted from sequence fragments are useful features for improving prediction accuracy.

Phosphorylation effects on cis/trans isomerization and the backbone conformation of serine-proline motifs: accelerated molecular dynamics analysis.

PubMed

Hamelberg, Donald; Shen, Tongye; McCammon, J Andrew

2005-02-16

The presence of serine/threonine-proline motifs in proteins provides a conformational switching mechanism of the backbone through the cis/trans isomerization of the peptidyl-prolyl (omega) bond. The reversible phosphorylation of the serine/threonine modulates this switching in regulatory proteins to alter signaling and transcription. However, the mechanism is not well understood. This is partly because cis/trans isomerization is a very slow process and, hence, difficult to study. We have used our accelerated molecular dynamics method to study the cis/trans proline isomerization, preferred backbone conformation of a serine-proline motif, and the effects of phosphorylation of the serine residue. We demonstrate that, unlike normal molecular dynamics, the accelerated molecular dynamics allows for the system to escape very easily from the trans isomer to cis isomer, and vice versa. Moreover, for both the unphosphorylated and phosphorylated peptides, the statistical thermodynamic properties are recaptured, and the results are consistent with experimental values. Isomerization of the proline omega bond is shown to be asymmetric and strongly dependent on the psi backbone angle before and after phosphorylation. The rates of escape decrease after phosphorylation. Also, the alpha-helical backbone conformation is more favored after phosphorylation. This accelerated molecular dynamics approach provides a general approach for enhancing the conformational transitions of molecular systems without having prior knowledge of the location of the minima and barriers on the potential-energy landscape.

Backbone amide 15N chemical shift tensors report on hydrogen bonding interactions in proteins: A magic angle spinning NMR study.

PubMed

Paramasivam, Sivakumar; Gronenborn, Angela M; Polenova, Tatyana

2018-08-01

Chemical shift tensors (CSTs) are an exquisite probe of local geometric and electronic structure. 15 N CST are very sensitive to hydrogen bonding, yet they have been reported for very few proteins to date. Here we present experimental results and statistical analysis of backbone amide 15 N CSTs for 100 residues of four proteins, two E. coli thioredoxin reassemblies (1-73-(U- 13 C, 15 N)/74-108-(U- 15 N) and 1-73-(U- 15 N)/74-108-(U- 13 C, 15 N)), dynein light chain 8 LC8, and CAP-Gly domain of the mammalian dynactin. The 15 N CSTs were measured by a symmetry-based CSA recoupling method, ROCSA. Our results show that the principal component δ 11 is very sensitive to the presence of hydrogen bonding interactions due to its unique orientation in the molecular frame. The downfield chemical shift change of backbone amide nitrogen nuclei with increasing hydrogen bond strength is manifested in the negative correlation of the principal components with hydrogen bond distance for both α-helical and β-sheet secondary structure elements. Our findings highlight the potential for the use of 15 N CSTs in protein structure refinement. Copyright © 2018 Elsevier Inc. All rights reserved.

High-resolution molecular structure of a peptide in an amyloid fibril determined by magic angle spinning NMR spectroscopy

NASA Astrophysics Data System (ADS)

Jaroniec, Christopher P.; Macphee, Cait E.; Bajaj, Vikram S.; McMahon, Michael T.; Dobson, Christopher M.; Griffin, Robert G.

2004-01-01

Amyloid fibrils are self-assembled filamentous structures associated with protein deposition conditions including Alzheimer's disease and the transmissible spongiform encephalopathies. Despite the immense medical importance of amyloid fibrils, no atomic-resolution structures are available for these materials, because the intact fibrils are insoluble and do not form diffraction-quality 3D crystals. Here we report the high-resolution structure of a peptide fragment of the amyloidogenic protein transthyretin, TTR(105-115), in its fibrillar form, determined by magic angle spinning NMR spectroscopy. The structure resolves not only the backbone fold but also the precise conformation of the side chains. Nearly complete 13C and 15N resonance assignments for TTR(105-115) formed the basis for the extraction of a set of distance and dihedral angle restraints. A total of 76 self-consistent experimental measurements, including 41 restraints on 19 backbone dihedral angles and 35 13C-15N distances between 3 and 6 Å were obtained from 2D and 3D NMR spectra recorded on three fibril samples uniformly 13C, 15N-labeled in consecutive stretches of four amino acids and used to calculate an ensemble of peptide structures. Our results indicate that TTR(105-115) adopts an extended -strand conformation in the amyloid fibrils such that both the main- and side-chain torsion angles are close to their optimal values. Moreover, the structure of this peptide in the fibrillar form has a degree of long-range order that is generally associated only with crystalline materials. These findings provide an explanation of the unusual stability and characteristic properties of this form of polypeptide assembly.

Assessing protein conformational sampling methods based on bivariate lag-distributions of backbone angles

PubMed Central

Maadooliat, Mehdi; Huang, Jianhua Z.

2013-01-01

Despite considerable progress in the past decades, protein structure prediction remains one of the major unsolved problems in computational biology. Angular-sampling-based methods have been extensively studied recently due to their ability to capture the continuous conformational space of protein structures. The literature has focused on using a variety of parametric models of the sequential dependencies between angle pairs along the protein chains. In this article, we present a thorough review of angular-sampling-based methods by assessing three main questions: What is the best distribution type to model the protein angles? What is a reasonable number of components in a mixture model that should be considered to accurately parameterize the joint distribution of the angles? and What is the order of the local sequence–structure dependency that should be considered by a prediction method? We assess the model fits for different methods using bivariate lag-distributions of the dihedral/planar angles. Moreover, the main information across the lags can be extracted using a technique called Lag singular value decomposition (LagSVD), which considers the joint distribution of the dihedral/planar angles over different lags using a nonparametric approach and monitors the behavior of the lag-distribution of the angles using singular value decomposition. As a result, we developed graphical tools and numerical measurements to compare and evaluate the performance of different model fits. Furthermore, we developed a web-tool (http://www.stat.tamu.edu/∼madoliat/LagSVD) that can be used to produce informative animations. PMID:22926831

Sterically allowed configuration space for amino acid dipeptides

NASA Astrophysics Data System (ADS)

Caballero, Diego; Maatta, Jukka; Sammalkorpi, Maria; O'Hern, Corey; Regan, Lynne

2014-03-01

Despite recent improvements in computational methods for protein design, we still lack a quantitative, predictive understanding of the intrinsic propensities for amino acids to be in particular backbone or side-chain conformations. This question has remained unsettled for years because of the discrepancies between different experimental approaches. To address it, I performed all-atom hard-sphere simulations of hydrophobic residues with stereo-chemical constraints and non-attractive steric interactions between non-bonded atoms for ALA, ILE, LEU and VAL dipeptide mimetics. For these hard-sphere MD simulations, I show that transitions between α-helix and β-sheet structures only occur when the bond angle τ(N -Cα - C) >110° , and the probability distribution of bond angles for structures in the `bridge' region of ϕ- ψ space is shifted to larger angles compared to that in other regions. In contrast, the relevant bond-angle distributions obtained from most molecular dynamics packages are broader and shifter to larger values. I encounter similar correlations between bond angles and side-chain dihedral angles. The success of these studies is an argument for re-incorporating local stereochemical constraints into computational protein design methodology.

On Ramachandran angles, closed strings and knots in protein structure

NASA Astrophysics Data System (ADS)

Chen, Si; Niemi, Antti J.

2016-08-01

The Ramachandran angles (φ,\\psi ) of a protein backbone form the vertices of a piecewise geodesic curve on the surface of a torus. When the ends of the curve are connected to each other similarly, by a geodesic, the result is a closed string that in general wraps around the torus a number of times both in the meridional and the longitudinal directions. The two wrapping numbers are global characteristics of the protein structure. A statistical analysis of the wrapping numbers in terms of crystallographic x-ray structures in the protein data bank (PDB) reveals that proteins have no net chirality in the ϕ direction but in the ψ direction, proteins prefer to display chirality. A comparison between the wrapping numbers and the concept of folding index discloses a non-linearity in their relationship. Thus these three integer valued invariants can be used in tandem, to scrutinize and classify the global loop structure of individual PDB proteins, in terms of the overall fold topology.

Development of techniques in magnetic resonance and structural studies of the prion protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bitter, Hans-Marcus L.

2000-07-01

Magnetic resonance is the most powerful analytical tool used by chemists today. Its applications range from determining structures of large biomolecules to imaging of human brains. Nevertheless, magnetic resonance remains a relatively young field, in which many techniques are currently being developed that have broad applications. In this dissertation, two new techniques are presented, one that enables the determination of torsion angles in solid-state peptides and proteins, and another that involves imaging of heterogenous materials at ultra-low magnetic fields. In addition, structural studies of the prion protein via solid-state NMR are described. More specifically, work is presented in which themore » dependence of chemical shifts on local molecular structure is used to predict chemical shift tensors in solid-state peptides with theoretical ab initio surfaces. These predictions are then used to determine the backbone dihedral angles in peptides. This method utilizes the theoretical chemicalshift tensors and experimentally determined chemical-shift anisotropies (CSAs) to predict the backbone and side chain torsion angles in alanine, leucine, and valine residues. Additionally, structural studies of prion protein fragments are described in which conformationally-dependent chemical-shift measurements were made to gain insight into the structural differences between the various conformational states of the prion protein. These studies are of biological and pathological interest since conformational changes in the prion protein are believed to cause prion diseases. Finally, an ultra-low field magnetic resonance imaging technique is described that enables imaging and characterization of heterogeneous and porous media. The notion of imaging gases at ultra-low fields would appear to be very difficult due to the prohibitively low polarization and spin densities as well as the low sensitivities of conventional Faraday coil detectors. However, Chapter 5 describes how gas imaging at ultra-low fields is realized by incorporating the high sensitivities of a dc superconducting quantum interference device (SQUID) with the high polarizations attainable through optica11y pumping 129Xe gas.« less

Effect of polarization on the stability of a helix dimer

NASA Astrophysics Data System (ADS)

Wang, Xing Y.; Zhang, John Z. H.

2011-01-01

Molecular dynamics (MD) simulations have been carried out to study helix-helix interaction using both standard AMBER and polarized force fields. Comparison of the two simulations shows that electrostatic polarization of intra-protein hydrogen bonds plays a significant role in stabilizing the structure of helix dimer. This stabilizing effect is clearly demonstrated by examining the monomer structure, helix crossing angle and stability of backbone hydrogen bonds under AMBER and PPC. Since reliable prediction of protein-protein structure is a significant challenge, the current study should help shed light on the importance of electrostatic polarization of protein in helix-helix interaction and helix bundle structures.

Local backbone structure prediction of proteins

PubMed Central

De Brevern, Alexandre G.; Benros, Cristina; Gautier, Romain; Valadié, Hélène; Hazout, Serge; Etchebest, Catherine

2004-01-01

Summary A statistical analysis of the PDB structures has led us to define a new set of small 3D structural prototypes called Protein Blocks (PBs). This structural alphabet includes 16 PBs, each one is defined by the (φ, Ψ) dihedral angles of 5 consecutive residues. The amino acid distributions observed in sequence windows encompassing these PBs are used to predict by a Bayesian approach the local 3D structure of proteins from the sole knowledge of their sequences. LocPred is a software which allows the users to submit a protein sequence and performs a prediction in terms of PBs. The prediction results are given both textually and graphically. PMID:15724288

An Evaluation of Explicit Receptor Flexibility in Molecular Docking Using Molecular Dynamics and Torsion Angle Molecular Dynamics.

PubMed

Armen, Roger S; Chen, Jianhan; Brooks, Charles L

2009-10-13

Incorporating receptor flexibility into molecular docking should improve results for flexible proteins. However, the incorporation of explicit all-atom flexibility with molecular dynamics for the entire protein chain may also introduce significant error and "noise" that could decrease docking accuracy and deteriorate the ability of a scoring function to rank native-like poses. We address this apparent paradox by comparing the success of several flexible receptor models in cross-docking and multiple receptor ensemble docking for p38α mitogen-activated protein (MAP) kinase. Explicit all-atom receptor flexibility has been incorporated into a CHARMM-based molecular docking method (CDOCKER) using both molecular dynamics (MD) and torsion angle molecular dynamics (TAMD) for the refinement of predicted protein-ligand binding geometries. These flexible receptor models have been evaluated, and the accuracy and efficiency of TAMD sampling is directly compared to MD sampling. Several flexible receptor models are compared, encompassing flexible side chains, flexible loops, multiple flexible backbone segments, and treatment of the entire chain as flexible. We find that although including side chain and some backbone flexibility is required for improved docking accuracy as expected, docking accuracy also diminishes as additional and unnecessary receptor flexibility is included into the conformational search space. Ensemble docking results demonstrate that including protein flexibility leads to to improved agreement with binding data for 227 active compounds. This comparison also demonstrates that a flexible receptor model enriches high affinity compound identification without significantly increasing the number of false positives from low affinity compounds.

An Evaluation of Explicit Receptor Flexibility in Molecular Docking Using Molecular Dynamics and Torsion Angle Molecular Dynamics

PubMed Central

Armen, Roger S.; Chen, Jianhan; Brooks, Charles L.

2009-01-01

Incorporating receptor flexibility into molecular docking should improve results for flexible proteins. However, the incorporation of explicit all-atom flexibility with molecular dynamics for the entire protein chain may also introduce significant error and “noise” that could decrease docking accuracy and deteriorate the ability of a scoring function to rank native-like poses. We address this apparent paradox by comparing the success of several flexible receptor models in cross-docking and multiple receptor ensemble docking for p38α mitogen-activated protein (MAP) kinase. Explicit all-atom receptor flexibility has been incorporated into a CHARMM-based molecular docking method (CDOCKER) using both molecular dynamics (MD) and torsion angle molecular dynamics (TAMD) for the refinement of predicted protein-ligand binding geometries. These flexible receptor models have been evaluated, and the accuracy and efficiency of TAMD sampling is directly compared to MD sampling. Several flexible receptor models are compared, encompassing flexible side chains, flexible loops, multiple flexible backbone segments, and treatment of the entire chain as flexible. We find that although including side chain and some backbone flexibility is required for improved docking accuracy as expected, docking accuracy also diminishes as additional and unnecessary receptor flexibility is included into the conformational search space. Ensemble docking results demonstrate that including protein flexibility leads to to improved agreement with binding data for 227 active compounds. This comparison also demonstrates that a flexible receptor model enriches high affinity compound identification without significantly increasing the number of false positives from low affinity compounds. PMID:20160879

DOE Office of Scientific and Technical Information (OSTI.GOV)

Driscoll, P.C.; Gronenborn, A.M.; Beress, L.

The three-dimensional solution structure of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata has been determined on the basis of 489 interproton and 24 hydrogen-bonding distance restraints supplemented by 23 {phi} backbone and 21 {sub {chi}1} side-chain torsion angle restraints derived from nuclear magnetic resonance (NMR) measurements. A total of 42 structures is calculated by a hybrid metric matrix distance geometry-dynamical simulated annealing approach. Both the backbone and side-chain atom positions are well defined. The average atomic rms difference between the 42 individual SA structures and the mean structure obtained by averaging their coordinates is 0.67more » {plus minus} 0.12 {angstrom} for the backbone atoms and 0.90 {plus minus} 0.17 {angstrom} for all atoms. The core of the protein is formed by a triple-stranded antiparallel {beta}-sheet composed of residues 14-16 (strand 1), 30-34 (strand 2), and 37-41 (strand 3) with an additional mini-antiparallel {beta}-sheet at the N-terminus (residues 6-9). The first and second strands of the triple-stranded antiparallel {beta}-sheet are connected by a long exposed loop. A number of side-chain interactions are discussed in light of the structure.« less

Access to aliphatic protons as reporters in non-deuterated proteins by solid-state NMR.

PubMed

Vasa, Suresh Kumar; Rovó, Petra; Giller, Karin; Becker, Stefan; Linser, Rasmus

2016-03-28

Interactions within proteins, with their surrounding, and with other molecules are mediated mostly by hydrogen atoms. In fully protonated, inhomogeneous, or larger proteins, however, aliphatic proton shifts tend to show little dispersion despite fast Magic-Angle Spinning. 3D correlations dispersing aliphatic proton shifts by their better resolved amide N/H shifts can alleviate this problem. Using inverse second-order cross-polarization (iSOCP), we here introduce dedicated and improved means to sensitively link site-specific chemical shift information from aliphatic protons with a backbone amide resolution. Thus, even in cases where protein deuteration is impossible, this approach may enable access to various aspects of protein functions that are reported on by protons.

Discrete Haar transform and protein structure.

PubMed

Morosetti, S

1997-12-01

The discrete Haar transform of the sequence of the backbone dihedral angles (phi and psi) was performed over a set of X-ray protein structures of high resolution from the Brookhaven Protein Data Bank. Afterwards, the new dihedral angles were calculated by the inverse transform, using a growing number of Haar functions, from the lower to the higher degree. New structures were obtained using these dihedral angles, with standard values for bond lengths and angles, and with omega = 0 degree. The reconstructed structures were compared with the experimental ones, and analyzed by visual inspection and statistical analysis. When half of the Haar coefficients were used, all the reconstructed structures were not yet collapsed to a tertiary folding, but they showed yet realized most of the secondary motifs. These results indicate a substantial separation of structural information in the space of Haar transform, with the secondary structural information mainly present in the Haar coefficients of lower degrees, and the tertiary one present in the higher degree coefficients. Because of this separation, the representation of the folded structures in the space of Haar transform seems a promising candidate to encompass the problem of premature convergence in genetic algorithms.

Determination of Structural Topology of a Membrane Protein in Lipid -Bilayers using Polarization Optimized Experiments (POE) for Static and MAS Solid State NMR Spectroscopy

PubMed Central

Mote, Kaustubh R.; Gopinath, T.; Veglia, Gianluigi

2013-01-01

The low sensitivity inherent to both the static and magic angle spinning techniques of solid-state NMR (ssNMR) spectroscopy has thus far limited the routine application of multidimensional experiments to determine the structure of membrane proteins in lipid bilayers. Here, we demonstrate the advantage of using a recently developed class of experiments, polarization optimized experiments (POE), for both static and MAS spectroscopy to achieve higher sensitivity and substantial time-savings for 2D and 3D experiments. We used sarcolipin, a single pass membrane protein, reconstituted in oriented bicelles (for oriented ssNMR) and multilamellar vesicles (for MAS ssNMR) as a benchmark. The restraints derived by these experiments are then combined into a hybrid energy function to allow simultaneous determination of structure and topology. The resulting structural ensemble converged to a helical conformation with a backbone RMSD ∼ 0.44 Å, a tilt angle of 24° ± 1°, and an azimuthal angle of 55° ± 6°. This work represents a crucial first step toward obtaining high-resolution structures of large membrane proteins using combined multidimensional O-ssNMR and MAS-ssNMR. PMID:23963722

Semiautomated model building for RNA crystallography using a directed rotameric approach.

PubMed

Keating, Kevin S; Pyle, Anna Marie

2010-05-04

Structured RNA molecules play essential roles in a variety of cellular processes; however, crystallographic studies of such RNA molecules present a large number of challenges. One notable complication arises from the low resolutions typical of RNA crystallography, which results in electron density maps that are imprecise and difficult to interpret. This problem is exacerbated by the lack of computational tools for RNA modeling, as many of the techniques commonly used in protein crystallography have no equivalents for RNA structure. This leads to difficulty and errors in the model building process, particularly in modeling of the RNA backbone, which is highly error prone due to the large number of variable torsion angles per nucleotide. To address this, we have developed a method for accurately building the RNA backbone into maps of intermediate or low resolution. This method is semiautomated, as it requires a crystallographer to first locate phosphates and bases in the electron density map. After this initial trace of the molecule, however, an accurate backbone structure can be built without further user intervention. To accomplish this, backbone conformers are first predicted using RNA pseudotorsions and the base-phosphate perpendicular distance. Detailed backbone coordinates are then calculated to conform both to the predicted conformer and to the previously located phosphates and bases. This technique is shown to produce accurate backbone structure even when starting from imprecise phosphate and base coordinates. A program implementing this methodology is currently available, and a plugin for the Coot model building program is under development.

Modeling 15N NMR chemical shift changes in protein backbone with pressure

NASA Astrophysics Data System (ADS)

La Penna, Giovanni; Mori, Yoshiharu; Kitahara, Ryo; Akasaka, Kazuyuki; Okamoto, Yuko

2016-08-01

Nitrogen chemical shift is a useful parameter for determining the backbone three-dimensional structure of proteins. Empirical models for fast calculation of N chemical shift are improving their reliability, but there are subtle effects that cannot be easily interpreted. Among these, the effects of slight changes in hydrogen bonds, both intramolecular and with water molecules in the solvent, are particularly difficult to predict. On the other hand, these hydrogen bonds are sensitive to changes in protein environment. In this work, the change of N chemical shift with pressure for backbone segments in the protein ubiquitin is correlated with the change in the population of hydrogen bonds involving the backbone amide group. The different extent of interaction of protein backbone with the water molecules in the solvent is put in evidence.

Increasing Sequence Diversity with Flexible Backbone Protein Design: The Complete Redesign of a Protein Hydrophobic Core

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murphy, Grant S.; Mills, Jeffrey L.; Miley, Michael J.

2015-10-15

Protein design tests our understanding of protein stability and structure. Successful design methods should allow the exploration of sequence space not found in nature. However, when redesigning naturally occurring protein structures, most fixed backbone design algorithms return amino acid sequences that share strong sequence identity with wild-type sequences, especially in the protein core. This behavior places a restriction on functional space that can be explored and is not consistent with observations from nature, where sequences of low identity have similar structures. Here, we allow backbone flexibility during design to mutate every position in the core (38 residues) of a four-helixmore » bundle protein. Only small perturbations to the backbone, 12 {angstrom}, were needed to entirely mutate the core. The redesigned protein, DRNN, is exceptionally stable (melting point >140C). An NMR and X-ray crystal structure show that the side chains and backbone were accurately modeled (all-atom RMSD = 1.3 {angstrom}).« less

pi-Turns: types, systematics and the context of their occurrence in protein structures

PubMed Central

Dasgupta, Bhaskar; Chakrabarti, Pinak

2008-01-01

Background For a proper understanding of protein structure and folding it is important to know if a polypeptide segment adopts a conformation inherent in the sequence or it depends on the context of its flanking secondary structures. Turns of various lengths have been studied and characterized starting from three-residue γ-turn to six-residue π-turn. The Schellman motif occurring at the C-terminal end of α-helices is a classical example of hydrogen bonded π-turn involving residues at (i) and (i+5) positions. Hydrogen bonded and non-hydrogen bonded β- and α-turns have been identified previously; likewise, a systematic characterization of π-turns would provide valuable insight into turn structures. Results An analysis of protein structures indicates that at least 20% of π-turns occur independent of the Schellman motif. The two categories of π-turns, designated as π-HB and SCH, have been further classified on the basis of backbone conformation and both have AAAa as the major class. They differ in the residue usage at position (i+1), the former having a large preference for Pro that is absent in the latter. As in the case of shorter length β- and α-turns, π-turns have also been identified not only on the basis of the existence of hydrogen bond, but also using the distance between terminal Cα-atoms, and this resulted in a comparable number of non-hydrogen-bonded π-turns (π-NHB). The presence of shorter β- and α-turns within all categories of π-turns, the subtle variations in backbone torsion angles along the turn residues, the location of the turns in the context of tertiary structures have been studied. Conclusion π-turns have been characterized, first using hydrogen bond and the distance between Cα atoms of the terminal residues, and then using backbone torsion angles. While the Schellman motif has a structural role in helix termination, many of the π-HB turns, being located on surface cavities, have functional role and there is also sequence conservation. PMID:18808671

pi-Turns: types, systematics and the context of their occurrence in protein structures.

PubMed

Dasgupta, Bhaskar; Chakrabarti, Pinak

2008-09-22

For a proper understanding of protein structure and folding it is important to know if a polypeptide segment adopts a conformation inherent in the sequence or it depends on the context of its flanking secondary structures. Turns of various lengths have been studied and characterized starting from three-residue gamma-turn to six-residue pi-turn. The Schellman motif occurring at the C-terminal end of alpha-helices is a classical example of hydrogen bonded pi-turn involving residues at (i) and (i+5) positions. Hydrogen bonded and non-hydrogen bonded beta- and alpha-turns have been identified previously; likewise, a systematic characterization of pi-turns would provide valuable insight into turn structures. An analysis of protein structures indicates that at least 20% of pi-turns occur independent of the Schellman motif. The two categories of pi-turns, designated as pi-HB and SCH, have been further classified on the basis of backbone conformation and both have AAAa as the major class. They differ in the residue usage at position (i+1), the former having a large preference for Pro that is absent in the latter. As in the case of shorter length beta- and alpha-turns, pi-turns have also been identified not only on the basis of the existence of hydrogen bond, but also using the distance between terminal C alpha-atoms, and this resulted in a comparable number of non-hydrogen-bonded pi-turns (pi-NHB). The presence of shorter beta- and alpha-turns within all categories of pi-turns, the subtle variations in backbone torsion angles along the turn residues, the location of the turns in the context of tertiary structures have been studied. pi-turns have been characterized, first using hydrogen bond and the distance between C alpha atoms of the terminal residues, and then using backbone torsion angles. While the Schellman motif has a structural role in helix termination, many of the pi-HB turns, being located on surface cavities, have functional role and there is also sequence conservation.

A protein-dependent side-chain rotamer library.

PubMed

Bhuyan, Md Shariful Islam; Gao, Xin

2011-12-14

Protein side-chain packing problem has remained one of the key open problems in bioinformatics. The three main components of protein side-chain prediction methods are a rotamer library, an energy function and a search algorithm. Rotamer libraries summarize the existing knowledge of the experimentally determined structures quantitatively. Depending on how much contextual information is encoded, there are backbone-independent rotamer libraries and backbone-dependent rotamer libraries. Backbone-independent libraries only encode sequential information, whereas backbone-dependent libraries encode both sequential and locally structural information. However, side-chain conformations are determined by spatially local information, rather than sequentially local information. Since in the side-chain prediction problem, the backbone structure is given, spatially local information should ideally be encoded into the rotamer libraries. In this paper, we propose a new type of backbone-dependent rotamer library, which encodes structural information of all the spatially neighboring residues. We call it protein-dependent rotamer libraries. Given any rotamer library and a protein backbone structure, we first model the protein structure as a Markov random field. Then the marginal distributions are estimated by the inference algorithms, without doing global optimization or search. The rotamers from the given library are then re-ranked and associated with the updated probabilities. Experimental results demonstrate that the proposed protein-dependent libraries significantly outperform the widely used backbone-dependent libraries in terms of the side-chain prediction accuracy and the rotamer ranking ability. Furthermore, without global optimization/search, the side-chain prediction power of the protein-dependent library is still comparable to the global-search-based side-chain prediction methods.

Computation-Guided Backbone Grafting of a Discontinuous Motif onto a Protein Scaffold

DOE Office of Scientific and Technical Information (OSTI.GOV)

Azoitei, Mihai L.; Correia, Bruno E.; Ban, Yih-En Andrew

2012-02-07

The manipulation of protein backbone structure to control interaction and function is a challenge for protein engineering. We integrated computational design with experimental selection for grafting the backbone and side chains of a two-segment HIV gp120 epitope, targeted by the cross-neutralizing antibody b12, onto an unrelated scaffold protein. The final scaffolds bound b12 with high specificity and with affinity similar to that of gp120, and crystallographic analysis of a scaffold bound to b12 revealed high structural mimicry of the gp120-b12 complex structure. The method can be generalized to design other functional proteins through backbone grafting.

Modeling {sup 15}N NMR chemical shift changes in protein backbone with pressure

DOE Office of Scientific and Technical Information (OSTI.GOV)

La Penna, Giovanni, E-mail: glapenna@iccom.cnr.it; Mori, Yoshiharu, E-mail: ymori@ims.ac.jp; Kitahara, Ryo, E-mail: ryo@ph.ritsumei.ac.jp

2016-08-28

Nitrogen chemical shift is a useful parameter for determining the backbone three-dimensional structure of proteins. Empirical models for fast calculation of N chemical shift are improving their reliability, but there are subtle effects that cannot be easily interpreted. Among these, the effects of slight changes in hydrogen bonds, both intramolecular and with water molecules in the solvent, are particularly difficult to predict. On the other hand, these hydrogen bonds are sensitive to changes in protein environment. In this work, the change of N chemical shift with pressure for backbone segments in the protein ubiquitin is correlated with the change inmore » the population of hydrogen bonds involving the backbone amide group. The different extent of interaction of protein backbone with the water molecules in the solvent is put in evidence.« less

High-resolution protein design with backbone freedom.

PubMed

Harbury, P B; Plecs, J J; Tidor, B; Alber, T; Kim, P S

1998-11-20

Recent advances in computational techniques have allowed the design of precise side-chain packing in proteins with predetermined, naturally occurring backbone structures. Because these methods do not model protein main-chain flexibility, they lack the breadth to explore novel backbone conformations. Here the de novo design of a family of alpha-helical bundle proteins with a right-handed superhelical twist is described. In the design, the overall protein fold was specified by hydrophobic-polar residue patterning, whereas the bundle oligomerization state, detailed main-chain conformation, and interior side-chain rotamers were engineered by computational enumerations of packing in alternate backbone structures. Main-chain flexibility was incorporated through an algebraic parameterization of the backbone. The designed peptides form alpha-helical dimers, trimers, and tetramers in accord with the design goals. The crystal structure of the tetramer matches the designed structure in atomic detail.

The effects of high concentrations of ionic liquid on GB1 protein structure and dynamics probed by high-resolution magic-angle-spinning NMR spectroscopy.

PubMed

Warner, Lisa; Gjersing, Erica; Follett, Shelby E; Elliott, K Wade; Dzyuba, Sergei V; Varga, Krisztina

2016-12-01

Ionic liquids have great potential in biological applications and biocatalysis, as some ionic liquids can stabilize proteins and enhance enzyme activity, while others have the opposite effect. However, on the molecular level, probing ionic liquid interactions with proteins, especially in solutions containing high concentration of ionic liquids, has been challenging. In the present work the 13 C, 15 N-enriched GB1 model protein was used to demonstrate applicability of high-resolution magic-angle-spinning (HR-MAS) NMR spectroscopy to investigate ionic liquid - protein interactions. Effect of an ionic liquid (1-butyl-3-methylimidazolium bromide, [C 4 -mim]Br) on GB1was studied over a wide range of the ionic liquid concentrations (0.6 to 3.5 M, which corresponds to 10%-60% v/v). Interactions between GB1 and [C 4 -mim]Br were observed from changes in the chemical shifts of the protein backbone as well as the changes in 15 N ps-ns dynamics and rotational correlation times. Site-specific interactions between the protein and [C 4 -mim]Br were assigned using 3D methods under HR-MAS conditions. Thus, HR-MAS NMR is a viable tool that could aid in elucidation of the molecular mechanism of ionic liquid - protein interactions.

Capturing non-local interactions by long short-term memory bidirectional recurrent neural networks for improving prediction of protein secondary structure, backbone angles, contact numbers and solvent accessibility.

PubMed

Heffernan, Rhys; Yang, Yuedong; Paliwal, Kuldip; Zhou, Yaoqi

2017-09-15

The accuracy of predicting protein local and global structural properties such as secondary structure and solvent accessible surface area has been stagnant for many years because of the challenge of accounting for non-local interactions between amino acid residues that are close in three-dimensional structural space but far from each other in their sequence positions. All existing machine-learning techniques relied on a sliding window of 10-20 amino acid residues to capture some 'short to intermediate' non-local interactions. Here, we employed Long Short-Term Memory (LSTM) Bidirectional Recurrent Neural Networks (BRNNs) which are capable of capturing long range interactions without using a window. We showed that the application of LSTM-BRNN to the prediction of protein structural properties makes the most significant improvement for residues with the most long-range contacts (|i-j| >19) over a previous window-based, deep-learning method SPIDER2. Capturing long-range interactions allows the accuracy of three-state secondary structure prediction to reach 84% and the correlation coefficient between predicted and actual solvent accessible surface areas to reach 0.80, plus a reduction of 5%, 10%, 5% and 10% in the mean absolute error for backbone ϕ , ψ , θ and τ angles, respectively, from SPIDER2. More significantly, 27% of 182724 40-residue models directly constructed from predicted C α atom-based θ and τ have similar structures to their corresponding native structures (6Å RMSD or less), which is 3% better than models built by ϕ and ψ angles. We expect the method to be useful for assisting protein structure and function prediction. The method is available as a SPIDER3 server and standalone package at http://sparks-lab.org . yaoqi.zhou@griffith.edu.au or yuedong.yang@griffith.edu.au. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Computational protein design with backbone plasticity

PubMed Central

MacDonald, James T.; Freemont, Paul S.

2016-01-01

The computational algorithms used in the design of artificial proteins have become increasingly sophisticated in recent years, producing a series of remarkable successes. The most dramatic of these is the de novo design of artificial enzymes. The majority of these designs have reused naturally occurring protein structures as ‘scaffolds’ onto which novel functionality can be grafted without having to redesign the backbone structure. The incorporation of backbone flexibility into protein design is a much more computationally challenging problem due to the greatly increased search space, but promises to remove the limitations of reusing natural protein scaffolds. In this review, we outline the principles of computational protein design methods and discuss recent efforts to consider backbone plasticity in the design process. PMID:27911735

Wetting of nonconserved residue-backbones: A feature indicative of aggregation associated regions of proteins.

PubMed

Pradhan, Mohan R; Pal, Arumay; Hu, Zhongqiao; Kannan, Srinivasaraghavan; Chee Keong, Kwoh; Lane, David P; Verma, Chandra S

2016-02-01

Aggregation is an irreversible form of protein complexation and often toxic to cells. The process entails partial or major unfolding that is largely driven by hydration. We model the role of hydration in aggregation using "Dehydrons." "Dehydrons" are unsatisfied backbone hydrogen bonds in proteins that seek shielding from water molecules by associating with ligands or proteins. We find that the residues at aggregation interfaces have hydrated backbones, and in contrast to other forms of protein-protein interactions, are under less evolutionary pressure to be conserved. Combining evolutionary conservation of residues and extent of backbone hydration allows us to distinguish regions on proteins associated with aggregation (non-conserved dehydron-residues) from other interaction interfaces (conserved dehydron-residues). This novel feature can complement the existing strategies used to investigate protein aggregation/complexation. © 2015 Wiley Periodicals, Inc.

Predicting β-turns and their types using predicted backbone dihedral angles and secondary structures

PubMed Central

2010-01-01

Background β-turns are secondary structure elements usually classified as coil. Their prediction is important, because of their role in protein folding and their frequent occurrence in protein chains. Results We have developed a novel method that predicts β-turns and their types using information from multiple sequence alignments, predicted secondary structures and, for the first time, predicted dihedral angles. Our method uses support vector machines, a supervised classification technique, and is trained and tested on three established datasets of 426, 547 and 823 protein chains. We achieve a Matthews correlation coefficient of up to 0.49, when predicting the location of β-turns, the highest reported value to date. Moreover, the additional dihedral information improves the prediction of β-turn types I, II, IV, VIII and "non-specific", achieving correlation coefficients up to 0.39, 0.33, 0.27, 0.14 and 0.38, respectively. Our results are more accurate than other methods. Conclusions We have created an accurate predictor of β-turns and their types. Our method, called DEBT, is available online at http://comp.chem.nottingham.ac.uk/debt/. PMID:20673368

Predicting beta-turns and their types using predicted backbone dihedral angles and secondary structures.

PubMed

Kountouris, Petros; Hirst, Jonathan D

2010-07-31

Beta-turns are secondary structure elements usually classified as coil. Their prediction is important, because of their role in protein folding and their frequent occurrence in protein chains. We have developed a novel method that predicts beta-turns and their types using information from multiple sequence alignments, predicted secondary structures and, for the first time, predicted dihedral angles. Our method uses support vector machines, a supervised classification technique, and is trained and tested on three established datasets of 426, 547 and 823 protein chains. We achieve a Matthews correlation coefficient of up to 0.49, when predicting the location of beta-turns, the highest reported value to date. Moreover, the additional dihedral information improves the prediction of beta-turn types I, II, IV, VIII and "non-specific", achieving correlation coefficients up to 0.39, 0.33, 0.27, 0.14 and 0.38, respectively. Our results are more accurate than other methods. We have created an accurate predictor of beta-turns and their types. Our method, called DEBT, is available online at http://comp.chem.nottingham.ac.uk/debt/.

Data on diverse roles of helix perturbations in membrane proteins.

PubMed

Shelar, Ashish; Bansal, Manju

2016-12-01

The various structural variations observed in TM helices of membrane proteins have been deconstructed into 9 distinct types of helix perturbations. These perturbations are defined by the deviation of TM helices from the predominantly observed linear α-helical conformation, to form 3 10 - and π-helices, as well as adopting curved and kinked geometries. The data presented here supplements the article 'Helix perturbations in Membrane Proteins Assist in Inter-helical Interactions and Optimal Helix Positioning in the Bilayer' (A. Shelar, M. Bansal, 2016) [1]. This data provides strong evidence for the role of various helix perturbations in influencing backbone torsion angles of helices, mediating inter-helical interactions, oligomer formation and accommodation of hydrophobic residues within the bilayer. The methodology used for creation of various datasets of membrane protein families (Sodium/Calcium exchanger and Heme Copper Oxidase) has also been mentioned.

Deriving high-resolution protein backbone structure propensities from all crystal data using the information maximization device.

PubMed

Solis, Armando D

2014-01-01

The most informative probability distribution functions (PDFs) describing the Ramachandran phi-psi dihedral angle pair, a fundamental descriptor of backbone conformation of protein molecules, are derived from high-resolution X-ray crystal structures using an information-theoretic approach. The Information Maximization Device (IMD) is established, based on fundamental information-theoretic concepts, and then applied specifically to derive highly resolved phi-psi maps for all 20 single amino acid and all 8000 triplet sequences at an optimal resolution determined by the volume of current data. The paper shows that utilizing the latent information contained in all viable high-resolution crystal structures found in the Protein Data Bank (PDB), totaling more than 77,000 chains, permits the derivation of a large number of optimized sequence-dependent PDFs. This work demonstrates the effectiveness of the IMD and the superiority of the resulting PDFs by extensive fold recognition experiments and rigorous comparisons with previously published triplet PDFs. Because it automatically optimizes PDFs, IMD results in improved performance of knowledge-based potentials, which rely on such PDFs. Furthermore, it provides an easy computational recipe for empirically deriving other kinds of sequence-dependent structural PDFs with greater detail and precision. The high-resolution phi-psi maps derived in this work are available for download.

Cyclic coordinate descent: A robotics algorithm for protein loop closure.

PubMed

Canutescu, Adrian A; Dunbrack, Roland L

2003-05-01

In protein structure prediction, it is often the case that a protein segment must be adjusted to connect two fixed segments. This occurs during loop structure prediction in homology modeling as well as in ab initio structure prediction. Several algorithms for this purpose are based on the inverse Jacobian of the distance constraints with respect to dihedral angle degrees of freedom. These algorithms are sometimes unstable and fail to converge. We present an algorithm developed originally for inverse kinematics applications in robotics. In robotics, an end effector in the form of a robot hand must reach for an object in space by altering adjustable joint angles and arm lengths. In loop prediction, dihedral angles must be adjusted to move the C-terminal residue of a segment to superimpose on a fixed anchor residue in the protein structure. The algorithm, referred to as cyclic coordinate descent or CCD, involves adjusting one dihedral angle at a time to minimize the sum of the squared distances between three backbone atoms of the moving C-terminal anchor and the corresponding atoms in the fixed C-terminal anchor. The result is an equation in one variable for the proposed change in each dihedral. The algorithm proceeds iteratively through all of the adjustable dihedral angles from the N-terminal to the C-terminal end of the loop. CCD is suitable as a component of loop prediction methods that generate large numbers of trial structures. It succeeds in closing loops in a large test set 99.79% of the time, and fails occasionally only for short, highly extended loops. It is very fast, closing loops of length 8 in 0.037 sec on average.

Ensemble models of proteins and protein domains based on distance distribution restraints.

PubMed

Jeschke, Gunnar

2016-04-01

Conformational ensembles of intrinsically disordered peptide chains are not fully determined by experimental observations. Uncertainty due to lack of experimental restraints and due to intrinsic disorder can be distinguished if distance distributions restraints are available. Such restraints can be obtained from pulsed dipolar electron paramagnetic resonance (EPR) spectroscopy applied to pairs of spin labels. Here, we introduce a Monte Carlo approach for generating conformational ensembles that are consistent with a set of distance distribution restraints, backbone dihedral angle statistics in known protein structures, and optionally, secondary structure propensities or membrane immersion depths. The approach is tested with simulated restraints for a terminal and an internal loop and for a protein with 69 residues by using sets of sparse restraints for underlying well-defined conformations and for published ensembles of a premolten globule-like and a coil-like intrinsically disordered protein. © 2016 Wiley Periodicals, Inc.

Physics-based method to validate and repair flaws in protein structures

PubMed Central

Martin, Osvaldo A.; Arnautova, Yelena A.; Icazatti, Alejandro A.; Scheraga, Harold A.; Vila, Jorge A.

2013-01-01

A method that makes use of information provided by the combination of 13Cα and 13Cβ chemical shifts, computed at the density functional level of theory, enables one to (i) validate, at the residue level, conformations of proteins and detect backbone or side-chain flaws by taking into account an ensemble average of chemical shifts over all of the conformations used to represent a protein, with a sensitivity of ∼90%; and (ii) provide a set of (χ1/χ2) torsional angles that leads to optimal agreement between the observed and computed 13Cα and 13Cβ chemical shifts. The method has been incorporated into the CheShift-2 protein validation Web server. To test the reliability of the provided set of (χ1/χ2) torsional angles, the side chains of all reported conformations of five NMR-determined protein models were refined by a simple routine, without using NOE-based distance restraints. The refinement of each of these five proteins leads to optimal agreement between the observed and computed 13Cα and 13Cβ chemical shifts for ∼94% of the flaws, on average, without introducing a significantly large number of violations of the NOE-based distance restraints for a distance range ≤ 0.5 Ǻ, in which the largest number of distance violations occurs. The results of this work suggest that use of the provided set of (χ1/χ2) torsional angles together with other observables, such as NOEs, should lead to a fast and accurate refinement of the side-chain conformations of protein models. PMID:24082119

Physics-based method to validate and repair flaws in protein structures.

PubMed

Martin, Osvaldo A; Arnautova, Yelena A; Icazatti, Alejandro A; Scheraga, Harold A; Vila, Jorge A

2013-10-15

A method that makes use of information provided by the combination of (13)C(α) and (13)C(β) chemical shifts, computed at the density functional level of theory, enables one to (i) validate, at the residue level, conformations of proteins and detect backbone or side-chain flaws by taking into account an ensemble average of chemical shifts over all of the conformations used to represent a protein, with a sensitivity of ∼90%; and (ii) provide a set of (χ1/χ2) torsional angles that leads to optimal agreement between the observed and computed (13)C(α) and (13)C(β) chemical shifts. The method has been incorporated into the CheShift-2 protein validation Web server. To test the reliability of the provided set of (χ1/χ2) torsional angles, the side chains of all reported conformations of five NMR-determined protein models were refined by a simple routine, without using NOE-based distance restraints. The refinement of each of these five proteins leads to optimal agreement between the observed and computed (13)C(α) and (13)C(β) chemical shifts for ∼94% of the flaws, on average, without introducing a significantly large number of violations of the NOE-based distance restraints for a distance range ≤ 0.5 , in which the largest number of distance violations occurs. The results of this work suggest that use of the provided set of (χ1/χ2) torsional angles together with other observables, such as NOEs, should lead to a fast and accurate refinement of the side-chain conformations of protein models.

How accurately do force fields represent protein side chain ensembles?

PubMed

Petrović, Dušan; Wang, Xue; Strodel, Birgit

2018-05-23

Although the protein backbone is the most fundamental part of the structure, the fine-tuning of side-chain conformations is important for protein function, for example, in protein-protein and protein-ligand interactions, and also in enzyme catalysis. While several benchmarks testing the performance of protein force fields for side chain properties have already been published, they often considered only a few force fields and were not tested against the same experimental observables; hence, they are not directly comparable. In this work, we explore the ability of twelve force fields, which are different flavors of AMBER, CHARMM, OPLS, or GROMOS, to reproduce average rotamer angles and rotamer populations obtained from extensive NMR studies of the 3 J and residual dipolar coupling constants for two small proteins: ubiquitin and GB3. Based on a total of 196 μs sampling time, our results reveal that all force fields identify the correct side chain angles, while the AMBER and CHARMM force fields clearly outperform the OPLS and GROMOS force fields in estimating rotamer populations. The three best force fields for representing the protein side chain dynamics are AMBER 14SB, AMBER 99SB*-ILDN, and CHARMM36. Furthermore, we observe that the side chain ensembles of buried amino acid residues are generally more accurately represented than those of the surface exposed residues. This article is protected by copyright. All rights reserved. © 2018 Wiley Periodicals, Inc.

Underestimated Halogen Bonds Forming with Protein Backbone in Protein Data Bank.

PubMed

Zhang, Qian; Xu, Zhijian; Shi, Jiye; Zhu, Weiliang

2017-07-24

Halogen bonds (XBs) are attracting increasing attention in biological systems. Protein Data Bank (PDB) archives experimentally determined XBs in biological macromolecules. However, no software for structure refinement in X-ray crystallography takes into account XBs, which might result in the weakening or even vanishing of experimentally determined XBs in PDB. In our previous study, we showed that side-chain XBs forming with protein side chains are underestimated in PDB on the basis of the phenomenon that the proportion of side-chain XBs to overall XBs decreases as structural resolution becomes lower and lower. However, whether the dominant backbone XBs forming with protein backbone are overlooked is still a mystery. Here, with the help of the ratio (R F ) of the observed XBs' frequency of occurrence to their frequency expected at random, we demonstrated that backbone XBs are largely overlooked in PDB, too. Furthermore, three cases were discovered possessing backbone XBs in high resolution structures while losing the XBs in low resolution structures. In the last two cases, even at 1.80 Å resolution, the backbone XBs were lost, manifesting the urgent need to consider XBs in the refinement process during X-ray crystallography study.

The effects of high concentrations of ionic liquid on GB1 protein structure and dynamics probed by high-resolution magic-angle-spinning NMR spectroscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Warner, Lisa; Gjersing, Erica; Follett, Shelby E.

Ionic liquids have great potential in biological applications and biocatalysis, as some ionic liquids can stabilize proteins and enhance enzyme activity, while others have the opposite effect. However, on the molecular level, probing ionic liquid interactions with proteins, especially in solutions containing high concentrations of ionic liquids, has been challenging. In the present work the 13C, 15N-enriched GB1 model protein was used to demonstrate applicability of high-resolution magic-angle-spinning (HR-MAS) NMR spectroscopy to investigate ionic liquid-protein interactions. Effect of an ionic liquid (1-butyl-3-methylimidazolium bromide, [C 4-mim]Br) on GB1was studied over a wide range of the ionic liquid concentrations (0.6-3.5 M, whichmore » corresponds to 10-60% v/v). Interactions between GB1 and [C 4-mim]Br were observed from changes in the chemical shifts of the protein backbone as well as the changes in 15N ps-ns dynamics and rotational correlation times. Site-specific interactions between the protein and [C 4-mim]Br were assigned using 3D methods under HR-MAS conditions. Furthermore, HR-MAS NMR is a viable tool that could aid in elucidation of molecular mechanisms of ionic liquid-protein interactions.« less

The effects of high concentrations of ionic liquid on GB1 protein structure and dynamics probed by high-resolution magic-angle-spinning NMR spectroscopy

DOE PAGES

Warner, Lisa; Gjersing, Erica; Follett, Shelby E.; ...

2016-08-11

Ionic liquids have great potential in biological applications and biocatalysis, as some ionic liquids can stabilize proteins and enhance enzyme activity, while others have the opposite effect. However, on the molecular level, probing ionic liquid interactions with proteins, especially in solutions containing high concentrations of ionic liquids, has been challenging. In the present work the 13C, 15N-enriched GB1 model protein was used to demonstrate applicability of high-resolution magic-angle-spinning (HR-MAS) NMR spectroscopy to investigate ionic liquid-protein interactions. Effect of an ionic liquid (1-butyl-3-methylimidazolium bromide, [C 4-mim]Br) on GB1was studied over a wide range of the ionic liquid concentrations (0.6-3.5 M, whichmore » corresponds to 10-60% v/v). Interactions between GB1 and [C 4-mim]Br were observed from changes in the chemical shifts of the protein backbone as well as the changes in 15N ps-ns dynamics and rotational correlation times. Site-specific interactions between the protein and [C 4-mim]Br were assigned using 3D methods under HR-MAS conditions. Furthermore, HR-MAS NMR is a viable tool that could aid in elucidation of molecular mechanisms of ionic liquid-protein interactions.« less

Multiple loop conformations of peptides predicted by molecular dynamics simulations are compatible with nuclear magnetic resonance.

PubMed

Carstens, Heiko; Renner, Christian; Milbradt, Alexander G; Moroder, Luis; Tavan, Paul

2005-03-29

The affinity and selectivity of protein-protein interactions can be fine-tuned by varying the size, flexibility, and amino acid composition of involved surface loops. As a model for such surface loops, we study the conformational landscape of an octapeptide, whose flexibility is chemically steered by a covalent ring closure integrating an azobenzene dye into and by a disulfide bridge additionally constraining the peptide backbone. Because the covalently integrated azobenzene dyes can be switched by light between a bent cis state and an elongated trans state, six cyclic peptide models of strongly different flexibilities are obtained. The conformational states of these peptide models are sampled by NMR and by unconstrained molecular dynamics (MD) simulations. Prototypical conformations and the free-energy landscapes in the high-dimensional space spanned by the phi/psi angles at the peptide backbone are obtained by clustering techniques from the MD trajectories. Multiple open-loop conformations are shown to be predicted by MD particularly in the very flexible cases and are shown to comply with the NMR data despite the fact that such open-loop conformations are missing in the refined NMR structures.

A Discontinuous Potential Model for Protein-Protein Interactions.

PubMed

Shao, Qing; Hall, Carol K

2016-01-01

Protein-protein interactions play an important role in many biologic and industrial processes. In this work, we develop a two-bead-per-residue model that enables us to account for protein-protein interactions in a multi-protein system using discontinuous molecular dynamics simulations. This model deploys discontinuous potentials to describe the non-bonded interactions and virtual bonds to keep proteins in their native state. The geometric and energetic parameters are derived from the potentials of mean force between sidechain-sidechain, sidechain-backbone, and backbone-backbone pairs. The energetic parameters are scaled with the aim of matching the second virial coefficient of lysozyme reported in experiment. We also investigate the performance of several bond-building strategies.

Protein backbone engineering as a strategy to advance foldamers toward the frontier of protein-like tertiary structure.

PubMed

Reinert, Zachary E; Horne, W Seth

2014-11-28

A variety of non-biological structural motifs have been incorporated into the backbone of natural protein sequences. In parallel work, diverse unnatural oligomers of de novo design (termed "foldamers") have been developed that fold in defined ways. In this Perspective article, we survey foundational studies on protein backbone engineering, with a focus on alterations made in the context of complex tertiary folds. We go on to summarize recent work illustrating the potential promise of these methods to provide a general framework for the construction of foldamer mimics of protein tertiary structures.

A discrete search algorithm for finding the structure of protein backbones and side chains.

PubMed

Sallaume, Silas; Martins, Simone de Lima; Ochi, Luiz Satoru; Da Silva, Warley Gramacho; Lavor, Carlile; Liberti, Leo

2013-01-01

Some information about protein structure can be obtained by using Nuclear Magnetic Resonance (NMR) techniques, but they provide only a sparse set of distances between atoms in a protein. The Molecular Distance Geometry Problem (MDGP) consists in determining the three-dimensional structure of a molecule using a set of known distances between some atoms. Recently, a Branch and Prune (BP) algorithm was proposed to calculate the backbone of a protein, based on a discrete formulation for the MDGP. We present an extension of the BP algorithm that can calculate not only the protein backbone, but the whole three-dimensional structure of proteins.

Prelude and Fugue, predicting local protein structure, early folding regions and structural weaknesses.

PubMed

Kwasigroch, Jean Marc; Rooman, Marianne

2006-07-15

Prelude&Fugue are bioinformatics tools aiming at predicting the local 3D structure of a protein from its amino acid sequence in terms of seven backbone torsion angle domains, using database-derived potentials. Prelude(&Fugue) computes all lowest free energy conformations of a protein or protein region, ranked by increasing energy, and possibly satisfying some interresidue distance constraints specified by the user. (Prelude&)Fugue detects sequence regions whose predicted structure is significantly preferred relative to other conformations in the absence of tertiary interactions. These programs can be used for predicting secondary structure, tertiary structure of short peptides, flickering early folding sequences and peptides that adopt a preferred conformation in solution. They can also be used for detecting structural weaknesses, i.e. sequence regions that are not optimal with respect to the tertiary fold. http://babylone.ulb.ac.be/Prelude_and_Fugue.

Normal Mode Analysis of Polytheonamide B

NASA Astrophysics Data System (ADS)

Mori, Takaharu; Kokubo, Hironori; Shimizu, Hirofumi; Iwamoto, Masayuki; Oiki, Shigetoshi; Okamoto, Yuko

2007-09-01

Polytheonamide B is a linear 48-residue peptide which forms a single β-helix structure with alternating d- and l-amino acids and contains methylated and hydroxy variants of proteinogenic amino acids. To investigate the dynamical properties of polytheonamide B we perform the normal mode analysis. Root-mean-square displacements of all backbone atoms, root-mean-square fluctuations of the backbone dihedral angles (φ,\\psi), and correlation factors for the Cα atom fluctuations and for the dihedral angle fluctuations are calculated. The normal mode analysis reveals that polytheonamide B shows the elastic rod behavior in the very low-frequency regions and that librational motions of backbone amide planes have the modes with relatively low frequencies, which is relevant to the function of polytheonamide B. In addition, these librational motions occur almost independently and weakly anticorrelate with those of the hydrogen-bonded neighboring amide planes. Calculations of the backbone fluctuations show that the flexibility of polytheonamide B is roughly uniform over the entire helix. We compare our results with those of gramicidin A, the analogue of polytheonamide B, to discuss the structures and functions, and obtain some common features in the flexibilities and dynamics of the backbone atoms. These results present important clues for clarifying the function of polytheonamide B at the atomic level.

Modeling helical proteins using residual dipolar couplings, sparse long-range distance constraints and a simple residue-based force field

PubMed Central

Eggimann, Becky L.; Vostrikov, Vitaly V.; Veglia, Gianluigi; Siepmann, J. Ilja

2013-01-01

We present a fast and simple protocol to obtain moderate-resolution backbone structures of helical proteins. This approach utilizes a combination of sparse backbone NMR data (residual dipolar couplings and paramagnetic relaxation enhancements) or EPR data with a residue-based force field and Monte Carlo/simulated annealing protocol to explore the folding energy landscape of helical proteins. By using only backbone NMR data, which are relatively easy to collect and analyze, and strategically placed spin relaxation probes, we show that it is possible to obtain protein structures with correct helical topology and backbone RMS deviations well below 4 Å. This approach offers promising alternatives for the structural determination of proteins in which nuclear Overha-user effect data are difficult or impossible to assign and produces initial models that will speed up the high-resolution structure determination by NMR spectroscopy. PMID:24639619

Experimental Protein Structure Verification by Scoring with a Single, Unassigned NMR Spectrum.

PubMed

Courtney, Joseph M; Ye, Qing; Nesbitt, Anna E; Tang, Ming; Tuttle, Marcus D; Watt, Eric D; Nuzzio, Kristin M; Sperling, Lindsay J; Comellas, Gemma; Peterson, Joseph R; Morrissey, James H; Rienstra, Chad M

2015-10-06

Standard methods for de novo protein structure determination by nuclear magnetic resonance (NMR) require time-consuming data collection and interpretation efforts. Here we present a qualitatively distinct and novel approach, called Comparative, Objective Measurement of Protein Architectures by Scoring Shifts (COMPASS), which identifies the best structures from a set of structural models by numerical comparison with a single, unassigned 2D (13)C-(13)C NMR spectrum containing backbone and side-chain aliphatic signals. COMPASS does not require resonance assignments. It is particularly well suited for interpretation of magic-angle spinning solid-state NMR spectra, but also applicable to solution NMR spectra. We demonstrate COMPASS with experimental data from four proteins--GB1, ubiquitin, DsbA, and the extracellular domain of human tissue factor--and with reconstructed spectra from 11 additional proteins. For all these proteins, with molecular mass up to 25 kDa, COMPASS distinguished the correct fold, most often within 1.5 Å root-mean-square deviation of the reference structure. Copyright © 2015 Elsevier Ltd. All rights reserved.

Rational Design of Orthogonal Multipolar Interactions with Fluorine in Protein–Ligand Complexes

DOE PAGES

Pollock, Jonathan; Borkin, Dmitry; Lund, George; ...

2015-08-19

Multipolar interactions involving fluorine and the protein backbone have been frequently observed in protein–ligand complexes. Such fluorine–backbone interactions may substantially contribute to the high affinity of small molecule inhibitors. Here we found that introduction of trifluoromethyl groups into two different sites in the thienopyrimidine class of menin–MLL inhibitors considerably improved their inhibitory activity. In both cases, trifluoromethyl groups are engaged in short interactions with the backbone of menin. In order to understand the effect of fluorine, we synthesized a series of analogues by systematically changing the number of fluorine atoms, and we determined high-resolution crystal structures of the complexes withmore » menin. Here, we found that introduction of fluorine at favorable geometry for interactions with backbone carbonyls may improve the activity of menin–MLL inhibitors as much as 5- to 10-fold. In order to facilitate the design of multipolar fluorine–backbone interactions in protein–ligand complexes, we developed a computational algorithm named FMAP, which calculates fluorophilic sites in proximity to the protein backbone. We demonstrated that FMAP could be used to rationalize improvement in the activity of known protein inhibitors upon introduction of fluorine. Furthermore, FMAP may also represent a valuable tool for designing new fluorine substitutions and support ligand optimization in drug discovery projects. Analysis of the menin–MLL inhibitor complexes revealed that the backbone in secondary structures is particularly accessible to the interactions with fluorine. Lastly, considering that secondary structure elements are frequently exposed at protein interfaces, we postulate that multipolar fluorine–backbone interactions may represent a particularly attractive approach to improve inhibitors of protein–protein interactions.« less

The Ramachandran Number: An Order Parameter for Protein Geometry

DOE PAGES

Mannige, Ranjan V.; Kundu, Joyjit; Whitelam, Stephen; ...

2016-08-04

Three-dimensional protein structures usually contain regions of local order, called secondary structure, such as α-helices and β-sheets. Secondary structure is characterized by the local rotational state of the protein backbone, quantified by two dihedral angles called Øand Ψ. Particular types of secondary structure can generally be described by a single (diffuse) location on a two-dimensional plot drawn in the space of the angles Ø andΨ, called a Ramachandran plot. By contrast, a recently-discovered nanomaterial made from peptoids, structural isomers of peptides, displays a secondary-structure motif corresponding to two regions on the Ramachandran plot [Mannige et al., Nature 526, 415 (2015)].more » In order to describe such 'higher-order' secondary structure in a compact way we introduce here a means of describing regions on the Ramachandran plot in terms of a single Ramachandran number, R, which is a structurally meaningful combination of Ø andΨ. We show that the potential applications of R are numerous: it can be used to describe the geometric content of protein structures, and can be used to draw diagrams that reveal, at a glance, the frequency of occurrence of regular secondary structures and disordered regions in large protein datasets. We propose that R might be used as an order parameter for protein geometry for a wide range of applications.« less

Reconstructing the free-energy landscape of Met-enkephalin using dihedral principal component analysis and well-tempered metadynamics

NASA Astrophysics Data System (ADS)

Sicard, François; Senet, Patrick

2013-06-01

Well-Tempered Metadynamics (WTmetaD) is an efficient method to enhance the reconstruction of the free-energy surface of proteins. WTmetaD guarantees a faster convergence in the long time limit in comparison with the standard metadynamics. It still suffers, however, from the same limitation, i.e., the non-trivial choice of pertinent collective variables (CVs). To circumvent this problem, we couple WTmetaD with a set of CVs generated from a dihedral Principal Component Analysis (dPCA) on the Ramachandran dihedral angles describing the backbone structure of the protein. The dPCA provides a generic method to extract relevant CVs built from internal coordinates, and does not depend on the alignment to an arbitrarily chosen reference structure as usual in Cartesian PCA. We illustrate the robustness of this method in the case of a reference model protein, the small and very diffusive Met-enkephalin pentapeptide. We propose a justification a posteriori of the considered number of CVs necessary to bias the metadynamics simulation in terms of the one-dimensional free-energy profiles associated with Ramachandran dihedral angles along the amino-acid sequence.

Reconstructing the free-energy landscape of Met-enkephalin using dihedral principal component analysis and well-tempered metadynamics.

PubMed

Sicard, François; Senet, Patrick

2013-06-21

Well-Tempered Metadynamics (WTmetaD) is an efficient method to enhance the reconstruction of the free-energy surface of proteins. WTmetaD guarantees a faster convergence in the long time limit in comparison with the standard metadynamics. It still suffers, however, from the same limitation, i.e., the non-trivial choice of pertinent collective variables (CVs). To circumvent this problem, we couple WTmetaD with a set of CVs generated from a dihedral Principal Component Analysis (dPCA) on the Ramachandran dihedral angles describing the backbone structure of the protein. The dPCA provides a generic method to extract relevant CVs built from internal coordinates, and does not depend on the alignment to an arbitrarily chosen reference structure as usual in Cartesian PCA. We illustrate the robustness of this method in the case of a reference model protein, the small and very diffusive Met-enkephalin pentapeptide. We propose a justification a posteriori of the considered number of CVs necessary to bias the metadynamics simulation in terms of the one-dimensional free-energy profiles associated with Ramachandran dihedral angles along the amino-acid sequence.

Smectic order and backbone anisotropy of a side-chain liquid crystalline polymer by Small-Angle Neutron Scattering

NASA Astrophysics Data System (ADS)

Noirez, L.; Pépy, G.; Keller, P.; Benguigui, L.

1991-07-01

We have simultaneously measured, for the first time, the extension of the polymer backbone of a side-chain liquid crystalline polymer and the intensity of the 001 Bragg reflection, which gives the smectic order parameter Psi as a function of temperature in the smectic phase. We have qualitatively demonstrated that the more the smectic phase is ordered, the more the polymer backbone is localized between the mesogenic layers. It is shown that the Landau theory allows us to relate the radius of gyration parallel to the magnetic field of the polymer backbone to the smectic order parameter. We also show that the Renz-Warner theory is suitable at low temperatures.

Engineering botulinum neurotoxin domains for activation by toxin light chain.

PubMed

Stancombe, Patrick R; Masuyer, Geoffrey; Birch-Machin, Ian; Beard, Matthew; Foster, Keith A; Chaddock, John A; Acharya, K Ravi

2012-02-01

Targeted secretion inhibitors (TSI) are a new class of biopharmaceuticals designed from a botulinum neurotoxin protein scaffold. The backbone consists of the 50-kDa endopeptidase light chain and translocation domain (N-terminal portion of the heavy chain), lacks neuronal toxicity, but retains the ability to target cytoplasmic soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. TSI are produced as single-chain proteins and then cleaved post-translationally to generate functional heterodimers. Precise proteolytic cleavage is essential to activate the protein to a dichain form. TSI are themselves highly specific proteases. We have exploited this activity to create self-activating enzymes by replacing the native proteolytic site with a substrate SNARE peptide for the TSI protease. We have also created cross-activating backbones. By replacing the proteolytic activation site in one backbone with the substrate SNARE peptide for another serotype, controlled activation is achieved. SNARE peptides encompassing the whole of the coiled-coil region enabled complete activation and assembly of the dichain backbone. These engineered TSI backbones are capable of translocating their enzymatic domains to target intracellular SNARE proteins. They are also investigative tools with which to further the understanding of endopeptidase activity of light chain in SNARE interactions. © 2011 Syntaxin Ltd. Journal compilation © 2011 FEBS.

Representing and comparing protein structures as paths in three-dimensional space

PubMed Central

Zhi, Degui; Krishna, S Sri; Cao, Haibo; Pevzner, Pavel; Godzik, Adam

2006-01-01

Background Most existing formulations of protein structure comparison are based on detailed atomic level descriptions of protein structures and bypass potential insights that arise from a higher-level abstraction. Results We propose a structure comparison approach based on a simplified representation of proteins that describes its three-dimensional path by local curvature along the generalized backbone of the polypeptide. We have implemented a dynamic programming procedure that aligns curvatures of proteins by optimizing a defined sum turning angle deviation measure. Conclusion Although our procedure does not directly optimize global structural similarity as measured by RMSD, our benchmarking results indicate that it can surprisingly well recover the structural similarity defined by structure classification databases and traditional structure alignment programs. In addition, our program can recognize similarities between structures with extensive conformation changes that are beyond the ability of traditional structure alignment programs. We demonstrate the applications of procedure to several contexts of structure comparison. An implementation of our procedure, CURVE, is available as a public webserver. PMID:17052359

Experimental Protein Structure Verification by Scoring with a Single, Unassigned NMR Spectrum

PubMed Central

Courtney, Joseph M.; Ye, Qing; Nesbitt, Anna E.; Tang, Ming; Tuttle, Marcus D.; Watt, Eric D.; Nuzzio, Kristin M.; Sperling, Lindsay J.; Comellas, Gemma; Peterson, Joseph R.; Morrissey, James H.; Rienstra, Chad M.

2016-01-01

Standard methods for de novo protein structure determination by nuclear magnetic resonance (NMR) require time-consuming data collection and interpretation efforts. Here we present a qualitatively distinct and novel approach, called Comparative, Objective Measurement of Protein Architectures by Scoring Shifts (COMPASS), which identifies the best structures from a set of structural models by numerical comparison with a single, unassigned 2D 13C-13C NMR spectrum containing backbone and side-chain aliphatic signals. COMPASS does not require resonance assignments. It is particularly well suited for interpretation of magic-angle spinning solid-state NMR spectra, but also applicable to solution NMR spectra. We demonstrate COMPASS with experimental data from four proteins—GB1, ubiquitin, DsbA, and the extracellular domain of human tissue factor—and with reconstructed spectra from 11 additional proteins. For all these proteins, with molecular mass up to 25 kDa, COMPASS distinguished the correct fold, most often within 1.5 Å root-mean-square deviation of the reference structure. PMID:26365800

An energy function for dynamics simulations of polypeptides in torsion angle space

NASA Astrophysics Data System (ADS)

Sartori, F.; Melchers, B.; Böttcher, H.; Knapp, E. W.

1998-05-01

Conventional simulation techniques to model the dynamics of proteins in atomic detail are restricted to short time scales. A simplified molecular description, in which high frequency motions with small amplitudes are ignored, can overcome this problem. In this protein model only the backbone dihedrals φ and ψ and the χi of the side chains serve as degrees of freedom. Bond angles and lengths are fixed at ideal geometry values provided by the standard molecular dynamics (MD) energy function CHARMM. In this work a Monte Carlo (MC) algorithm is used, whose elementary moves employ cooperative rotations in a small window of consecutive amide planes, leaving the polypeptide conformation outside of this window invariant. A single of these window MC moves generates local conformational changes only. But, the application of many such moves at different parts of the polypeptide backbone leads to global conformational changes. To account for the lack of flexibility in the protein model employed, the energy function used to evaluate conformational energies is split into sequentially neighbored and sequentially distant contributions. The sequentially neighbored part is represented by an effective (φ,ψ)-torsion potential. It is derived from MD simulations of a flexible model dipeptide using a conventional MD energy function. To avoid exaggeration of hydrogen bonding strengths, the electrostatic interactions involving hydrogen atoms are scaled down at short distances. With these adjustments of the energy function, the rigid polypeptide model exhibits the same equilibrium distributions as obtained by conventional MD simulation with a fully flexible molecular model. Also, the same temperature dependence of the stability and build-up of α helices of 18-alanine as found in MD simulations is observed using the adapted energy function for MC simulations. Analyses of transition frequencies demonstrate that also dynamical aspects of MD trajectories are faithfully reproduced. Finally, it is demonstrated that even for high temperature unfolded polypeptides the MC simulation is more efficient by a factor of 10 than conventional MD simulations.

The introduction of strain and its effects on the structure and stability of T4 lysozyme.

PubMed

Liu, R; Baase, W A; Matthews, B W

2000-01-07

In order to try to better understand the role played by strain in the structure and stability of a protein a series of "small-to-large" mutations was made within the core of T4 lysozyme. Three different alanine residues, one involved in backbone contacts, one in side-chain contacts, and the third adjacent to a small cavity, were each replaced with subsets of the larger residues, Val, Leu, Ile, Met, Phe and Trp. As expected, the protein is progressively destabilized as the size of the introduced side-chain becomes larger. There does, however, seem to be a limit to the destabilization, suggesting that a protein of a given size may be capable of maintaining only a certain amount of strain. The changes in stability vary greatly from site to site. Substitution of larger residues for both Ala42 and Ala98 substantially destabilize the protein, even though the primary contacts in one case are predominantly with side-chain atoms and in the other with backbone. The results suggest that it is neither practical nor meaningful to try to separate the effects of introduced strain on side-chains from the effects on the backbone. Substitutions at Ala129 are much less destabilizing than at sites 42 or 98. This is most easily understood in terms of the pre-existing cavity, which provides partial space to accommodate the introduced side-chains. Crystal structures were obtained for a number of the mutants. These show that the changes in structure to accommodate the introduced side-chains usually consist of essentially rigid-body displacements of groups of linked atoms, achieved through relatively small changes in torsion angles. On rare occasions, a side-chain close to the site of substitution may change to a different rotamer. When such rotomer changes occur, they permit the structure to dissipate strain by a response that is plastic rather than elastic. In one case, a surface loop moves 1.2 A, not in direct response to a mutation, but in an interaction mediated via an intermolecular contact. It illustrates how the structure of a protein can be modified by crystal contacts. Copyright 2000 Academic Press.

Dihedral angle principal component analysis of molecular dynamics simulations.

PubMed

Altis, Alexandros; Nguyen, Phuong H; Hegger, Rainer; Stock, Gerhard

2007-06-28

It has recently been suggested by Mu et al. [Proteins 58, 45 (2005)] to use backbone dihedral angles instead of Cartesian coordinates in a principal component analysis of molecular dynamics simulations. Dihedral angles may be advantageous because internal coordinates naturally provide a correct separation of internal and overall motion, which was found to be essential for the construction and interpretation of the free energy landscape of a biomolecule undergoing large structural rearrangements. To account for the circular statistics of angular variables, a transformation from the space of dihedral angles {phi(n)} to the metric coordinate space {x(n)=cos phi(n),y(n)=sin phi(n)} was employed. To study the validity and the applicability of the approach, in this work the theoretical foundations underlying the dihedral angle principal component analysis (dPCA) are discussed. It is shown that the dPCA amounts to a one-to-one representation of the original angle distribution and that its principal components can readily be characterized by the corresponding conformational changes of the peptide. Furthermore, a complex version of the dPCA is introduced, in which N angular variables naturally lead to N eigenvalues and eigenvectors. Applying the methodology to the construction of the free energy landscape of decaalanine from a 300 ns molecular dynamics simulation, a critical comparison of the various methods is given.

Dihedral angle principal component analysis of molecular dynamics simulations

NASA Astrophysics Data System (ADS)

Altis, Alexandros; Nguyen, Phuong H.; Hegger, Rainer; Stock, Gerhard

2007-06-01

It has recently been suggested by Mu et al. [Proteins 58, 45 (2005)] to use backbone dihedral angles instead of Cartesian coordinates in a principal component analysis of molecular dynamics simulations. Dihedral angles may be advantageous because internal coordinates naturally provide a correct separation of internal and overall motion, which was found to be essential for the construction and interpretation of the free energy landscape of a biomolecule undergoing large structural rearrangements. To account for the circular statistics of angular variables, a transformation from the space of dihedral angles {φn} to the metric coordinate space {xn=cosφn,yn=sinφn} was employed. To study the validity and the applicability of the approach, in this work the theoretical foundations underlying the dihedral angle principal component analysis (dPCA) are discussed. It is shown that the dPCA amounts to a one-to-one representation of the original angle distribution and that its principal components can readily be characterized by the corresponding conformational changes of the peptide. Furthermore, a complex version of the dPCA is introduced, in which N angular variables naturally lead to N eigenvalues and eigenvectors. Applying the methodology to the construction of the free energy landscape of decaalanine from a 300ns molecular dynamics simulation, a critical comparison of the various methods is given.

Slow dynamics of a protein backbone in molecular dynamics simulation revealed by time-structure based independent component analysis

NASA Astrophysics Data System (ADS)

Naritomi, Yusuke; Fuchigami, Sotaro

2013-12-01

We recently proposed the method of time-structure based independent component analysis (tICA) to examine the slow dynamics involved in conformational fluctuations of a protein as estimated by molecular dynamics (MD) simulation [Y. Naritomi and S. Fuchigami, J. Chem. Phys. 134, 065101 (2011)]. Our previous study focused on domain motions of the protein and examined its dynamics by using rigid-body domain analysis and tICA. However, the protein changes its conformation not only through domain motions but also by various types of motions involving its backbone and side chains. Some of these motions might occur on a slow time scale: we hypothesize that if so, we could effectively detect and characterize them using tICA. In the present study, we investigated slow dynamics of the protein backbone using MD simulation and tICA. The selected target protein was lysine-, arginine-, ornithine-binding protein (LAO), which comprises two domains and undergoes large domain motions. MD simulation of LAO in explicit water was performed for 1 μs, and the obtained trajectory of Cα atoms in the backbone was analyzed by tICA. This analysis successfully provided us with slow modes for LAO that represented either domain motions or local movements of the backbone. Further analysis elucidated the atomic details of the suggested local motions and confirmed that these motions truly occurred on the expected slow time scale.

Small Angle Neutron Scattering experiments on ``side-on fixed"" liquid crystal polyacrylates

NASA Astrophysics Data System (ADS)

Leroux, N.; Keller, P.; Achard, M. F.; Noirez, L.; Hardouin, F.

1993-08-01

Small Angle Neutron Scattering experiments were carried out on liquid crystalline “side-on fixed” polyacrylates : we observe that the polymer backbone adopts a prolate conformation in the nematic phase. Such anisotropy of the global backbone is larger for smaller spacer length. In every case we measure at low temperatures a large chain extension as previously described in polysiloxanes. Par diffusion des neutrons aux petits angles nous observons que la chaîne de polyacrylates “en haltère” adopte une conformation type prolate en phase nématique. Son anisotropie est d'autant plus grande que l'espaceur est plus court. Dans tous les cas, nous retrouvons à basse température la forte extension de la chaîne polymère qui fut d'abord révélée dans les polysiloxanes.

Solution NMR and molecular dynamics reveal a persistent alpha helix within the dynamic region of PsbQ from photosystem II of higher plants.

PubMed

Rathner, Petr; Rathner, Adriana; Horničáková, Michaela; Wohlschlager, Christian; Chandra, Kousik; Kohoutová, Jaroslava; Ettrich, Rüdiger; Wimmer, Reinhard; Müller, Norbert

2015-09-01

The extrinsic proteins of photosystem II of higher plants and green algae PsbO, PsbP, PsbQ, and PsbR are essential for stable oxygen production in the oxygen evolving center. In the available X-ray crystallographic structure of higher plant PsbQ residues S14-Y33 are missing. Building on the backbone NMR assignment of PsbQ, which includes this "missing link", we report the extended resonance assignment including side chain atoms. Based on nuclear Overhauser effect spectra a high resolution solution structure of PsbQ with a backbone RMSD of 0.81 Å was obtained from torsion angle dynamics. Within the N-terminal residues 1-45 the solution structure deviates significantly from the X-ray crystallographic one, while the four-helix bundle core found previously is confirmed. A short α-helix is observed in the solution structure at the location where a β-strand had been proposed in the earlier crystallographic study. NMR relaxation data and unrestrained molecular dynamics simulations corroborate that the N-terminal region behaves as a flexible tail with a persistent short local helical secondary structure, while no indications of forming a β-strand are found. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

Quantitative assessments of the distinct contributions of polypeptide backbone amides versus sidechain groups to chain expansion via chemical denaturation

PubMed Central

Holehouse, Alex S.; Garai, Kanchan; Lyle, Nicholas; Vitalis, Andreas; Pappu, Rohit V.

2015-01-01

In aqueous solutions with high concentrations of chemical denaturants such as urea and guanidinium chloride (GdmCl) proteins expand to populate heterogeneous conformational ensembles. These denaturing environments are thought to be good solvents for generic protein sequences because properties of conformational distributions align with those of canonical random coils. Previous studies showed that water is a poor solvent for polypeptide backbones and therefore backbones form collapsed globular structures in aqueous solvents. Here, we ask if polypeptide backbones can intrinsically undergo the requisite chain expansion in aqueous solutions with high concentrations of urea and GdmCl. We answer this question using a combination of molecular dynamics simulations and fluorescence correlation spectroscopy. We find that the degree of backbone expansion is minimal in aqueous solutions with high concentrations denaturants. Instead, polypeptide backbones sample conformations that are denaturant-specific mixtures of coils and globules, with a persistent preference for globules. Therefore, typical denaturing environments cannot be classified as good solvents for polypeptide backbones. How then do generic protein sequences expand in denaturing environments? To answer this question, we investigated the effects of sidechains using simulations of two archetypal sequences with amino acid compositions that are mixtures of charged, hydrophobic, and polar groups. We find that sidechains lower the effective concentration of backbone amides in water leading to an intrinsic expansion of polypeptide backbones in the absence of denaturants. Additional dilution of the effective concentration of backbone amides is achieved through preferential interactions with denaturants. These effects lead to conformational statistics in denaturing environments that are congruent with those of canonical random coils. Our results highlight the role of sidechain-mediated interactions as determinants of the conformational properties of unfolded states in water and in influencing chain expansion upon denaturation. PMID:25664638

Golden rule for buttressing vulnerable soluble proteins.

PubMed

Fernández, Ariel; Berry, R Stephen

2010-05-07

Local weaknesses in the structure of soluble proteins have received little attention. The structure may be inherently weak at sites where hydration of the protein backbone is locally hampered by formation of an intramolecular hydrogen bond which in turn is not fully stabilized through burial within a hydrophobic environment. The result is insufficient compensation for the thermodynamic cost of dehydrating the backbone polar groups. This work shows that these structural deficiencies, the unburied backbone hydrogen bonds, are compensated in natural proteins by disulfide bonds that are needed to maintain the structural integrity. Examination of all PDB-reported soluble structures reveals that, after suitable normalization, the number of disulfide bonds, X, correlates tightly with the number of unburied backbone hydrogen bonds, Y, beyond the baseline level Y = 20, revealing a simple balance relation: Y = 5X + 20. This equation introduces a 1:5 ratio associated with the buttressing of soluble proteins with structural deficiencies. The results are justified on thermodynamic grounds and have implications for biomolecular engineering as they introduce two constants of universal applicability determining the architecture of soluble proteins.

NMR and SAXS characterization of the denatured state of the chemotactic protein Che Y: Implications for protein folding initiation

PubMed Central

Garcia, Pascal; Serrano, Luis; Durand, Dominique; Rico, Manuel; Bruix, Marta

2001-01-01

The denatured state of a double mutant of the chemotactic protein CheY (F14N/V83T) has been analyzed in the presence of 5 M urea, using small angle X-ray scattering (SAXS) and heteronuclear magnetic resonance. SAXS studies show that the denatured protein follows a wormlike chain model. Its backbone can be described as a chain composed of rigid elements connected by flexible links. A comparison of the contour length obtained for the chain at 5 M urea with the one expected for a fully expanded chain suggests that ∼25% of the residues are involved in residual structures. Conformational shifts of the α-protons, heteronuclear 15N-{1H} NOEs and 15N relaxation properties have been used to identify some regions in the protein that deviate from a random coil behavior. According to these NMR data, the protein can be divided into two subdomains, which largely coincide with the two folding subunits identified in a previous kinetic study of the folding of the protein. The first of these subdomains, spanning residues 1–70, is shown here to exhibit a restricted mobility as compared to the rest of the protein. Two regions, one in each subdomain, were identified as deviating from the random coil chemical shifts. Peptides corresponding to these sequences were characterized by NMR and their backbone 1H chemical shifts were compared to those in the intact protein under identical denaturing conditions. For the region located in the first subdomain, this comparison shows that the observed deviation from random coil parameters is caused by interactions with the rest of the molecule. The restricted flexibility of the first subdomain and the transient collapse detected in that subunit are consistent with the conclusions obtained by applying the protein engineering method to the characterization of the folding reaction transition state. PMID:11369848

15N CSA tensors and 15N-1H dipolar couplings of protein hydrophobic core residues investigated by static solid-state NMR

NASA Astrophysics Data System (ADS)

Vugmeyster, Liliya; Ostrovsky, Dmitry; Fu, Riqiang

2015-10-01

In this work, we assess the usefulness of static 15N NMR techniques for the determination of the 15N chemical shift anisotropy (CSA) tensor parameters and 15N-1H dipolar splittings in powder protein samples. By using five single labeled samples of the villin headpiece subdomain protein in a hydrated lyophilized powder state, we determine the backbone 15N CSA tensors at two temperatures, 22 and -35 °C, in order to get a snapshot of the variability across the residues and as a function of temperature. All sites probed belonged to the hydrophobic core and most of them were part of α-helical regions. The values of the anisotropy (which include the effect of the dynamics) varied between 130 and 156 ppm at 22 °C, while the values of the asymmetry were in the 0.32-0.082 range. The Leu-75 and Leu-61 backbone sites exhibited high mobility based on the values of their temperature-dependent anisotropy parameters. Under the assumption that most differences stem from dynamics, we obtained the values of the motional order parameters for the 15N backbone sites. While a simple one-dimensional line shape experiment was used for the determination of the 15N CSA parameters, a more advanced approach based on the ;magic sandwich; SAMMY pulse sequence (Nevzorov and Opella, 2003) was employed for the determination of the 15N-1H dipolar patterns, which yielded estimates of the dipolar couplings. Accordingly, the motional order parameters for the dipolar interaction were obtained. It was found that the order parameters from the CSA and dipolar measurements are highly correlated, validating that the variability between the residues is governed by the differences in dynamics. The values of the parameters obtained in this work can serve as reference values for developing more advanced magic-angle spinning recoupling techniques for multiple labeled samples.

Modeling backbone flexibility to achieve sequence diversity: The design of novel alpha-helical ligands for Bcl-xL

PubMed Central

Fu, Xiaoran; Apgar, James R.; Keating, Amy E.

2007-01-01

Computational protein design can be used to select sequences that are compatible with a fixed-backbone template. This strategy has been used in numerous instances to engineer novel proteins. However, the fixed-backbone assumption severely restricts the sequence space that is accessible via design. For challenging problems, such as the design of functional proteins, this may not be acceptable. In this paper, we present a method for introducing backbone flexibility into protein design calculations and apply it to the design of diverse helical BH3 ligands that bind to the anti-apoptotic protein Bcl-xL, a member of the Bcl-2 protein family. We demonstrate how normal mode analysis can be used to sample different BH3 backbones, and show that this leads to a larger and more diverse set of low-energy solutions than can be achieved using a native high-resolution Bcl-xL complex crystal structure as a template. We tested several of the designed solutions experimentally and found that this approach worked well when normal mode calculations were used to deform a native BH3 helix structure, but less well when they were used to deform an idealized helix. A subsequent round of design and testing identified a likely source of the problem as inadequate sampling of the helix pitch. In all, we tested seventeen designed BH3 peptide sequences, including several point mutants. Of these, eight bound well to Bcl-xL and four others showed weak but detectable binding. The successful designs showed a diversity of sequences that would have been difficult or impossible to achieve using only a fixed backbone. Thus, introducing backbone flexibility via normal mode analysis effectively broadened the set of sequences identified by computational design, and provided insight into positions important for binding Bcl-xL. PMID:17597151

On the relationship between NMR-derived amide order parameters and protein backbone entropy changes

PubMed Central

Sharp, Kim A.; O’Brien, Evan; Kasinath, Vignesh; Wand, A. Joshua

2015-01-01

Molecular dynamics simulations are used to analyze the relationship between NMR-derived squared generalized order parameters of amide NH groups and backbone entropy. Amide order parameters (O2NH) are largely determined by the secondary structure and average values appear unrelated to the overall flexibility of the protein. However, analysis of the more flexible subset (O2NH < 0.8) shows that these report both on the local flexibility of the protein and on a different component of the conformational entropy than that reported by the side chain methyl axis order parameters, O2axis. A calibration curve for backbone entropy vs. O2NH is developed which accounts for both correlations between amide group motions of different residues, and correlations between backbone and side chain motions. This calibration curve can be used with experimental values of O2NH changes obtained by NMR relaxation measurements to extract backbone entropy changes, e.g. upon ligand binding. In conjunction with our previous calibration for side chain entropy derived from measured O2axis values this provides a prescription for determination of the total protein conformational entropy changes from NMR relaxation measurements. PMID:25739366

On the relationship between NMR-derived amide order parameters and protein backbone entropy changes.

PubMed

Sharp, Kim A; O'Brien, Evan; Kasinath, Vignesh; Wand, A Joshua

2015-05-01

Molecular dynamics simulations are used to analyze the relationship between NMR-derived squared generalized order parameters of amide NH groups and backbone entropy. Amide order parameters (O(2) NH ) are largely determined by the secondary structure and average values appear unrelated to the overall flexibility of the protein. However, analysis of the more flexible subset (O(2) NH  < 0.8) shows that these report both on the local flexibility of the protein and on a different component of the conformational entropy than that reported by the side chain methyl axis order parameters, O(2) axis . A calibration curve for backbone entropy vs. O(2) NH is developed, which accounts for both correlations between amide group motions of different residues, and correlations between backbone and side chain motions. This calibration curve can be used with experimental values of O(2) NH changes obtained by NMR relaxation measurements to extract backbone entropy changes, for example, upon ligand binding. In conjunction with our previous calibration for side chain entropy derived from measured O(2) axis values this provides a prescription for determination of the total protein conformational entropy changes from NMR relaxation measurements. © 2015 Wiley Periodicals, Inc.

Quantitative analysis of backbone dynamics in a crystalline protein from nitrogen-15 spin-lattice relaxation.

PubMed

Giraud, Nicolas; Blackledge, Martin; Goldman, Maurice; Böckmann, Anja; Lesage, Anne; Penin, François; Emsley, Lyndon

2005-12-28

A detailed analysis of nitrogen-15 longitudinal relaxation times in microcrystalline proteins is presented. A theoretical model to quantitatively interpret relaxation times is developed in terms of motional amplitude and characteristic time scale. Different averaging schemes are examined in order to propose an analysis of relaxation curves that takes into account the specificity of MAS experiments. In particular, it is shown that magic angle spinning averages the relaxation rate experienced by a single spin over one rotor period, resulting in individual relaxation curves that are dependent on the orientation of their corresponding carousel with respect to the rotor axis. Powder averaging thus leads to a nonexponential behavior in the observed decay curves. We extract dynamic information from experimental decay curves, using a diffusion in a cone model. We apply this study to the analysis of spin-lattice relaxation rates of the microcrystalline protein Crh at two different fields and determine differential dynamic parameters for several residues in the protein.

Experimental verification of force fields for molecular dynamics simulations using Gly-Pro-Gly-Gly.

PubMed

Aliev, Abil E; Courtier-Murias, Denis

2010-09-30

Experimental NMR verification of MD simulations using 12 different force fields (AMBER, CHARMM, GROMOS, and OPLS-AA) and 5 different water models has been undertaken to identify reliable MD protocols for structure and dynamics elucidations of small open chain peptides containing Gly and Pro. A conformationally flexible tetrapeptide Gly-Pro-Gly-Gly was selected for NMR (3)J-coupling, chemical shift, and internuclear distance measurements, followed by their calculations using 2 μs long MD simulations in water. In addition, Ramachandran population maps for Pro-2 and Gly-3 residues of GPGG obtained from MD simulations were used for detailed comparisons with similar maps from the protein data bank (PDB) for large number of Gly and Pro residues in proteins. The MD simulations revealed strong dependence of the populations and geometries of preferred backbone and side chain conformations, as well as the time scales of the peptide torsional transitions on the force field used. On the basis of the analysis of the measured and calculated data, AMBER99SB is identified as the most reliable force field for reproducing NMR measured parameters, which are dependent on the peptide backbone and the Pro side chain geometries and dynamics. Ramachandran maps showing the dependence of conformational populations as a function of backbone ϕ/ψ angles for Pro-2 and Gly-3 residues of GPGG from MD simulations using AMBER99SB, AMBER03, and CHARMM were found to resemble similar maps for Gly and Pro residues from the PDB survey. Three force fields (AMBER99, AMBER99ϕ, and AMBER94) showed the least satisfactory agreement with both the solution NMR and the PDB survey data. The poor performance of these force fields is attributed to their propensity to overstabilize helical peptide backbone conformations at the Pro-2 and Gly-3 residues. On the basis of the similarity of the MD and PDB Ramachandran plots, the following sequence of transitions is suggested for the Gly backbone conformation: α(L) ⇆ β(PR) ⇆ β(S) ⇆ β(P) ⇆ α, where backbone secondary structures α(L) and α are associated with helices and turns, β(P) and β(PR) correspond to the left- and right-handed polyproline II structures and β(S) denotes the fully stretched backbone conformation. Compared to the force field dependence, less significant, but noteworthy, variations in the populations of the peptide backbone conformations were observed. For different solvent models considered, a correlation was noted between the number of torsional transitions in GPGG and the water self-diffusion coefficient on using TIP3P, TIP4P, and TIP5P models. In addition to MD results, we also report DFT derived Karplus relationships for Gly and Pro residues using B972 and B3LYP functionals.

Coupling Protein Side-Chain and Backbone Flexibility Improves the Re-design of Protein-Ligand Specificity.

PubMed

Ollikainen, Noah; de Jong, René M; Kortemme, Tanja

2015-01-01

Interactions between small molecules and proteins play critical roles in regulating and facilitating diverse biological functions, yet our ability to accurately re-engineer the specificity of these interactions using computational approaches has been limited. One main difficulty, in addition to inaccuracies in energy functions, is the exquisite sensitivity of protein-ligand interactions to subtle conformational changes, coupled with the computational problem of sampling the large conformational search space of degrees of freedom of ligands, amino acid side chains, and the protein backbone. Here, we describe two benchmarks for evaluating the accuracy of computational approaches for re-engineering protein-ligand interactions: (i) prediction of enzyme specificity altering mutations and (ii) prediction of sequence tolerance in ligand binding sites. After finding that current state-of-the-art "fixed backbone" design methods perform poorly on these tests, we develop a new "coupled moves" design method in the program Rosetta that couples changes to protein sequence with alterations in both protein side-chain and protein backbone conformations, and allows for changes in ligand rigid-body and torsion degrees of freedom. We show significantly increased accuracy in both predicting ligand specificity altering mutations and binding site sequences. These methodological improvements should be useful for many applications of protein-ligand design. The approach also provides insights into the role of subtle conformational adjustments that enable functional changes not only in engineering applications but also in natural protein evolution.

HARMONY: a server for the assessment of protein structures

PubMed Central

Pugalenthi, G.; Shameer, K.; Srinivasan, N.; Sowdhamini, R.

2006-01-01

Protein structure validation is an important step in computational modeling and structure determination. Stereochemical assessment of protein structures examine internal parameters such as bond lengths and Ramachandran (φ,ψ) angles. Gross structure prediction methods such as inverse folding procedure and structure determination especially at low resolution can sometimes give rise to models that are incorrect due to assignment of misfolds or mistracing of electron density maps. Such errors are not reflected as strain in internal parameters. HARMONY is a procedure that examines the compatibility between the sequence and the structure of a protein by assigning scores to individual residues and their amino acid exchange patterns after considering their local environments. Local environments are described by the backbone conformation, solvent accessibility and hydrogen bonding patterns. We are now providing HARMONY through a web server such that users can submit their protein structure files and, if required, the alignment of homologous sequences. Scores are mapped on the structure for subsequent examination that is useful to also recognize regions of possible local errors in protein structures. HARMONY server is located at PMID:16844999

The Effect of Natural Osmolyte Mixtures on the Temperature-Pressure Stability of the Protein RNase A

NASA Astrophysics Data System (ADS)

Arns, Loana; Schuabb, Vitor; Meichsner, Shari; Berghaus, Melanie; Winter, Roland

2018-05-01

In biological cells, osmolytes appear as complex mixtures with variable compositions, depending on the particular environmental conditions of the organism. Based on various spectroscopic, thermodynamic and small-angle scattering data, we explored the effect of two different natural osmolyte mixtures, which are found in shallow-water and deep-sea shrimps, on the temperature and pressure stability of a typical monomeric protein, RNase A. Both natural osmolyte mixtures stabilize the protein against thermal and pressure denaturation. This effect seems to be mainly caused by the major osmolyte components of the osmolyte mixtures, i.e. by glycine and trimethylamine-N-oxide (TMAO), respectively. A minor compaction of the structure, in particular in the unfolded state, seems to be largely due to TMAO. Differences in thermodynamic properties observed for glycine and TMAO, and hence also for the two osmolyte mixtures, are most likely due to different solvation properties and interactions with the protein. Different from TMAO, glycine seems to interact with the amino acid side chains and/or the backbone of the protein, thus competing with hydration water and leading to a less hydrated protein surface.

A probabilistic and continuous model of protein conformational space for template-free modeling.

PubMed

Zhao, Feng; Peng, Jian; Debartolo, Joe; Freed, Karl F; Sosnick, Tobin R; Xu, Jinbo

2010-06-01

One of the major challenges with protein template-free modeling is an efficient sampling algorithm that can explore a huge conformation space quickly. The popular fragment assembly method constructs a conformation by stringing together short fragments extracted from the Protein Data Base (PDB). The discrete nature of this method may limit generated conformations to a subspace in which the native fold does not belong. Another worry is that a protein with really new fold may contain some fragments not in the PDB. This article presents a probabilistic model of protein conformational space to overcome the above two limitations. This probabilistic model employs directional statistics to model the distribution of backbone angles and 2(nd)-order Conditional Random Fields (CRFs) to describe sequence-angle relationship. Using this probabilistic model, we can sample protein conformations in a continuous space, as opposed to the widely used fragment assembly and lattice model methods that work in a discrete space. We show that when coupled with a simple energy function, this probabilistic method compares favorably with the fragment assembly method in the blind CASP8 evaluation, especially on alpha or small beta proteins. To our knowledge, this is the first probabilistic method that can search conformations in a continuous space and achieves favorable performance. Our method also generated three-dimensional (3D) models better than template-based methods for a couple of CASP8 hard targets. The method described in this article can also be applied to protein loop modeling, model refinement, and even RNA tertiary structure prediction.

Hydrogen-bonded turns in proteins: The case for a recount

PubMed Central

Panasik, Nick; Fleming, Patrick J.; Rose, George D.

2005-01-01

β-Turns are sites at which proteins change their overall chain direction, and they occur with high frequency in globular proteins. The Protein Data Bank has many instances of conformations that resemble β-turns but lack the characteristic N–H(i) → O=C(i − 3) hydrogen bond of an authentic β-turn. Here, we identify potential hydrogen-bonded β-turns in the coil library, a Web-accessible database utility comprised of all residues not in repetitive secondary structure, neither α-helix nor β-sheet (http://www.roselab.jhu.edu/coil). In particular, candidate turns were identified as four-residue segments satisfying highly relaxed geometric criteria but lacking a strictly defined hydrogen bond. Such candidates were then subjected to a minimization protocol to determine whether slight changes in torsion angles are sufficient to shift the conformation into reference-quality geometry without deviating significantly from the original structure. This approach of applying constrained minimization to known structures reveals a substantial population of previously unidentified, stringently defined, hydrogen-bonded β-turns. In particular, 33% of coil library residues were classified as β-turns prior to minimization. After minimization, 45% of such residues could be classified as β-turns, with another 8% in 310 helixes (which closely resemble type III β-turns). Of the remaining coil library residues, 37% have backbone dihedral angles in left-handed polyproline II structure. PMID:16251367

Hydrogen-bonded turns in proteins: the case for a recount.

PubMed

Panasik, Nick; Fleming, Patrick J; Rose, George D

2005-11-01

Beta-turns are sites at which proteins change their overall chain direction, and they occur with high frequency in globular proteins. The Protein Data Bank has many instances of conformations that resemble beta-turns but lack the characteristic N-H(i) --> O=C(i - 3) hydrogen bond of an authentic beta-turn. Here, we identify potential hydrogen-bonded beta-turns in the coil library, a Web-accessible database utility comprised of all residues not in repetitive secondary structure, neither alpha-helix nor beta-sheet (http://www.roselab.jhu.edu/coil). In particular, candidate turns were identified as four-residue segments satisfying highly relaxed geometric criteria but lacking a strictly defined hydrogen bond. Such candidates were then subjected to a minimization protocol to determine whether slight changes in torsion angles are sufficient to shift the conformation into reference-quality geometry without deviating significantly from the original structure. This approach of applying constrained minimization to known structures reveals a substantial population of previously unidentified, stringently defined, hydrogen-bonded beta-turns. In particular, 33% of coil library residues were classified as beta-turns prior to minimization. After minimization, 45% of such residues could be classified as beta-turns, with another 8% in 3(10) helixes (which closely resemble type III beta-turns). Of the remaining coil library residues, 37% have backbone dihedral angles in left-handed polyproline II structure.

The puckering free-energy surface of proline

NASA Astrophysics Data System (ADS)

Wu, Di

2013-03-01

Proline has two preferred puckering states, which are often characterized by the pseudorotation phase angle and amplitude. Although proline's five endocyclic torsion angles can be utilized to calculate the phase angle and amplitude, it is not clear if there is any direct correlation between each torsion angle and the proline-puckering pathway. Here we have designed five proline puckering pathways utilizing each torsion angle χj (j = 1˜5) as the reaction coordinate. By examining the free-energy surfaces of the five puckering pathways, we find they can be categorized into two groups. The χ2 pathway (χ2 is about the Cβ—Cγ bond) is especially meaningful in describing proline puckering: it changes linearly with the puckering amplitude and symmetrically with the phase angle. Our results show that this conclusion applies to both trans and cis proline conformations. We have also analyzed the correlations of proline puckering and its backbone torsion angles ϕ and ψ. We show proline has preferred puckering states at the specific regions of ϕ, ψ angles. Interestingly, the shapes of ψ-χ2 free-energy surfaces are similar among the trans proline in water, cis proline in water and cis proline in the gas phase, but they differ substantially from that of the trans proline in the gas phase. Our calculations are conducted using molecular simulations; we also verify our results using the proline conformations selected from the Protein Data Bank. In addition, we have compared our results with those calculated by the quantum mechanical methods.

A new default restraint library for the protein backbone in Phenix: a conformation-dependent geometry goes mainstream

DOE PAGES

Moriarty, Nigel W.; Tronrud, Dale E.; Adams, Paul D.; ...

2016-01-01

Chemical restraints are a fundamental part of crystallographic protein structure refinement. In response to mounting evidence that conventional restraints have shortcomings, it has previously been documented that using backbone restraints that depend on the protein backbone conformation helps to address these shortcomings and improves the performance of refinements [Moriartyet al.(2014),FEBS J.281, 4061–4071]. It is important that these improvements be made available to all in the protein crystallography community. Toward this end, a change in the default geometry library used byPhenixis described here. Tests are presented showing that this change will not generate increased numbers of outliers during validation, or depositionmore » in the Protein Data Bank, during the transition period in which some validation tools still use the conventional restraint libraries.« less

A new default restraint library for the protein backbone in Phenix: a conformation-dependent geometry goes mainstream

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moriarty, Nigel W.; Tronrud, Dale E.; Adams, Paul D.

Chemical restraints are a fundamental part of crystallographic protein structure refinement. In response to mounting evidence that conventional restraints have shortcomings, it has previously been documented that using backbone restraints that depend on the protein backbone conformation helps to address these shortcomings and improves the performance of refinements [Moriartyet al.(2014),FEBS J.281, 4061–4071]. It is important that these improvements be made available to all in the protein crystallography community. Toward this end, a change in the default geometry library used byPhenixis described here. Tests are presented showing that this change will not generate increased numbers of outliers during validation, or depositionmore » in the Protein Data Bank, during the transition period in which some validation tools still use the conventional restraint libraries.« less

Peptide models XLV: conformational properties of N-formyl-L-methioninamide and its relevance to methionine in proteins.

PubMed

Láng, András; Csizmadia, Imre G; Perczel, András

2005-02-15

The conformational space of the most biologically significant backbone folds of a suitable methionine peptide model was explored by density functional computational method. Using a medium [6-31G(d)] and a larger basis set [6-311++G(2d,2p)], the systematic exploration of low-energy backbone structures restricted for the "L-region" in the Ramachandran map of N-formyl-L-methioninamide results in conformers corresponding to the building units of an extended backbone structure (betaL), an inverse gamma-turn (gammaL), or a right-handed helical structure (alphaL). However, no poly-proline II type (epsilonL) fold was found, indicating that this conformer has no intrinsic stability, and highlighting the effect of molecular environment in stabilizing this backbone structure. This is in agreement with the abundance of the epsilonL-type backbone conformation of methionine found in proteins. Stability properties (DeltaE) and distinct backbone-side-chain interactions support the idea that specific intramolecular contacts are operative in the selection of the lowest energy conformers. Apart from the number of different folds, all stable conformers are within a 10 kcal x mol(-1) energy range, indicating the highly flexible behavior of methionine. This conformational feature can be important in supporting catalytic processes, facilitating protein folding and dimerization via metal ion binding. In both of the biological examples discussed (HIV-1 reverse transcriptase and PcoC copper-resistant protein), the conformational properties of Met residues were found to be of key importance. Spatial proximity to other types of residues or the same type of residue seems to be crucial for the structural integrity of a protein, whether Met is buried or exposed.

Conformational diversity and computational enzyme design

PubMed Central

Lassila, Jonathan K.

2010-01-01

The application of computational protein design methods to the design of enzyme active sites offers potential routes to new catalysts and new reaction specificities. Computational design methods have typically treated the protein backbone as a rigid structure for the sake of computational tractability. However, this fixed-backbone approximation introduces its own special challenges for enzyme design and it contrasts with an emerging picture of natural enzymes as dynamic ensembles with multiple conformations and motions throughout a reaction cycle. This review considers the impact of conformational variation and dynamics on computational enzyme design and it highlights new approaches to addressing protein conformational diversity in enzyme design including recent advances in multistate design, backbone flexibility, and computational library design. PMID:20829099

High-resolution orientation and depth of insertion of the voltage-sensing S4 helix of a potassium channel in lipid bilayers.

PubMed

Doherty, Tim; Su, Yongchao; Hong, Mei

2010-08-27

The opening and closing of voltage-gated potassium (Kv) channels are controlled by several conserved Arg residues in the S4 helix of the voltage-sensing domain. The interaction of these positively charged Arg residues with the lipid membrane has been of intense interest for understanding how membrane proteins fold to allow charged residues to insert into lipid bilayers against free-energy barriers. Using solid-state NMR, we have now determined the orientation and insertion depth of the S4 peptide of the KvAP channel in lipid bilayers. Two-dimensional (15)N correlation experiments of macroscopically oriented S4 peptide in phospholipid bilayers revealed a tilt angle of 40 degrees and two possible rotation angles differing by 180 degrees around the helix axis. Remarkably, the tilt angle and one of the two rotation angles are identical to those of the S4 helix in the intact voltage-sensing domain, suggesting that interactions between the S4 segment and other helices of the voltage-sensing domain are not essential for the membrane topology of the S4 helix. (13)C-(31)P distances between the S4 backbone and the lipid (31)P indicate a approximately 9 A local thinning and 2 A average thinning of the DMPC (1,2-dimyristoyl-sn-glycero-3-phosphochloline)/DMPG (1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol) bilayer, consistent with neutron diffraction data. Moreover, a short distance of 4.6 A from the guanidinium C(zeta) of the second Arg to (31)P indicates the existence of guanidinium phosphate hydrogen bonding and salt bridges. These data suggest that the structure of the Kv gating helix is mainly determined by protein-lipid interactions instead of interhelical protein-protein interactions, and the S4 amino acid sequence encodes sufficient information for the membrane topology of this crucial gating helix. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Knowledge-Guided Docking of WW Domain Proteins and Flexible Ligands

NASA Astrophysics Data System (ADS)

Lu, Haiyun; Li, Hao; Banu Bte Sm Rashid, Shamima; Leow, Wee Kheng; Liou, Yih-Cherng

Studies of interactions between protein domains and ligands are important in many aspects such as cellular signaling. We present a knowledge-guided approach for docking protein domains and flexible ligands. The approach is applied to the WW domain, a small protein module mediating signaling complexes which have been implicated in diseases such as muscular dystrophy and Liddle’s syndrome. The first stage of the approach employs a substring search for two binding grooves of WW domains and possible binding motifs of peptide ligands based on known features. The second stage aligns the ligand’s peptide backbone to the two binding grooves using a quasi-Newton constrained optimization algorithm. The backbone-aligned ligands produced serve as good starting points to the third stage which uses any flexible docking algorithm to perform the docking. The experimental results demonstrate that the backbone alignment method in the second stage performs better than conventional rigid superposition given two binding constraints. It is also shown that using the backbone-aligned ligands as initial configurations improves the flexible docking in the third stage. The presented approach can also be applied to other protein domains that involve binding of flexible ligand to two or more binding sites.

Improving the accuracy of protein stability predictions with multistate design using a variety of backbone ensembles.

PubMed

Davey, James A; Chica, Roberto A

2014-05-01

Multistate computational protein design (MSD) with backbone ensembles approximating conformational flexibility can predict higher quality sequences than single-state design with a single fixed backbone. However, it is currently unclear what characteristics of backbone ensembles are required for the accurate prediction of protein sequence stability. In this study, we aimed to improve the accuracy of protein stability predictions made with MSD by using a variety of backbone ensembles to recapitulate the experimentally measured stability of 85 Streptococcal protein G domain β1 sequences. Ensembles tested here include an NMR ensemble as well as those generated by molecular dynamics (MD) simulations, by Backrub motions, and by PertMin, a new method that we developed involving the perturbation of atomic coordinates followed by energy minimization. MSD with the PertMin ensembles resulted in the most accurate predictions by providing the highest number of stable sequences in the top 25, and by correctly binning sequences as stable or unstable with the highest success rate (≈90%) and the lowest number of false positives. The performance of PertMin ensembles is due to the fact that their members closely resemble the input crystal structure and have low potential energy. Conversely, the NMR ensemble as well as those generated by MD simulations at 500 or 1000 K reduced prediction accuracy due to their low structural similarity to the crystal structure. The ensembles tested herein thus represent on- or off-target models of the native protein fold and could be used in future studies to design for desired properties other than stability. Copyright © 2013 Wiley Periodicals, Inc.

Free-energy landscape of RNA hairpins constructed via dihedral angle principal component analysis.

PubMed

Riccardi, Laura; Nguyen, Phuong H; Stock, Gerhard

2009-12-31

To systematically construct a low-dimensional free-energy landscape of RNA systems from a classical molecular dynamics simulation, various versions of the principal component analysis (PCA) are compared: the cPCA using the Cartesian coordinates of all atoms, the dPCA using the sine/cosine-transformed six backbone dihedral angles as well as the glycosidic torsional angle chi and the pseudorotational angle P, the aPCA which ignores the circularity of the 6 + 2 dihedral angles of the RNA, and the dPCA(etatheta), which approximates the 6 backbone dihedral angles by 2 pseudotorsional angles eta and theta. As representative examples, a 10-nucleotide UUCG hairpin and the 36-nucleotide segment SL1 of the Psi site of HIV-1 are studied by classical molecular dynamics simulation, using the Amber all-atom force field and explicit solvent. It is shown that the conformational heterogeneity of the RNA hairpins can only be resolved by an angular PCA such as the dPCA but not by the cPCA using Cartesian coordinates. Apart from possible artifacts due to the coupling of overall and internal motion, this is because the details of hydrogen bonding and stacking interactions but also of global structural rearrangements of the RNA are better discriminated by dihedral angles. In line with recent experiments, it is found that the free energy landscape of RNA hairpins is quite rugged and contains various metastable conformational states which may serve as an intermediate for unfolding.

Rapid analysis of protein backbone resonance assignments using cryogenic probes, a distributed Linux-based computing architecture, and an integrated set of spectral analysis tools.

PubMed

Monleón, Daniel; Colson, Kimberly; Moseley, Hunter N B; Anklin, Clemens; Oswald, Robert; Szyperski, Thomas; Montelione, Gaetano T

2002-01-01

Rapid data collection, spectral referencing, processing by time domain deconvolution, peak picking and editing, and assignment of NMR spectra are necessary components of any efficient integrated system for protein NMR structure analysis. We have developed a set of software tools designated AutoProc, AutoPeak, and AutoAssign, which function together with the data processing and peak-picking programs NMRPipe and Sparky, to provide an integrated software system for rapid analysis of protein backbone resonance assignments. In this paper we demonstrate that these tools, together with high-sensitivity triple resonance NMR cryoprobes for data collection and a Linux-based computer cluster architecture, can be combined to provide nearly complete backbone resonance assignments and secondary structures (based on chemical shift data) for a 59-residue protein in less than 30 hours of data collection and processing time. In this optimum case of a small protein providing excellent spectra, extensive backbone resonance assignments could also be obtained using less than 6 hours of data collection and processing time. These results demonstrate the feasibility of high throughput triple resonance NMR for determining resonance assignments and secondary structures of small proteins, and the potential for applying NMR in large scale structural proteomics projects.

Reversibly constraining spliceosome-substrate complexes by engineering disulfide crosslinks.

PubMed

McCarthy, Patrick; Garside, Erin; Meschede-Krasa, Yonatan; MacMillan, Andrew; Pomeranz Krummel, Daniel

2017-08-01

The spliceosome is a highly dynamic mega-Dalton enzyme, formed in part by assembly of U snRNPs onto its pre-mRNA substrate transcripts. Early steps in spliceosome assembly are challenging to study biochemically and structurally due to compositional and conformational dynamics. We detail an approach to covalently and reversibly constrain or trap non-covalent pre-mRNA/protein spliceosome complexes. This approach involves engineering a single disulfide bond between a thiol-bearing cysteine sidechain and a proximal backbone phosphate of the pre-mRNA, site-specifically modified with an N-thioalkyl moiety. When distance and angle between reactants is optimal, the sidechain will react with the single N-thioalkyl to form a crosslink upon oxidation. We provide protocols detailing how this has been applied successfully to trap an 11-subunit RNA-protein assembly, the human U1 snRNP, in complex with a pre-mRNA. Copyright © 2017 Elsevier Inc. All rights reserved.

ROMP- and RAFT-Based Guanidinium-Containing Polymers as Scaffolds for Protein Mimic Synthesis.

PubMed

Sarapas, Joel M; Backlund, Coralie M; deRonde, Brittany M; Minter, Lisa M; Tew, Gregory N

2017-05-17

Cell-penetrating peptides are an important class of molecules with promising applications in bioactive cargo delivery. A diverse series of guanidinium-containing polymeric cell-penetrating peptide mimics (CPPMs) with varying backbone chemistries was synthesized and assessed for delivery of both GFP and fluorescently tagged siRNA. Specifically, we examined CPPMs based on norbornene, methacrylate, and styrene backbones to determine how backbone structure impacted internalization of these two cargoes. Either charge content or degree of polymerization was held constant at 20, with diguanidinium norbornene molecules being polymerized to both 10 and 20 repeat units. Generally, homopolymer CPPMs delivered low amounts of siRNA into Jurkat T cells, with no apparent backbone dependence; however, by adding a short hydrophobic methyl methacrylate block to the guanidinium-rich methacrylate polymer, siRNA delivery to nearly the entire cell population was achieved. Protein internalization yielded similar results for most of the CPPMs, though the block polymer was unable to deliver proteins. In contrast, the styrene-based CPPM yielded the highest internalization for GFP (≈40 % of cells affected), showing that indeed backbone chemistry impacts protein delivery, specifically through the incorporation of an aromatic group. These results demonstrate that an understanding of how polymer structure affects cargo-dependent internalization is critical to designing new, more effective CPPMs. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Small Angle Neutron Scattering (sans) Studies on "SIDE-END FIXED" and "SIDE-ON FIXED" Liquid Crystal Polymers

NASA Astrophysics Data System (ADS)

Hardouin, F.; Noirez, L.; Keller, P.; Leroux, N.; Cotton, J. P.

The following sections are included: * Introduction * Experimental * Results and discussion * Determination of the backbone conformation in the nematic and smectic A phases of "side-end fixed" L.C. polymethacrylates (PMA) or polyacrylates (PA) * Determination of the global and backbone conformation in the nematic and smectic A phases of "side-end fixed" L.C. polysiloxanes (PMS) * Determination of the backbone conformation in the unique nematic phase (without smectic A phase) or in the reentrant nematic phase (below smectic A phase) of "side-end fixed" L.C. polyacrylates (PA) * Determination of the global conformation in the nematic phase of "side-on fixed" L.C. polysiloxanes (PMS) * Determination of the global conformation in the nematic phase of "diluted side-on fixed" L.C. copolysiloxanes * Determination of the backbone conformation in the nematic phase of "side-on fixed" L.C. polyacrylates * Conclusions * References

Electrochemistry-Assisted Top-Down Characterization of Disulfide-Containing Proteins

PubMed Central

Zhang, Yun; Cui, Weidong; Zhang, Hao; Dewald, Howard D.; Chen, Hao

2013-01-01

Covalent disulfide bond linkage in a protein represents an important challenge for mass spectrometry (MS)-based top-down protein structure analysis as it reduces the backbone cleavage efficiency for MS/MS dissociation. This study presents a strategy for solving this critical issue via integrating electrochemistry (EC) online with top-down MS approach. In this approach, proteins undergo electrolytic reduction in an electrochemical cell to break disulfide bonds and then online ionized into gaseous ions for analysis by electron-capture dissociation (ECD) and collision-induced dissociation (CID). The electrochemical reduction of proteins allows to remove disulfide bond constraints and also leads to increased charge numbers of the resulting protein ions. As a result, sequence coverage was significantly enhanced, as exemplified by β-lactoglobulin A (24 vs. 73 backbone cleavages before and after electrolytic reduction, respectively) and lysozyme (5 vs. 66 backbone cleavages before and after electrolytic reduction, respectively). This methodology is fast and does not need chemical reductants, which would have an important impact in high-throughput proteomics research. PMID:22448817

Electrochemistry-assisted top-down characterization of disulfide-containing proteins.

PubMed

Zhang, Yun; Cui, Weidong; Zhang, Hao; Dewald, Howard D; Chen, Hao

2012-04-17

Covalent disulfide bond linkage in a protein represents an important challenge for mass spectrometry (MS)-based top-down protein structure analysis as it reduces the backbone cleavage efficiency for MS/MS dissociation. This study presents a strategy for solving this critical issue via integrating electrochemistry (EC) online with a top-down MS approach. In this approach, proteins undergo electrolytic reduction in an electrochemical cell to break disulfide bonds and then undergo online ionization into gaseous ions for analysis by electron-capture dissociation (ECD) and collision-induced dissociation (CID). The electrochemical reduction of proteins allows one to remove disulfide bond constraints and also leads to increased charge numbers of the resulting protein ions. As a result, sequence coverage was significantly enhanced, as exemplified by β-lactoglobulin A (24 vs 75 backbone cleavages before and after electrolytic reduction, respectively) and lysozyme (5 vs 66 backbone cleavages before and after electrolytic reduction, respectively). This methodology is fast and does not need chemical reductants, which would have an important impact in high-throughput proteomics research.

Molecular mechanical studies of DNA flexibility: Coupled backbone torsion angles and base-pair openings

PubMed Central

Keepers, Joe W.; Kollman, Peter A.; Weiner, Paul K.; James, Thomas L.

1982-01-01

Molecular mechanics studies have been carried out on “B-DNA-like” structures of [d(C-G-C-G-A-A-T-T-C-G-C-G)]2 and [d(A)]12·[d(T)]12. Each of the backbone torsion angles (ψ, φ, ω, ω′, φ′) has been “forced” to alternative values from the normal B-DNA values (g+, t, g-, g-, t conformations). Compensating torsion angle changes preserve most of the base stacking energy in the double helix. In a second part of the study, one purine N3-pyrimidine N1 distance at a time has been forced to a value of 6 Å in an attempt to simulate the base opening motions required to rationalize proton exchange data for DNA. When the 6-Å constraint is removed, many of the structures revert to the normal Watson-Crick hydrogen-bonded structure, but a number are trapped in structures ≈5 kcal/mol higher in energy than the starting B-DNA structure. The relative energy of these structures, some of which involve a non-Watson-Crick thymine C2(carbonyl)[unk]adenine 6NH2 hydrogen bond, are qualitatively consistent with the ΔH for a “base pair-open state” suggested by Mandal et al. of 4-6 kcal/mol [Mandal, C., Kallenbach, N. R. & Englander, S. W. (1979) J. Mol. Biol. 135, 391-411]. The picture of DNA flexibility emerging from this study depicts the backbone as undergoing rapid motion between local torsional minima on a nanosecond time scale. Backbone motion is mainly localized within a dinucleoside segment and generally not conformationally coupled along the chain or across the base pairs. Base motions are much smaller in magnitude than backbone motions. Base sliding allows imino N—H exchange, but it is localized, and only a small fraction of the N—H groups is exposed at any one time. Stacking and hydrogen bonding cause a rigid core of bases in the center of the molecule accounting for the hydrodynamic properties of DNA. PMID:6957879

Pressure response of protein backbone structure. Pressure-induced amide 15N chemical shifts in BPTI.

PubMed Central

Akasaka, K.; Li, H.; Yamada, H.; Li, R.; Thoresen, T.; Woodward, C. K.

1999-01-01

The effect of pressure on amide 15N chemical shifts was studied in uniformly 15N-labeled basic pancreatic trypsin inhibitor (BPTI) in 90%1H2O/10%2H2O, pH 4.6, by 1H-15N heteronuclear correlation spectroscopy between 1 and 2,000 bar. Most 15N signals were low field shifted linearly and reversibly with pressure (0.468 +/- 0.285 ppm/2 kbar), indicating that the entire polypeptide backbone structure is sensitive to pressure. A significant variation of shifts among different amide groups (0-1.5 ppm/2 kbar) indicates a heterogeneous response throughout within the three-dimensional structure of the protein. A tendency toward low field shifts is correlated with a decrease in hydrogen bond distance on the order of 0.03 A/2 kbar for the bond between the amide nitrogen atom and the oxygen atom of either carbonyl or water. The variation of 15N shifts is considered to reflect site-specific changes in phi, psi angles. For beta-sheet residues, a decrease in psi angles by 1-2 degrees/2 kbar is estimated. On average, shifts are larger for helical and loop regions (0.553 +/- 0.343 and 0.519 +/- 0.261 ppm/2 kbar, respectively) than for beta-sheet (0.295 +/- 0.195 ppm/2 kbar), suggesting that the pressure-induced structural changes (local compressibilities) are larger in helical and loop regions than in beta-sheet. Because compressibility is correlated with volume fluctuation, the result is taken to indicate that the volume fluctuation is larger in helical and loop regions than in beta-sheet. An important aspect of the volume fluctuation inferred from pressure shifts is that they include motions in slower time ranges (less than milliseconds) in which many biological processes may take place. PMID:10548039

Solution structure and thermodynamics of 2',5' RNA intercalation.

PubMed

Horowitz, Eric D; Lilavivat, Seth; Holladay, Benjamin W; Germann, Markus W; Hud, Nicholas V

2009-04-29

As a means to explore the influence of the nucleic acid backbone on the intercalative binding of ligands to DNA and RNA, we have determined the solution structure of a proflavine-bound 2',5'-linked octamer duplex with the sequence GCCGCGGC. This structure represents the first NMR structure of an intercalated RNA duplex, of either backbone structural isomer. By comparison with X-ray crystal structures, we have identified similarities and differences between intercalated 3',5' and 2',5'-linked RNA duplexes. First, the two forms of RNA have different sugar pucker geometries at the intercalated nucleotide steps, yet have the same interphosphate distances. Second, as in intercalated 3',5' RNA, the phosphate backbone angle zeta at the 2',5' RNA intercalation site prefers to be in the trans conformation, whereas unintercalated 2',5' and 3',5' RNA prefer the -gauche conformation. These observations provide new insights regarding the transitions required for intercalation of a phosphodiester-ribose backbone and suggest a possible contribution of the backbone to the origin of the nearest-neighbor exclusion principle. Thermodynamic studies presented for intercalation of both structural RNA isomers also reveal a surprising sensitivity of intercalator binding enthalpy and entropy to the details of RNA backbone structure.

Short-distance probes for protein backbone structure based on energy transfer between bimane and transition metal ions

PubMed Central

Taraska, Justin W.; Puljung, Michael C.; Zagotta, William N.

2009-01-01

The structure and dynamics of proteins underlies the workings of virtually every biological process. Existing biophysical methods are inadequate to measure protein structure at atomic resolution, on a rapid time scale, with limited amounts of protein, and in the context of a cell or membrane. FRET can measure distances between two probes, but depends on the orientation of the probes and typically works only over long distances comparable with the size of many proteins. Also, common probes used for FRET can be large and have long, flexible attachment linkers that position dyes far from the protein backbone. Here, we improve and extend a fluorescence method called transition metal ion FRET that uses energy transfer to transition metal ions as a reporter of short-range distances in proteins with little orientation dependence. This method uses a very small cysteine-reactive dye monobromobimane, with virtually no linker, and various transition metal ions bound close to the peptide backbone as the acceptor. We show that, unlike larger fluorophores and longer linkers, this donor–acceptor pair accurately reports short-range distances and changes in backbone distances. We further extend the method by using cysteine-reactive metal chelators, which allow the technique to be used in protein regions of unknown secondary structure or when native metal ion binding sites are present. This improved method overcomes several of the key limitations of classical FRET for intramolecular distance measurements. PMID:19805285

Toward structural dynamics: protein motions viewed by chemical shift modulations and direct detection of C'N multiple-quantum relaxation.

PubMed

Mori, Mirko; Kateb, Fatiha; Bodenhausen, Geoffrey; Piccioli, Mario; Abergel, Daniel

2010-03-17

Multiple quantum relaxation in proteins reveals unexpected relationships between correlated or anti-correlated conformational backbone dynamics in alpha-helices or beta-sheets. The contributions of conformational exchange to the relaxation rates of C'N coherences (i.e., double- and zero-quantum coherences involving backbone carbonyl (13)C' and neighboring amide (15)N nuclei) depend on the kinetics of slow exchange processes, as well as on the populations of the conformations and chemical shift differences of (13)C' and (15)N nuclei. The relaxation rates of C'N coherences, which reflect concerted fluctuations due to slow chemical shift modulations (CSMs), were determined by direct (13)C detection in diamagnetic and paramagnetic proteins. In well-folded proteins such as lanthanide-substituted calbindin (CaLnCb), copper,zinc superoxide dismutase (Cu,Zn SOD), and matrix metalloproteinase (MMP12), slow conformational exchange occurs along the entire backbone. Our observations demonstrate that relaxation rates of C'N coherences arising from slow backbone dynamics have positive signs (characteristic of correlated fluctuations) in beta-sheets and negative signs (characteristic of anti-correlated fluctuations) in alpha-helices. This extends the prospects of structure-dynamics relationships to slow time scales that are relevant for protein function and enzymatic activity.

Structural test of the parameterized-backbone method for protein design.

PubMed

Plecs, Joseph J; Harbury, Pehr B; Kim, Peter S; Alber, Tom

2004-09-03

Designing new protein folds requires a method for simultaneously optimizing the conformation of the backbone and the side-chains. One approach to this problem is the use of a parameterized backbone, which allows the systematic exploration of families of structures. We report the crystal structure of RH3, a right-handed, three-helix coiled coil that was designed using a parameterized backbone and detailed modeling of core packing. This crystal structure was determined using another rationally designed feature, a metal-binding site that permitted experimental phasing of the X-ray data. RH3 adopted the intended fold, which has not been observed previously in biological proteins. Unanticipated structural asymmetry in the trimer was a principal source of variation within the RH3 structure. The sequence of RH3 differs from that of a previously characterized right-handed tetramer, RH4, at only one position in each 11 amino acid sequence repeat. This close similarity indicates that the design method is sensitive to the core packing interactions that specify the protein structure. Comparison of the structures of RH3 and RH4 indicates that both steric overlap and cavity formation provide strong driving forces for oligomer specificity.

Structure and backbone dynamics of a microcrystalline metalloprotein by solid-state NMR.

PubMed

Knight, Michael J; Pell, Andrew J; Bertini, Ivano; Felli, Isabella C; Gonnelli, Leonardo; Pierattelli, Roberta; Herrmann, Torsten; Emsley, Lyndon; Pintacuda, Guido

2012-07-10

We introduce a new approach to improve structural and dynamical determination of large metalloproteins using solid-state nuclear magnetic resonance (NMR) with (1)H detection under ultrafast magic angle spinning (MAS). The approach is based on the rapid and sensitive acquisition of an extensive set of (15)N and (13)C nuclear relaxation rates. The system on which we demonstrate these methods is the enzyme Cu, Zn superoxide dismutase (SOD), which coordinates a Cu ion available either in Cu(+) (diamagnetic) or Cu(2+) (paramagnetic) form. Paramagnetic relaxation enhancements are obtained from the difference in rates measured in the two forms and are employed as structural constraints for the determination of the protein structure. When added to (1)H-(1)H distance restraints, they are shown to yield a twofold improvement of the precision of the structure. Site-specific order parameters and timescales of motion are obtained by a gaussian axial fluctuation (GAF) analysis of the relaxation rates of the diamagnetic molecule, and interpreted in relation to backbone structure and metal binding. Timescales for motion are found to be in the range of the overall correlation time in solution, where internal motions characterized here would not be observable.

Folding 19 proteins to their native state and stability of large proteins from a coarse-grained model.

PubMed

Kapoor, Abhijeet; Travesset, Alex

2014-03-01

We develop an intermediate resolution model, where the backbone is modeled with atomic resolution but the side chain with a single bead, by extending our previous model (Proteins (2013) DOI: 10.1002/prot.24269) to properly include proline, preproline residues and backbone rigidity. Starting from random configurations, the model properly folds 19 proteins (including a mutant 2A3D sequence) into native states containing β sheet, α helix, and mixed α/β. As a further test, the stability of H-RAS (a 169 residue protein, critical in many signaling pathways) is investigated: The protein is stable, with excellent agreement with experimental B-factors. Despite that proteins containing only α helices fold to their native state at lower backbone rigidity, and other limitations, which we discuss thoroughly, the model provides a reliable description of the dynamics as compared with all atom simulations, but does not constrain secondary structures as it is typically the case in more coarse-grained models. Further implications are described. Copyright © 2013 Wiley Periodicals, Inc.

A tensegrity model for hydrogen bond networks in proteins.

PubMed

Bywater, Robert P

2017-05-01

Hydrogen-bonding networks in proteins considered as structural tensile elements are in balance separately from any other stabilising interactions that may be in operation. The hydrogen bond arrangement in the network is reminiscent of tensegrity structures in architecture and sculpture. Tensegrity has been discussed before in cells and tissues and in proteins. In contrast to previous work only hydrogen bonds are studied here. The other interactions within proteins are either much stronger - covalent bonds connecting the atoms in the molecular skeleton or weaker forces like the so-called hydrophobic interactions. It has been demonstrated that the latter operate independently from hydrogen bonds. Each category of interaction must, if the protein is to have a stable structure, balance out. The hypothesis here is that the entire hydrogen bond network is in balance without any compensating contributions from other types of interaction. For sidechain-sidechain, sidechain-backbone and backbone-backbone hydrogen bonds in proteins, tensegrity balance ("closure") is required over the entire length of the polypeptide chain that defines individually folding units in globular proteins ("domains") as well as within the repeating elements in fibrous proteins that consist of extended chain structures. There is no closure to be found in extended structures that do not have repeating elements. This suggests an explanation as to why globular domains, as well as the repeat units in fibrous proteins, have to have a defined number of residues. Apart from networks of sidechain-sidechain hydrogen bonds there are certain key points at which this closure is achieved in the sidechain-backbone hydrogen bonds and these are associated with demarcation points at the start or end of stretches of secondary structure. Together, these three categories of hydrogen bond achieve the closure that is necessary for the stability of globular protein domains as well as repeating elements in fibrous proteins.

Predicting the tolerated sequences for proteins and protein interfaces using RosettaBackrub flexible backbone design.

PubMed

Smith, Colin A; Kortemme, Tanja

2011-01-01

Predicting the set of sequences that are tolerated by a protein or protein interface, while maintaining a desired function, is useful for characterizing protein interaction specificity and for computationally designing sequence libraries to engineer proteins with new functions. Here we provide a general method, a detailed set of protocols, and several benchmarks and analyses for estimating tolerated sequences using flexible backbone protein design implemented in the Rosetta molecular modeling software suite. The input to the method is at least one experimentally determined three-dimensional protein structure or high-quality model. The starting structure(s) are expanded or refined into a conformational ensemble using Monte Carlo simulations consisting of backrub backbone and side chain moves in Rosetta. The method then uses a combination of simulated annealing and genetic algorithm optimization methods to enrich for low-energy sequences for the individual members of the ensemble. To emphasize certain functional requirements (e.g. forming a binding interface), interactions between and within parts of the structure (e.g. domains) can be reweighted in the scoring function. Results from each backbone structure are merged together to create a single estimate for the tolerated sequence space. We provide an extensive description of the protocol and its parameters, all source code, example analysis scripts and three tests applying this method to finding sequences predicted to stabilize proteins or protein interfaces. The generality of this method makes many other applications possible, for example stabilizing interactions with small molecules, DNA, or RNA. Through the use of within-domain reweighting and/or multistate design, it may also be possible to use this method to find sequences that stabilize particular protein conformations or binding interactions over others.

RosettaRemodel: A Generalized Framework for Flexible Backbone Protein Design

PubMed Central

Huang, Po-Ssu; Ban, Yih-En Andrew; Richter, Florian; Andre, Ingemar; Vernon, Robert; Schief, William R.; Baker, David

2011-01-01

We describe RosettaRemodel, a generalized framework for flexible protein design that provides a versatile and convenient interface to the Rosetta modeling suite. RosettaRemodel employs a unified interface, called a blueprint, which allows detailed control over many aspects of flexible backbone protein design calculations. RosettaRemodel allows the construction and elaboration of customized protocols for a wide range of design problems ranging from loop insertion and deletion, disulfide engineering, domain assembly, loop remodeling, motif grafting, symmetrical units, to de novo structure modeling. PMID:21909381

Protein Structure Classification and Loop Modeling Using Multiple Ramachandran Distributions.

PubMed

Najibi, Seyed Morteza; Maadooliat, Mehdi; Zhou, Lan; Huang, Jianhua Z; Gao, Xin

2017-01-01

Recently, the study of protein structures using angular representations has attracted much attention among structural biologists. The main challenge is how to efficiently model the continuous conformational space of the protein structures based on the differences and similarities between different Ramachandran plots. Despite the presence of statistical methods for modeling angular data of proteins, there is still a substantial need for more sophisticated and faster statistical tools to model the large-scale circular datasets. To address this need, we have developed a nonparametric method for collective estimation of multiple bivariate density functions for a collection of populations of protein backbone angles. The proposed method takes into account the circular nature of the angular data using trigonometric spline which is more efficient compared to existing methods. This collective density estimation approach is widely applicable when there is a need to estimate multiple density functions from different populations with common features. Moreover, the coefficients of adaptive basis expansion for the fitted densities provide a low-dimensional representation that is useful for visualization, clustering, and classification of the densities. The proposed method provides a novel and unique perspective to two important and challenging problems in protein structure research: structure-based protein classification and angular-sampling-based protein loop structure prediction.

Backbone conformational preferences of an intrinsically disordered protein in solution.

PubMed

Espinoza-Fonseca, L Michel; Ilizaliturri-Flores, Ian; Correa-Basurto, José

2012-06-01

We have performed a 4-μs molecular dynamics simulation to investigate the native conformational preferences of the intrinsically disordered kinase-inducible domain (KID) of the transcription factor CREB in solution. There is solid experimental evidence showing that KID does not possess a bound-like structure in solution; however, it has been proposed that coil-to-helix transitions upon binding to its binding partner (CBP) are template-driven. While these studies indicate that IDPs possess a bias towards the bound structure, they do not provide direct evidence on the time-dependent conformational preferences of IDPs in atomic detail. Our simulation captured intrinsic conformational characteristics of KID that are in good agreement with experimental data such as a very small percentage of helical structure in its segment α(B) and structural disorder in solution. We used dihedral principal component analysis dPCA to map the conformations of KID in the microsecond timescale. By using principal components as reaction coordinates, we further constructed dPCA-based free energy landscapes of KID. Analysis of the free energy landscapes showed that KID is best characterized as a conformational ensemble of rapidly interconverting conformations. Interestingly, we found that despite the conformational heterogeneity of the backbone and the absence of substantial secondary structure, KID does not randomly sample the conformational space in solution: analysis of the (Φ, Ψ) dihedral angles showed that several individual residues of KID possess a strong bias toward the helical region of the Ramachandran plot. We suggest that the intrinsic conformational preferences of KID provide a bias toward the folded state without having to populate bound-like conformations before binding. Furthermore, we argue that these conformational preferences do not represent actual structural constraints which drive binding through a single pathway, which allows for specific interactions with multiple binding partners. Based on this evidence, we propose that the backbone conformational preferences of KID provide a thermodynamic advantage for folding and binding without negatively affecting the kinetics of binding. We further discuss the relation of our results to previous studies to rationalize the functional implications of the conformational preferences of IDPs, such as the optimization of structural disorder in protein-protein interactions. This study illustrates the importance in obtaining atomistic information of intrinsically disordered proteins in real time to reveal functional features arising from their complex conformational space.

Effects of disulfide bridges and backbone connectivity on water sorption by protein matrices.

PubMed

Kim, Sang Beom; Singh, Rakesh S; Paul, Prem K C; Debenedetti, Pablo G

2017-08-11

Understanding the water sorption behavior of protein powders is important in applications such as the preservation of protein-based pharmaceuticals. Most globular proteins exhibit a characteristic sigmoidal water adsorption isotherm at ambient conditions. However, it is not well understood how water sorption behavior is influenced by intrinsic factors that are related to structural properties of proteins. We investigate computationally how structural constraints on proteins influence the water sorption isotherms of amorphous protein powders. Specifically, we study the effects of non-local disulfide linkages and backbone connectivity using pheromone ER-23 and lysozyme as model proteins. We find that non-local disulfide linkages can significantly restrict structural changes during hydration and dehydration, and this in turn greatly reduces the extent of hysteresis between the adsorption and desorption branches. Upon removing the backbone connectivity by breaking all peptide bonds in lysozyme, we find that the hysteresis shifts towards the lower humidity regime, and the water uptake capacity is significantly enhanced. We attribute these changes to the higher aggregation propensity of the constraint-free amino acids in dehydrated condition, and the formation of a spanning water network at high hydration levels.

Peierls-Nabarro barrier and protein loop propagation

NASA Astrophysics Data System (ADS)

Sieradzan, Adam K.; Niemi, Antti; Peng, Xubiao

2014-12-01

When a self-localized quasiparticle excitation propagates along a discrete one-dimensional lattice, it becomes subject to a dissipation that converts the kinetic energy into lattice vibrations. Eventually the kinetic energy no longer enables the excitation to cross over the minimum energy barrier between neighboring sites, and the excitation becomes localized within a lattice cell. In the case of a protein, the lattice structure consists of the Cα backbone. The self-localized quasiparticle excitation is the elemental building block of loops. It can be modeled by a kink that solves a variant of the discrete nonlinear Schrödinger equation. We study the propagation of such a kink in the case of the protein G related albumin-binding domain, using the united residue coarse-grained molecular-dynamics force field. We estimate the height of the energy barriers that the kink needs to cross over in order to propagate along the backbone lattice. We analyze how these barriers give rise to both stresses and reliefs, which control the kink movement. For this, we deform a natively folded protein structure by parallel translating the kink along the backbone away from its native position. We release the transposed kink, and we follow how it propagates along the backbone toward the native location. We observe that the dissipative forces that are exerted on the kink by the various energy barriers have a pivotal role in determining how a protein folds toward its native state.

Conformational Entropy of FK506 Binding to FKBP12 Determined by Nuclear Magnetic Resonance Relaxation and Molecular Dynamics Simulations.

PubMed

Solomentsev, Gleb; Diehl, Carl; Akke, Mikael

2018-03-06

FKBP12 (FK506 binding protein 12 kDa) is an important drug target. Nuclear magnetic resonance (NMR) order parameters, describing amplitudes of motion on the pico- to nanosecond time scale, can provide estimates of changes in conformational entropy upon ligand binding. Here we report backbone and methyl-axis order parameters of the apo and FK506-bound forms of FKBP12, based on 15 N and 2 H NMR relaxation. Binding of FK506 to FKBP12 results in localized changes in order parameters, notably for the backbone of residues E54 and I56 and the side chains of I56, I90, and I91, all positioned in the binding site. The order parameters increase slightly upon FK506 binding, indicating an unfavorable entropic contribution to binding of TΔ S = -18 ± 2 kJ/mol at 293 K. Molecular dynamics simulations indicate a change in conformational entropy, associated with all dihedral angles, of TΔ S = -26 ± 9 kJ/mol. Both these values are significant compared to the total entropy of binding determined by isothermal titration calorimetry and referenced to a reactant concentration of 1 mM ( TΔ S = -29 ± 1 kJ/mol). Our results reveal subtle differences in the response to ligand binding compared to that of the previously studied rapamycin-FKBP12 complex, despite the high degree of structural homology between the two complexes and their nearly identical ligand-FKBP12 interactions. These results highlight the delicate dependence of protein dynamics on drug interactions, which goes beyond the view provided by static structures, and reinforce the notion that protein conformational entropy can make important contributions to the free energy of ligand binding.

Accurate protein structure modeling using sparse NMR data and homologous structure information.

PubMed

Thompson, James M; Sgourakis, Nikolaos G; Liu, Gaohua; Rossi, Paolo; Tang, Yuefeng; Mills, Jeffrey L; Szyperski, Thomas; Montelione, Gaetano T; Baker, David

2012-06-19

While information from homologous structures plays a central role in X-ray structure determination by molecular replacement, such information is rarely used in NMR structure determination because it can be incorrect, both locally and globally, when evolutionary relationships are inferred incorrectly or there has been considerable evolutionary structural divergence. Here we describe a method that allows robust modeling of protein structures of up to 225 residues by combining (1)H(N), (13)C, and (15)N backbone and (13)Cβ chemical shift data, distance restraints derived from homologous structures, and a physically realistic all-atom energy function. Accurate models are distinguished from inaccurate models generated using incorrect sequence alignments by requiring that (i) the all-atom energies of models generated using the restraints are lower than models generated in unrestrained calculations and (ii) the low-energy structures converge to within 2.0 Å backbone rmsd over 75% of the protein. Benchmark calculations on known structures and blind targets show that the method can accurately model protein structures, even with very remote homology information, to a backbone rmsd of 1.2-1.9 Å relative to the conventional determined NMR ensembles and of 0.9-1.6 Å relative to X-ray structures for well-defined regions of the protein structures. This approach facilitates the accurate modeling of protein structures using backbone chemical shift data without need for side-chain resonance assignments and extensive analysis of NOESY cross-peak assignments.

TROSY of side-chain amides in large proteins

PubMed Central

Liu, Aizhuo; Yao, Lishan; Li, Yue; Yan, Honggao

2012-01-01

By using the mixed solvent of 50% H2O/50% D2O and employing deuterium decoupling, TROSY experiments exclusively detect NMR signals from semideuterated isotopomers of carboxamide groups with high sensitivities for proteins with molecular weights up to 80 kDa. This isotopomer-selective strategy extends TROSY experiments from exclusively detecting backbone to both backbone and side-chain amides, particularly in large proteins. Because of differences in both TROSY effect and dynamics between 15N–HE{DZ} and 15N–HZ{DE} isotopomers of the same carboxamide, the 15N transverse magnetization of the latter relaxes significantly faster than that of the former, which provides a direct and reliable stereospecific distinction between the two configurations. The TROSY effects on the 15N–HE{DZ} isotopomers of side-chain amides are as significant as on backbone amides. PMID:17347000

Neutron scattering studies of molecular conformations in liquid crystal polymers

NASA Astrophysics Data System (ADS)

Noirez, L.; Moussa, F.; Cotton, J. P.; Keller, P.; Pépy, G.

1991-03-01

A comblike liquid crystal polymer (LPC) is a polymer on which mesogenic molecules have been grafted. It exhibits a succession of liquid crystal phases. Usually the equilibrium conformation of an ordinary polymeric chain corresponds to a maximum entropy, i.e., to an isotropic spherical coil. How does the backbone of a LCP behave in the nematic and smectic field? Small-angle neutron scattering may answer this question. Such measurements are presented here on four different polymers as a function of temperature. An anisotropy of the backbone conformation is found in all these studied compounds, much more pronounced in the smectic phase than in the nematic phase: the backbone spreads more or less perpendicularly to its hanging cores. A comparison with existing theories and a discussion of these results is outlined.

Evaluating and learning from RNA pseudotorsional space: quantitative validation of a reduced representation for RNA structure.

PubMed

Wadley, Leven M; Keating, Kevin S; Duarte, Carlos M; Pyle, Anna Marie

2007-09-28

Quantitatively describing RNA structure and conformational elements remains a formidable problem. Seven standard torsion angles and the sugar pucker are necessary to characterize the conformation of an RNA nucleotide completely. Progress has been made toward understanding the discrete nature of RNA structure, but classifying simple and ubiquitous structural elements such as helices and motifs remains a difficult task. One approach for describing RNA structure in a simple, mathematically consistent, and computationally accessible manner involves the invocation of two pseudotorsions, eta (C4'(n-1), P(n), C4'(n), P(n+1)) and theta (P(n), C4'(n), P(n+1), C4'(n+1)), which can be used to describe RNA conformation in much the same way that varphi and psi are used to describe backbone configuration of proteins. Here, we conduct an exploration and statistical evaluation of pseudotorsional space and of the Ramachandran-like eta-theta plot. We show that, through the rigorous quantitative analysis of the eta-theta plot, the pseudotorsional descriptors eta and theta, together with sugar pucker, are sufficient to describe RNA backbone conformation fully in most cases. These descriptors are also shown to contain considerable information about nucleotide base conformation, revealing a previously uncharacterized interplay between backbone and base orientation. A window function analysis is used to discern statistically relevant regions of density in the eta-theta scatter plot and then nucleotides in colocalized clusters in the eta-theta plane are shown to have similar 3-D structures through RMSD analysis of the RNA structural constituents. We find that major clusters in the eta-theta plot are few, underscoring the discrete nature of RNA backbone conformation. Like the Ramachandran plot, the eta-theta plot is a valuable system for conceptualizing biomolecular conformation, it is a useful tool for analyzing RNA tertiary structures, and it is a vital component of new approaches for solving the 3-D structures of large RNA molecules and RNA assemblies.

Multistate approaches in computational protein design

PubMed Central

Davey, James A; Chica, Roberto A

2012-01-01

Computational protein design (CPD) is a useful tool for protein engineers. It has been successfully applied towards the creation of proteins with increased thermostability, improved binding affinity, novel enzymatic activity, and altered ligand specificity. Traditionally, CPD calculations search and rank sequences using a single fixed protein backbone template in an approach referred to as single-state design (SSD). While SSD has enjoyed considerable success, certain design objectives require the explicit consideration of multiple conformational and/or chemical states. Cases where a “multistate” approach may be advantageous over the SSD approach include designing conformational changes into proteins, using native ensembles to mimic backbone flexibility, and designing ligand or oligomeric association specificities. These design objectives can be efficiently tackled using multistate design (MSD), an emerging methodology in CPD that considers any number of protein conformational or chemical states as inputs instead of a single protein backbone template, as in SSD. In this review article, recent examples of the successful design of a desired property into proteins using MSD are described. These studies employing MSD are divided into two categories—those that utilized multiple conformational states, and those that utilized multiple chemical states. In addition, the scoring of competing states during negative design is discussed as a current challenge for MSD. PMID:22811394

Mixed pyruvate labeling enables backbone resonance assignment of large proteins using a single experiment.

PubMed

Robson, Scott A; Takeuchi, Koh; Boeszoermenyi, Andras; Coote, Paul W; Dubey, Abhinav; Hyberts, Sven; Wagner, Gerhard; Arthanari, Haribabu

2018-01-24

Backbone resonance assignment is a critical first step in the investigation of proteins by NMR. This is traditionally achieved with a standard set of experiments, most of which are not optimal for large proteins. Of these, HNCA is the most sensitive experiment that provides sequential correlations. However, this experiment suffers from chemical shift degeneracy problems during the assignment procedure. We present a strategy that increases the effective resolution of HNCA and enables near-complete resonance assignment using this single HNCA experiment. We utilize a combination of 2- 13 C and 3- 13 C pyruvate as the carbon source for isotope labeling, which suppresses the one bond ( 1 J αβ ) coupling providing enhanced resolution for the Cα resonance and amino acid-specific peak shapes that arise from the residual coupling. Using this approach, we can obtain near-complete (>85%) backbone resonance assignment of a 42 kDa protein using a single HNCA experiment.

Optical backbone-sidechain charge transfer transitions in proteins sensitive to secondary structure and modifications.

PubMed

Mandal, I; Paul, S; Venkatramani, R

2018-04-17

The absorption of light by proteins can induce charge transfer (CT) transitions in the UV-visible range of the electromagnetic spectrum. Metal-ligand complexes or active site prosthetic groups which absorb in the visible region exhibit prominent CT transitions. Furthermore, the protein backbone also exhibits CT transitions in the far UV range. In this manuscript, we present a detailed computational study of new near UV-visible CT transitions that involve amino acids with charged side chains. Specifically, using time dependent density functional theory calculations, we examine the absorption spectra of naturally charged amino acids (Lys, Glu, Arg, Asp and His), extracted from solution phase protein structures generated by classical molecular dynamics simulations, and phosphorylated amino acids (Tyr, Thr and Ser) from experimentally determined protein structures. We show that amino acids with charged sidechains present a directed electronic donor-bridge-acceptor paradigm, with the lowest energy optical excitations demonstrating peptide backbone-sidechain charge separations. The UV-visible spectral range of the backbone-sidechain CT transitions is determined by the chemical nature of the donor, bridge and acceptor groups within each amino acid, amino acid conformation and the protein secondary structure where the amino acids are located. Photoinduced CT occurs in opposite directions for the anionic and cationic amino acids along the ground state dipole moment vector for the chromophores. We find that photoinduced charge separation is more facile for the anionic amino acids (Asp, Glu, pSer, pThr and pTyr) relative to that for the cationic amino acids (Lys, Arg and Hsp). Our results provide a foundation for the development of spectroscopic markers based on the recently proposed Protein Charge Transfer Spectra (ProCharTS) which are relevant for the study of DNA-binding or intrinsically disordered proteins that are rich in charged amino acids.

Backbone and sidechain methyl Ile (δ1), Leu and Val chemical shift assignments of RDE-4 (1-243), an RNA interference initiation protein in C. elegans.

PubMed

Chiliveri, Sai Chaitanya; Kumar, Sonu; Marelli, Udaya Kiran; Deshmukh, Mandar V

2012-10-01

The RNAi pathway of several organisms requires presence of double stranded RNA binding proteins for functioning of Dicer in gene regulation. In C. elegans, a double stranded RNA binding protein, RDE-4 (385 aa, 44 kDa) recognizes long exogenous dsRNA and initiates the RNAi pathway. We have achieved complete backbone and stereospecific methyl sidechain Ile (δ1), Leu and Val chemical shifts of first 243 amino acids of RDE-4, namely RDE-4ΔC.

On contribution of known atomic partial charges of protein backbone in electrostatic potential density maps.

PubMed

Wang, Jimin

2017-06-01

Partial charges of atoms in a molecule and electrostatic potential (ESP) density for that molecule are known to bear a strong correlation. In order to generate a set of point-field force field parameters for molecular dynamics, Kollman and coworkers have extracted atomic partial charges for each of all 20 amino acids using restrained partial charge-fitting procedures from theoretical ESP density obtained from condensed-state quantum mechanics. The magnitude of atomic partial charges for neutral peptide backbone they have obtained is similar to that of partial atomic charges for ionized carboxylate side chain atoms. In this study, the effect of these known atomic partial charges on ESP is examined using computer simulations and compared with the experimental ESP density recently obtained for proteins using electron microscopy. It is found that the observed ESP density maps are most consistent with the simulations that include atomic partial charges of protein backbone. Therefore, atomic partial charges are integral part of atomic properties in protein molecules and should be included in model refinement. © 2017 The Protein Society.

Solution structure of an artificial Fe8S8 ferredoxin: the D13C variant of Bacillus schlegelii Fe7S8 ferredoxin.

PubMed

Aono, S; Bentrop, D; Bertini, I; Cosenza, G; Luchinat, C

1998-12-01

The solution structure of the D13C variant of the thermostable Fe7S8 ferredoxin from Bacillus schlegelii has been determined by 1H-NMR spectroscopy in its oxidized form. In a variable-temperature NMR study the D13C variant was as thermostable (up to 90 degrees C) as the wild-type protein (WT). Seventy-five out of 77 amino acid residues and 81% of all theoretically expected proton resonances in the D13C Fe8S8 protein have been assigned. Its structure was determined through torsion angle dynamics calculations with the program DYANA, using 935 meaningful NOEs (from a total of 1251), hydrogen bond constraints, and NMR-derived dihedral angle constraints for the cluster-ligating cysteines. Afterwards, restrained energy minimization and restrained molecular dynamics were applied to each conformer of the family. The final family of 20 structures has RMSD values from the mean structure of 0.055 nm for the backbone atoms and of 0.099 nm for all heavy atoms. The overall folding of the WT is maintained in the mutant, except for the immediate vicinity of the new cysteine, which becomes much more similar to native Fe8S8 proteins. The two residues at positions 11 and 12, which constitute an insertion with respect to all known Fe8S8 proteins, assume a conformation that does not prevent the preceding and following residues from folding like in native Fe8S8 proteins. Clear evidence for the existence of two conformations involving almost half of the amino acid residues was found. The two conformations are structurally indistinguishable. Temperature-dependent NMR experiments show that one of them is thermodynamically more stable than the other.

Force Field for Peptides and Proteins based on the Classical Drude Oscillator

PubMed Central

Lopes, Pedro E.M.; Huang, Jing; Shim, Jihyun; Luo, Yun; Li, Hui; Roux, Benoît; MacKerell, Alexander D.

2013-01-01

Presented is a polarizable force field based on a classical Drude oscillator framework, currently implemented in the programs CHARMM and NAMD, for modeling and molecular dynamics (MD) simulation studies of peptides and proteins. Building upon parameters for model compounds representative of the functional groups in proteins, the development of the force field focused on the optimization of the parameters for the polypeptide backbone and the connectivity between the backbone and side chains. Optimization of the backbone electrostatic parameters targeted quantum mechanical conformational energies, interactions with water, molecular dipole moments and polarizabilities and experimental condensed phase data for short polypeptides such as (Ala)5. Additional optimization of the backbone φ, ψ conformational preferences included adjustments of the tabulated two-dimensional spline function through the CMAP term. Validation of the model included simulations of a collection of peptides and proteins. This 1st generation polarizable model is shown to maintain the folded state of the studied systems on the 100 ns timescale in explicit solvent MD simulations. The Drude model typically yields larger RMS differences as compared to the additive CHARMM36 force field (C36) and shows additional flexibility as compared to the additive model. Comparison with NMR chemical shift data shows a small degradation of the polarizable model with respect to the additive, though the level of agreement may be considered satisfactory, while for residues shown to have significantly underestimated S2 order parameters in the additive model, improvements are calculated with the polarizable model. Analysis of dipole moments associated with the peptide backbone and tryptophan side chains show the Drude model to have significantly larger values than those present in C36, with the dipole moments of the peptide backbone enhanced to a greater extent in sheets versus helices and the dipoles of individual moieties observed to undergo significant variations during the MD simulations. Although there are still some limitations, the presented model, termed Drude-2013, is anticipated to yield a molecular picture of peptide and protein structure and function that will be of increased physical validity and internal consistency in a computationally accessible fashion. PMID:24459460

Free energy landscapes of peptides by enhanced conformational sampling.

PubMed

Nakajima, N; Higo, J; Kidera, A; Nakamura, H

2000-02-11

The free energy landscapes of peptide conformations in water have been observed by the enhanced conformational sampling method, applying the selectively enhanced multicanonical molecular dynamics simulations. The conformations of the peptide dimers, -Gly-Gly-, -Gly-Ala-, -Gly-Ser-, -Ala-Gly-, -Asn-Gly-, -Pro-Gly-, -Pro-Ala-, and -Ala-Ala-, which were all blocked with N-terminal acetyl and C-terminal N-methyl groups, were individually sampled with the explicit TIP3P water molecules. From each simulation trajectory, we obtained the canonical ensemble at 300 K, from which the individual three-dimensional landscape was drawn by the potential of mean force using the three reaction coordinates: the backbone dihedral angle, psi, of the first amino acid, the backbone dihedral angle, phi, of the second amino acid, and the distance between the carbonyl oxygen of the N-terminal acetyl group and the C-terminal amide proton. The most stable state and several meta-stable states correspond to extended conformations and typical beta-turn conformations, and their free energy values were accounted for from the potentials of mean force at the states. In addition, the contributions from the intra-molecular energies of peptides and those from the hydration effects were analyzed. Consequently, the stable beta-turn conformations in the free energy landscape were consistent with the empirically preferred beta-turn types for each amino acid sequence. The thermodynamic values for the hydration effect were decomposed and they correlated well with the empirical values estimated from the solvent accessible surface area of each molecular conformation during the trajectories. The origin of the architecture of protein local fragments was analyzed from the viewpoint of the free energy and its decomposed factors. Copyright 2000 Academic Press.

Influence of the strength of the smectic order on the backbone anisotropy of side-chain liquid crystal polymers as revealed by SANS

NASA Astrophysics Data System (ADS)

Noirez, L.; Keller, P.; Cotton, J. P.

1992-06-01

It is proposed that the strength of the smectic order determines the backbone anisotropy of side-chain liquid crystal polymers. Here this strength increases with the length of the alkyl terminal group of the mesogens. Two liquid crystal polymethacrylates differing only by the mesogenic tails —OCH3 and —OC4H9 are considered. The backbone anisotropy of these polymers is measured by small angle neutron scattering (SANS) whereas the smectic order is evaluated from the intensity of the 001 Bragg peak. Il est proposé que la qualité de l'ordre smectique détermine l'anisotropie du squelette de polymères mésomorphes en peigne confinés dans les lamelles. Ici l'ordre smectique est augmenté en allongeant le groupe alkyl terminal des mésogènes. Nous étudions deux polyméthacrylates cristal liquide qui ne différent que par leurs groupes terminaux : —OCH3 et —OC4H9. L'anisotropie du squellete est mesurée par diffusion de neutrons aux petits angles tandis que l'ordre smectique est évalué à l'aide de l'intensité du pic de Bragg 001.

Use of 13Cα Chemical-Shifts in Protein Structure Determination

PubMed Central

Vila, Jorge A.; Ripoll, Daniel R.; Scheraga, Harold A.

2008-01-01

A physics-based method, aimed at determining protein structures by using NOE-derived distances together with observed and computed 13C chemical shifts, is proposed. The approach makes use of 13Cα chemical shifts, computed at the density functional level of theory, to obtain torsional constraints for all backbone and side-chain torsional angles without making a priori use of the occupancy of any region of the Ramachandran map by the amino acid residues. The torsional constraints are not fixed but are changed dynamically in each step of the procedure, following an iterative self-consistent approach intended to identify a set of conformations for which the computed 13Cα chemical shifts match the experimental ones. A test is carried out on a 76-amino acid all-α-helical protein, namely the B. Subtilis acyl carrier protein. It is shown that, starting from randomly generated conformations, the final protein models are more accurate than an existing NMR-derived structure model of this protein, in terms of both the agreement between predicted and observed 13Cα chemical shifts and some stereochemical quality indicators, and of similar accuracy as one of the protein models solved at a high level of resolution. The results provide evidence that this methodology can be used not only for structure determination but also for additional protein structure refinement of NMR-derived models deposited in the Protein Data Bank. PMID:17516673

Site-specific protein backbone and side-chain NMR chemical shift and relaxation analysis of human vinexin SH3 domain using a genetically encoded {sup 15}N/{sup 19}F-labeled unnatural amino acid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Pan; School of Life Science, University of Science and Technology of China, Hefei, Anhui 230026; Xi, Zhaoyong

Research highlights: {yields} Chemical synthesis of {sup 15}N/{sup 19}F-trifluomethyl phenylalanine. {yields} Site-specific incorporation of {sup 15}N/{sup 19}F-trifluomethyl phenylalanine to SH3. {yields} Site-specific backbone and side chain chemical shift and relaxation analysis. {yields} Different internal motions at different sites of SH3 domain upon ligand binding. -- Abstract: SH3 is a ubiquitous domain mediating protein-protein interactions. Recent solution NMR structural studies have shown that a proline-rich peptide is capable of binding to the human vinexin SH3 domain. Here, an orthogonal amber tRNA/tRNA synthetase pair for {sup 15}N/{sup 19}F-trifluoromethyl-phenylalanine ({sup 15}N/{sup 19}F-tfmF) has been applied to achieve site-specific labeling of SH3 at threemore » different sites. One-dimensional solution NMR spectra of backbone amide ({sup 15}N){sup 1}H and side-chain {sup 19}F were obtained for SH3 with three different site-specific labels. Site-specific backbone amide ({sup 15}N){sup 1}H and side-chain {sup 19}F chemical shift and relaxation analysis of SH3 in the absence or presence of a peptide ligand demonstrated different internal motions upon ligand binding at the three different sites. This site-specific NMR analysis might be very useful for studying large-sized proteins or protein complexes.« less

A stoichiometry driven universal spatial organization of backbones of folded proteins: are there Chargaff's rules for protein folding?

PubMed

Mittal, A; Jayaram, B; Shenoy, Sandhya; Bawa, Tejdeep Singh

2010-10-01

Protein folding is at least a six decade old problem, since the times of Pauling and Anfinsen. However, rules of protein folding remain elusive till date. In this work, rigorous analyses of several thousand crystal structures of folded proteins reveal a surprisingly simple unifying principle of backbone organization in protein folding. We find that protein folding is a direct consequence of a narrow band of stoichiometric occurrences of amino-acids in primary sequences, regardless of the size and the fold of a protein. We observe that "preferential interactions" between amino-acids do not drive protein folding, contrary to all prevalent views. We dedicate our discovery to the seminal contribution of Chargaff which was one of the major keys to elucidation of the stoichiometry-driven spatially organized double helical structure of DNA.

Photophysical Properties of Pt(II) Polypyridines with Five- versus Six-Membered Chelate Rings: Trade-Offs in Angle Strain.

PubMed

Natoli, Sean N; Hight, Lauren M; Zeller, Matthias; McMillin, David R

2018-06-04

This report describes the synthesis and characterization of a series of eight [Pt(NNN)X] + complexes where the tridentate NNN ligand is (2,2'-bipyrid-6-yl)(pyrid-2-yl)sulfide (btp) or methyl(2,2'-bipyrid-6-yl)(pyrid-2-yl)amine (bmap) and X is OMe, Cl, phenylethynyl (C 2 Ph), or cyclohexylethynyl (C 2 Cy). The expectation was that inserting a heteroatom into the backbone of 2,2':6',2″-terpyridine (trpy) would expand the overall intraligand bite angle, introduce ILCT character into the excited states, and improve the photophysical properties. Crystal structures of [Pt(bmap)C 2 Ph] + and [Pt(btp)Cl] + reveal that atom insertion into the trpy backbone successfully expands the bite angle of the ligand by 8-10°. However, the impact on the photophysics is minimal. Indeed, of the eight systems investigated, only the [Pt(bmap)C 2 Ph] + and [Pt(btp)C 2 Ph] + complexes display appreciable emission in fluid solution, and they exhibit shorter emission lifetimes than [Pt(trpy)C 2 Ph] + . One reason is that the bond angle preferences of platinum and the inserted heteroatom induce the six-membered rings to deviate from planarity and adopt a boat-like conformation, impairing charge delocalization within the ligand. In addition, angle strain induces the donor atoms about platinum to assume a pseudotetrahedral arrangement, which offsets any benefit due to the increase in overall bite angle by promoting deactivation via d-d excited states. The results reveal that, in order to improve the luminescence of a [Pt(NNN)X] + system, one must take care to avoid trading one kind of angle strain for another.

Experimentally driven atomistic model of 1,2 polybutadiene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gkourmpis, Thomas, E-mail: thomas.gkourmpis@borealisgroup.com; Mitchell, Geoffrey R.; Centre for Rapid and Sustainable Product Development, Institute Polytechnic Leiria, Marinha Grande

2014-02-07

We present an efficient method of combining wide angle neutron scattering data with detailed atomistic models, allowing us to perform a quantitative and qualitative mapping of the organisation of the chain conformation in both glass and liquid phases. The structural refinement method presented in this work is based on the exploitation of the intrachain features of the diffraction pattern and its intimate linkage with atomistic models by the use of internal coordinates for bond lengths, valence angles, and torsion rotations. Atomic connectivity is defined through these coordinates that are in turn assigned by pre-defined probability distributions, thus allowing for themore » models in question to be built stochastically. Incremental variation of these coordinates allows for the construction of models that minimise the differences between the observed and calculated structure factors. We present a series of neutron scattering data of 1,2 polybutadiene at the region 120–400 K. Analysis of the experimental data yields bond lengths for Cî—¸C and C î—» C of 1.54 Å and 1.35 Å, respectively. Valence angles of the backbone were found to be at 112° and the torsion distributions are characterised by five rotational states, a three-fold trans-skew± for the backbone and gauche± for the vinyl group. Rotational states of the vinyl group were found to be equally populated, indicating a largely atactic chan. The two backbone torsion angles exhibit different behaviour with respect to temperature of their trans population, with one of them adopting an almost all trans sequence. Consequently, the resulting configuration leads to a rather persistent chain, something indicated by the value of the characteristic ratio extrapolated from the model. We compare our results with theoretical predictions, computer simulations, RIS models and previously reported experimental results.« less

Relating Trp-Glu dipeptide fluorescence to molecular conformation: the role of the discrete Chi 1 and Chi 2 angles.

PubMed

Eisenberg, Azaria Solomon; Juszczak, Laura J

2013-07-05

Molecular dynamics (MD), coupled with fluorescence data for charged dipeptides of tryptophanyl glutamic acid (Trp-Glu), reveal a detailed picture of how specific conformation affects fluorescence. Fluorescence emission spectra and time-resolved emission measurements have been collected for all four charged species. MD simulations 20 to 30 ns in length have also been carried out for the Trp-Glu species, as simulation provides aqueous phase conformational data that can be correlated with the fluorescence data. The calculations show that each dipeptide species is characterized by a similar set of six, discrete Chi 1, Chi 2 dihedral angle pairs. The preferred Chi 1 angles--60°, 180°, and 300°--play the significant role in positioning the terminal amine relative to the indole ring. A Chi 1 angle of 60° results in the arching of the backbone over the indole ring and no interaction of the ring with the terminal amine. Chi 1 values of 180° and 300° result in an extension of the backbone away from the indole ring and a NH3 cation-π interaction with indole. This interaction is believed responsible for charge transfer quenching. Two fluorescence lifetimes and their corresponding amplitudes correlate with the Chi 1 angle probability distribution for all four charged Trp-Glu dipeptides. Fluorescence emission band maxima are also consistent with the proposed pattern of terminal amine cation quenching of fluorescence. Copyright © 2013 Wiley Periodicals, Inc.

Topological side-chain classification of beta-turns: ideal motifs for peptidomimetic development.

PubMed

Tran, Tran Trung; McKie, Jim; Meutermans, Wim D F; Bourne, Gregory T; Andrews, Peter R; Smythe, Mark L

2005-08-01

Beta-turns are important topological motifs for biological recognition of proteins and peptides. Organic molecules that sample the side chain positions of beta-turns have shown broad binding capacity to multiple different receptors, for example benzodiazepines. Beta-turns have traditionally been classified into various types based on the backbone dihedral angles (phi2, psi2, phi3 and psi3). Indeed, 57-68% of beta-turns are currently classified into 8 different backbone families (Type I, Type II, Type I', Type II', Type VIII, Type VIa1, Type VIa2 and Type VIb and Type IV which represents unclassified beta-turns). Although this classification of beta-turns has been useful, the resulting beta-turn types are not ideal for the design of beta-turn mimetics as they do not reflect topological features of the recognition elements, the side chains. To overcome this, we have extracted beta-turns from a data set of non-homologous and high-resolution protein crystal structures. The side chain positions, as defined by C(alpha)-C(beta) vectors, of these turns have been clustered using the kth nearest neighbor clustering and filtered nearest centroid sorting algorithms. Nine clusters were obtained that cluster 90% of the data, and the average intra-cluster RMSD of the four C(alpha)-C(beta) vectors is 0.36. The nine clusters therefore represent the topology of the side chain scaffold architecture of the vast majority of beta-turns. The mean structures of the nine clusters are useful for the development of beta-turn mimetics and as biological descriptors for focusing combinatorial chemistry towards biologically relevant topological space.

SCit: web tools for protein side chain conformation analysis.

PubMed

Gautier, R; Camproux, A-C; Tufféry, P

2004-07-01

SCit is a web server providing services for protein side chain conformation analysis and side chain positioning. Specific services use the dependence of the side chain conformations on the local backbone conformation, which is described using a structural alphabet that describes the conformation of fragments of four-residue length in a limited library of structural prototypes. Based on this concept, SCit uses sets of rotameric conformations dependent on the local backbone conformation of each protein for side chain positioning and the identification of side chains with unlikely conformations. The SCit web server is accessible at http://bioserv.rpbs.jussieu.fr/SCit.

NMR studies of the backbone flexibility and structure of human growth hormone: a comparison of high and low pH conformations.

PubMed

Kasimova, Marina R; Kristensen, Søren M; Howe, Peter W A; Christensen, Thorkild; Matthiesen, Finn; Petersen, Jørgen; Sørensen, Hans H; Led, Jens J

2002-05-03

(15)N NMR relaxation parameters and amide (1)H/(2)H-exchange rates have been used to characterize the structural flexibility of human growth hormone (rhGH) at neutral and acidic pH. Our results show that the rigidity of the molecule is strongly affected by the solution conditions. At pH 7.0 the backbone dynamics parameters of rhGH are uniform along the polypeptide chain and their values are similar to those of other folded proteins. In contrast, at pH 2.7 the overall backbone flexibility increases substantially compared to neutral pH and the average order parameter approaches the lower limit expected for a folded protein. However, a significant variation of the backbone dynamics through the molecule indicates that under acidic conditions the mobility of the residues becomes more dependent on their location within the secondary structure units. In particular, the order parameters of certain loop regions decrease dramatically and become comparable to those found in unfolded proteins. Furthermore, the HN-exchange rates at low pH reveal that the residues most protected from exchange are clustered at one end of the helical bundle, forming a stable nucleus. We suggest that this nucleus maintains the overall fold of the protein under destabilizing conditions. We therefore conclude that the acid state of rhGH consists of a structurally conserved, but dynamically more flexible helical core surrounded by an aura of highly mobile, unstructured loops. However, in spite of its prominent flexibility the acid state of rhGH cannot be considered a "molten globule" state because of its high stability. It appears from our work that under certain conditions, a protein can tolerate a considerable increase in flexibility of its backbone, along with an increased penetration of water into its core, while still maintaining a stable folded conformation.

Membrane Curvature Sensing by Amphipathic Helices Is Modulated by the Surrounding Protein Backbone.

PubMed

Doucet, Christine M; Esmery, Nina; de Saint-Jean, Maud; Antonny, Bruno

2015-01-01

Membrane curvature is involved in numerous biological pathways like vesicle trafficking, endocytosis or nuclear pore complex assembly. In addition to its topological role, membrane curvature is sensed by specific proteins, enabling the coordination of biological processes in space and time. Amongst membrane curvature sensors are the ALPS (Amphipathic Lipid Packing Sensors). ALPS motifs are short peptides with peculiar amphipathic properties. They are found in proteins targeted to distinct curved membranes, mostly in the early secretory pathway. For instance, the ALPS motif of the golgin GMAP210 binds trafficking vesicles, while the ALPS motif of Nup133 targets nuclear pores. It is not clear if, besides curvature sensitivity, ALPS motifs also provide target specificity, or if other domains in the surrounding protein backbone are involved. To elucidate this aspect, we studied the subcellular localization of ALPS motifs outside their natural protein context. The ALPS motifs of GMAP210 or Nup133 were grafted on artificial fluorescent probes. Importantly, ALPS motifs are held in different positions and these contrasting architectures were mimicked by the fluorescent probes. The resulting chimeras recapitulated the original proteins localization, indicating that ALPS motifs are sufficient to specifically localize proteins. Modulating the electrostatic or hydrophobic content of Nup133 ALPS motif modified its avidity for cellular membranes but did not change its organelle targeting properties. In contrast, the structure of the backbone surrounding the helix strongly influenced targeting. In particular, introducing an artificial coiled-coil between ALPS and the fluorescent protein increased membrane curvature sensitivity. This coiled-coil domain also provided membrane curvature sensitivity to the amphipathic helix of Sar1. The degree of curvature sensitivity within the coiled-coil context remains correlated to the natural curvature sensitivity of the helices. This suggests that the chemistry of ALPS motifs is a key parameter for membrane curvature sensitivity, which can be further modulated by the surrounding protein backbone.

PepComposer: computational design of peptides binding to a given protein surface

PubMed Central

Obarska-Kosinska, Agnieszka; Iacoangeli, Alfredo; Lepore, Rosalba; Tramontano, Anna

2016-01-01

There is a wide interest in designing peptides able to bind to a specific region of a protein with the aim of interfering with a known interaction or as starting point for the design of inhibitors. Here we describe PepComposer, a new pipeline for the computational design of peptides binding to a given protein surface. PepComposer only requires the target protein structure and an approximate definition of the binding site as input. We first retrieve a set of peptide backbone scaffolds from monomeric proteins that harbor the same backbone arrangement as the binding site of the protein of interest. Next, we design optimal sequences for the identified peptide scaffolds. The method is fully automatic and available as a web server at http://biocomputing.it/pepcomposer/webserver. PMID:27131789

On contribution of known atomic partial charges of protein backbone in electrostatic potential density maps

PubMed Central

2017-01-01

Abstract Partial charges of atoms in a molecule and electrostatic potential (ESP) density for that molecule are known to bear a strong correlation. In order to generate a set of point‐field force field parameters for molecular dynamics, Kollman and coworkers have extracted atomic partial charges for each of all 20 amino acids using restrained partial charge‐fitting procedures from theoretical ESP density obtained from condensed‐state quantum mechanics. The magnitude of atomic partial charges for neutral peptide backbone they have obtained is similar to that of partial atomic charges for ionized carboxylate side chain atoms. In this study, the effect of these known atomic partial charges on ESP is examined using computer simulations and compared with the experimental ESP density recently obtained for proteins using electron microscopy. It is found that the observed ESP density maps are most consistent with the simulations that include atomic partial charges of protein backbone. Therefore, atomic partial charges are integral part of atomic properties in protein molecules and should be included in model refinement. PMID:28370507

Thermodynamic contribution of backbone conformational entropy in the binding between SH3 domain and proline-rich motif.

PubMed

Zeng, Danyun; Shen, Qingliang; Cho, Jae-Hyun

2017-02-26

Biological functions of intrinsically disordered proteins (IDPs), and proteins containing intrinsically disordered regions (IDRs) are often mediated by short linear motifs, like proline-rich motifs (PRMs). Upon binding to their target proteins, IDPs undergo a disorder-to-order transition which is accompanied by a large conformational entropy penalty. Hence, the molecular mechanisms underlying control of conformational entropy are critical for understanding the binding affinity and selectivity of IDPs-mediated protein-protein interactions (PPIs). Here, we investigated the backbone conformational entropy change accompanied by binding of the N-terminal SH3 domain (nSH3) of CrkII and PRM derived from guanine nucleotide exchange factor 1 (C3G). In particular, we focused on the estimation of conformational entropy change of disordered PRM upon binding to the nSH3 domain. Quantitative characterization of conformational dynamics of disordered peptides like PRMs is limited. Hence, we combined various methods, including NMR model-free analysis, δ2D, DynaMine, and structure-based calculation of entropy loss. This study demonstrates that the contribution of backbone conformational entropy change is significant in the PPIs mediated by IDPs/IDRs. Copyright © 2017 Elsevier Inc. All rights reserved.

Study on fluorescence of Maillard reaction compounds in breakfast cereals.

PubMed

Delgado-Andrade, Cristina; Rufián-Henares, José A; Morales, Francisco J

2006-09-01

During the advanced stage of the Maillard reaction (MR) in food processing and cooking, Amadori rearrangement products undergo dehydration and fission and fluorescent substances are formed. Free and total (free + linked to the protein backbone) fluorescence (FIC) due to Maillard compounds in 60 commercial breakfast cereals was evaluated. Pronase was used for efficient release of linked fluorescent Maillard compounds from the protein backbone. Results were correlated with some heat-induced markers of the extent of the MR or sugar caramelisation during cereal processing, such as hydroxymethylfurfural, furfural, glucosilisomaltol and furosine. The effect of sample composition (dietary-fibre added, protein, etc.) on levels of FIC, expressed as fluorescence intensity (FI) per milligram of sample, is discussed. FIC is significantly correlated to the protein content of the sample and fluorescent Maillard compounds are mainly linked to the protein backbone. The ratio of total-FIC to free-FIC was 10.4-fold for corn-based, wheat-based and multicereal-based breakfast cereals but significantly higher in rice-based samples. Addition of dietary fibre or honey increased the FIC values. Data support the usefulness of FIC measurement as an unspecific heat-induced marker in breakfast cereals.

Development of a new physics-based internal coordinate mechanics force field and its application to protein loop modeling.

PubMed

Arnautova, Yelena A; Abagyan, Ruben A; Totrov, Maxim

2011-02-01

We report the development of internal coordinate mechanics force field (ICMFF), new force field parameterized using a combination of experimental data for crystals of small molecules and quantum mechanics calculations. The main features of ICMFF include: (a) parameterization for the dielectric constant relevant to the condensed state (ε = 2) instead of vacuum, (b) an improved description of hydrogen-bond interactions using duplicate sets of van der Waals parameters for heavy atom-hydrogen interactions, and (c) improved backbone covalent geometry and energetics achieved using novel backbone torsional potentials and inclusion of the bond angles at the C(α) atoms into the internal variable set. The performance of ICMFF was evaluated through loop modeling simulations for 4-13 residue loops. ICMFF was combined with a solvent-accessible surface area solvation model optimized using a large set of loop decoys. Conformational sampling was carried out using the biased probability Monte Carlo method. Average/median backbone root-mean-square deviations of the lowest energy conformations from the native structures were 0.25/0.21 Å for four residues loops, 0.84/0.46 Å for eight residue loops, and 1.16/0.73 Å for 12 residue loops. To our knowledge, these results are significantly better than or comparable with those reported to date for any loop modeling method that does not take crystal packing into account. Moreover, the accuracy of our method is on par with the best previously reported results obtained considering the crystal environment. We attribute this success to the high accuracy of the new ICM force field achieved by meticulous parameterization, to the optimized solvent model, and the efficiency of the search method. © 2010 Wiley-Liss, Inc.

Conformational variety for the ansa chain of rifamycins: Comparison of observed crystal structures and molecular dynamics simulations

NASA Astrophysics Data System (ADS)

Bacchi, Alessia; Pelizzi, Giancarlo

1999-07-01

The antibiotic activity (via inhibition of DNA-dependent RNA polymerase, DDRP) of rifamycins has been correlated to the conformation of the ansa chain, which can be described by means of 17 torsion angles defined along the ansa backbone. It has been shown that favourable or unfavourable conformations of the ansa chain in rifamycin crystals are generally diagnostic of activity or inactivity against isolated DDRP. The principles of structure correlation suggest that the torsional variety observed in rifamycin crystals should mimic the dynamic flexibility of the ansa chain in solution. Twenty-six crystal structures of rifamycins are grouped into two classes (active and non-active). For each class the variance of the 17 ansa backbone torsion angles is analysed. Active compounds show a well-defined common pattern, while non-active molecules are more scattered, mainly due to steric constraints forcing the molecules into unfavourable conformations. The experimental distributions of torsion angles are compared to the torsional freedom of the ansa chain simulated by molecular dynamics calculations performed at different temperatures and conditions on rifamycin S and rifamycin O, which represent a typical active and a typical sterically constrained molecule, respectively. It is shown that the torsional variety found in the crystalline state samples the dynamic behaviour of the ansa chain for active compounds. The methods of circular statistics are illustrated to describe torsion angle distributions.

Effect of Backbone Chemistry on the Structure of Polyurea Films Deposited by Molecular Layer Deposition

DOE PAGES

Bergsman, David S.; Closser, Richard G.; Tassone, Christopher J.; ...

2017-01-01

An experimental investigation into the growth of polyurea films by molecular layer deposition was performed by examining trends in the growth rate, crystallinity, and orientation of chains as a function of backbone flexibility. Growth curves obtained for films containing backbones of aliphatic and phenyl groups indicate that an increase in backbone flexibility leads to a reduction in growth rate from 4 to 1 Å/cycle. Crystallinity measurements collected using grazing incidence X-ray diffraction and Fourier transform infrared spectroscopy suggest that some chains form paracrystalline, out-of-plane stacks of polymer segments with packing distances ranging from 4.4 to 3.7 Å depending on themore » monomer size. Diffraction intensity is largely a function of the homogeneity of the backbone. Near-edge X-ray absorption fine structure measurements for thin and thick samples show an average chain orientation of ~25° relative to the substrate across all samples, suggesting that changes in growth rate are not caused by differences in chain angle but instead may be caused by differences in the frequency of chain terminations. In conclusion, these results suggest a model of molecular layer deposition-based chain growth in which films consist of a mixture of upward growing chains and horizontally aligned layers of paracrystalline polymer segments.« less

Charge transport properties of poly(dA)-poly(dT) DNA in variation of backbone disorder and amplitude of base-pair twisting motion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rahmi, Kinanti Aldilla, E-mail: kinanti.aldilla@ui.ac.id; Yudiarsah, Efta

By using tight binding Hamiltonian model, charge transport properties of poly(dA)-poly(dT) DNA in variation of backbone disorder and amplitude of base-pair twisting motion is studied. The DNA chain used is 32 base pairs long poly(dA)-poly(dT) molecule. The molecule is contacted to electrode at both ends. The influence of environment on charge transport in DNA is modeled as variation of backbone disorder. The twisting motion amplitude is taking into account by assuming that the twisting angle distributes following Gaussian distribution function with zero average and standard deviation proportional to square root of temperature and inversely proportional to the twisting motion frequency.more » The base-pair twisting motion influences both the onsite energy of the bases and electron hopping constant between bases. The charge transport properties are studied by calculating current using Landauer-Buttiker formula from transmission probabilities which is calculated by transfer matrix methods. The result shows that as the backbone disorder increases, the maximum current decreases. By decreasing the twisting motion frequency, the current increases rapidly at low voltage, but the current increases slower at higher voltage. The threshold voltage can increase or decrease with increasing backbone disorder and increasing twisting frequency.« less

Urea-mediated protein denaturation: a consensus view.

PubMed

Das, Atanu; Mukhopadhyay, Chaitali

2009-09-24

We have performed all-atom molecular dynamics simulations of three structurally similar small globular proteins in 8 M urea and compared the results with pure aqueous simulations. Protein denaturation is preceded by an initial loss of water from the first solvation shell and consequent in-flow of urea toward the protein. Urea reaches the first solvation shell of the protein mainly due to electrostatic interaction with a considerable contribution coming from the dispersion interaction. Urea shifts the equilibrium from the native to denatured ensemble by making the protein-protein contact less stable than protein-urea contact, which is just the reverse of the condition in pure water, where protein-protein contact is more stable than protein-water contact. We have also seen that water follows urea and reaches the protein interior at later stages of denaturation, while urea preferentially and efficiently solvates different parts of the protein. Solvation of the protein backbone via hydrogen bonding, favorable electrostatic interaction with hydrophilic residues, and dispersion interaction with hydrophobic residues are the key steps through which urea intrudes the core of the protein and denatures it. Why urea is preferred over water for binding to the protein backbone and how urea orients itself toward the protein backbone have been identified comprehensively. All the key components of intermolecular forces are found to play a significant part in urea-induced protein denaturation and also toward the stability of the denatured state ensemble. Changes in water network/structure and dynamical properties and higher degree of solvation of the hydrophobic residues validate the presence of "indirect mechanism" along with the "direct mechanism" and reinforce the effect of urea on protein.

Complementarity of stability patches at the interfaces of protein complexes: Implication for the structural organization of energetic hot spots.

PubMed

Kuttner, Yosef Y; Engel, Stanislav

2018-02-01

A rational design of protein complexes with defined functionalities and of drugs aimed at disrupting protein-protein interactions requires fundamental understanding of the mechanisms underlying the formation of specific protein complexes. Efforts to develop efficient small-molecule or protein-based binders often exploit energetic hot spots on protein surfaces, namely, the interfacial residues that provide most of the binding free energy in the complex. The molecular basis underlying the unusually high energy contribution of the hot spots remains obscure, and its elucidation would facilitate the design of interface-targeted drugs. To study the nature of the energetic hot spots, we analyzed the backbone dynamic properties of contact surfaces in several protein complexes. We demonstrate that, in most complexes, the backbone dynamic landscapes of interacting surfaces form complementary "stability patches," in which static areas from the opposing surfaces superimpose, and that these areas are predominantly located near the geometric center of the interface. We propose that a diminished enthalpy-entropy compensation effect augments the degree to which residues positioned within the complementary stability patches contribute to complex affinity, thereby giving rise to the energetic hot spots. These findings offer new insights into the nature of energetic hot spots and the role that backbone dynamics play in facilitating intermolecular recognition. Mapping the interfacial stability patches may provide guidance for protein engineering approaches aimed at improving the stability of protein complexes and could facilitate the design of ligands that target complex interfaces. © 2017 Wiley Periodicals, Inc.

Local Structure and Ion Transport in Glassy Poly(ethylene oxide styrene) Copolymers

NASA Astrophysics Data System (ADS)

Yang, Han-Chang; Mays, Jimmy; Sokolov, Alexei P.; Winey, Karen I.

2014-03-01

Polymer electrolytes have attracted attention for a wide variety of applications in energy production such as lithium-ion batteries and fuel cells. The concept of free volume provides important information about ion mobility and chain dynamics in the polymer matrix. Researchers have recently demonstrated that ion transport in glassy polymer can be improved by designing a system with high free volume. We have studied the effect of temperature and humidity on the intermolecular correlations of poly(ethylene oxide styrene-block-styrene) (PEOSt- b-St) block copolymer and poly(ethylene oxide styrene) (PEOSt) homopolymer using in situ multi-angle x-ray scattering across a wide range of scattering angles (q = 0.007-1.5 Å-1) . An increase in backbone-to-backbone distance is observed, indicating an increase in free volume between different polymer main chains. Structural characterization of the polymer segments will be discussed together with conductivity and dielectric results to better understand the ion transport mechanism in the local environment of the polymer system. Department of Chemistry, University of Tennessee.

Probing the Action of Chemical Denaturant on an Intrinsically Disordered Protein by Simulation and Experiment.

PubMed

Zheng, Wenwei; Borgia, Alessandro; Buholzer, Karin; Grishaev, Alexander; Schuler, Benjamin; Best, Robert B

2016-09-14

Chemical denaturants are the most commonly used agents for unfolding proteins and are thought to act by better solvating the unfolded state. Improved solvation is expected to lead to an expansion of unfolded chains with increasing denaturant concentration, providing a sensitive probe of the denaturant action. However, experiments have so far yielded qualitatively different results concerning the effects of chemical denaturation. Studies using Förster resonance energy transfer (FRET) and other methods found an increase in radius of gyration with denaturant concentration, but most small-angle X-ray scattering (SAXS) studies found no change. This discrepancy therefore challenges our understanding of denaturation mechanism and more generally the accuracy of these experiments as applied to unfolded or disordered proteins. Here, we use all-atom molecular simulations to investigate the effect of urea and guanidinium chloride on the structure of the intrinsically disordered protein ACTR, which can be studied by experiment over a wide range of denaturant concentration. Using unbiased molecular simulations with a carefully calibrated denaturant model, we find that the protein chain indeed swells with increasing denaturant concentration. This is due to the favorable association of urea or guanidinium chloride with the backbone of all residues and with the side-chains of almost all residues, with denaturant-water transfer free energies inferred from this association in reasonable accord with experimental estimates. Interactions of the denaturants with the backbone are dominated by hydrogen bonding, while interactions with side-chains include other contributions. By computing FRET efficiencies and SAXS intensities at each denaturant concentration, we show that the simulation trajectories are in accord with both experiments on this protein, demonstrating that there is no fundamental inconsistency between the two types of experiment. Agreement with experiment also supports the picture of chemical denaturation described in our simulations, driven by weak association of denaturant with the protein. Our simulations support some assumptions needed for each experiment to accurately reflect changes in protein size, namely, that the commonly used FRET chromophores do not qualitatively alter the results and that possible effects such as preferential solvent partitioning into the interior of the chain do not interfere with the determination of radius of gyration from the SAXS experiments.

Protein-Backbone Thermodynamics across the Membrane Interface.

PubMed

Bereau, Tristan; Kremer, Kurt

2016-07-07

The thermodynamics of insertion of a protein in a membrane depends on the fine interplay between backbone and side-chain contributions interacting with the lipid environment. Using computer simulations, we probe how different descriptions of the backbone glycyl unit affect the thermodynamics of insertion of individual residues, dipeptides, and entire transmembrane helices. Due to the lack of reference data, we first introduce an efficient methodology to estimate atomistic potential of mean force (PMF) curves from a series of representative and uncorrelated coarse-grained (CG) snapshots. We find strong discrepancies between two CG models, Martini and PLUM, against reference atomistic PMFs and experiments. Atomistic simulations suggest a weak free energy of insertion between water and a POPC membrane for the glycyl unit, in overall agreement with experimental results despite severe assumptions in our calculations. We show that refining the backbone contribution in PLUM significantly improves the PMF of insertion of the WALP16 transmembrane peptide. An improper balance between the glycyl backbone and the attached side chain will lead to energetic artifacts, rationalizing Martini's overstabilization of WALP's adsorbed interfacial state. It illustrates difficulties associated with free-energy-based parametrizations of single-residue models, as the relevant free energy of partitioning used for force-field parametrization does not arise from the entire residue but rather the solvent-accessible chemical groups.

SCit: web tools for protein side chain conformation analysis

PubMed Central

Gautier, R.; Camproux, A.-C.; Tufféry, P.

2004-01-01

SCit is a web server providing services for protein side chain conformation analysis and side chain positioning. Specific services use the dependence of the side chain conformations on the local backbone conformation, which is described using a structural alphabet that describes the conformation of fragments of four-residue length in a limited library of structural prototypes. Based on this concept, SCit uses sets of rotameric conformations dependent on the local backbone conformation of each protein for side chain positioning and the identification of side chains with unlikely conformations. The SCit web server is accessible at http://bioserv.rpbs.jussieu.fr/SCit. PMID:15215438

Insights into positive and negative requirements for protein-protein interactions by crystallographic analysis of the beta-lactamase inhibitory proteins BLIP, BLIP-I, and BLP.

PubMed

Gretes, Michael; Lim, Daniel C; de Castro, Liza; Jensen, Susan E; Kang, Sung Gyun; Lee, Kye Joon; Strynadka, Natalie C J

2009-06-05

Beta-lactamase inhibitory protein (BLIP) binds a variety of beta-lactamase enzymes with wide-ranging specificity. Its binding mechanism and interface interactions are a well-established model system for the characterization of protein-protein interactions. Published studies have examined the binding of BLIP to diverse target beta-lactamases (e.g., TEM-1, SME-1, and SHV-1). However, apart from point mutations of amino acid residues, variability on the inhibitor side of this enzyme-inhibitor interface has remained unexplored. Thus, we present crystal structures of two likely BLIP relatives: (1) BLIP-I (solved alone and in complex with TEM-1), which has beta-lactamase inhibitory activity very similar to that of BLIP; and (2) beta-lactamase-inhibitory-protein-like protein (BLP) (in two apo forms, including an ultra-high-resolution structure), which is unable to inhibit any tested beta-lactamase. Despite categorical differences in species of origin and function, BLIP-I and BLP share nearly identical backbone conformations, even at loop regions differing in BLIP. We describe interacting residues and provide a comparative structural analysis of the interactions formed at the interface of BLIP-I.TEM-1 versus those formed at the interface of BLIP.TEM-1. Along with initial attempts to functionally characterize BLP, we examine its amino acid residues that structurally correspond to BLIP/BLIP-I binding hotspots to explain its inability to bind and inhibit TEM-1. We conclude that the BLIP family fold is a robust and flexible scaffold that permits the formation of high-affinity protein-protein interactions while remaining highly selective. Comparison of the two naturally occurring, distinct binding interfaces built upon this scaffold (BLIP and BLIP-I) shows that there is substantial variation possible in the subnanomolar binding interaction with TEM-1. The corresponding (non-TEM-1-binding) BLP surface shows that numerous favorable backbone-backbone/backbone-side-chain interactions with a protein partner can be negated by the presence of a few, strongly unfavorable interactions, especially electrostatic repulsions.

Structure of the Bacillus subtilis phage SPO1-encoded type II DNA-binding protein TF1 in solution.

PubMed

Jia, X; Grove, A; Ivancic, M; Hsu, V L; Geiduscheck, E P; Kearns, D R

1996-10-25

The solution structure of a type II DNA-binding protein, the bacteriophage SPO1-encoded transcription factor 1 (TF1), was determined using NMR spectroscopy. Selective 2H-labeling, 13C-labeling and isotopic heterodimers were used to distinguish contacts between and within monomers of the dimeric protein. A total of 1914 distance and dihedral angle constraints derived from NMR experiments were used in structure calculations using restrained molecular dynamics and simulated annealing protocols. The ensemble of 30 calculated structures has a root-mean-square deviation (r.m.s.d.) of 0.9 A, about the average structure for the backbone atoms, and 1.2 A for all heavy-atoms of the dimeric core (helices 1 and 2) and the beta-sheets. A severe helix distortion at residues 92-93 in the middle of helix 3 is associated with r.m.s.d. of approximately 1.5 A for the helix 3 backbone. Deviations of approximately 5 A or larger are noted for the very flexible beta-ribbon arms that constitute part of a proposed DNA-binding region. A structural model of TF1 has been calculated based on the previously reported crystal structure of the homologous HU protein and this model was used as the starting structure for calculations. A comparison between the calculated average solution structure of TF1 and a solution structure of HU indicates a similarity in the dimeric core (excluding the nine amino acid residue tail) with pairwise deviations of 2 to 3 A. The largest deviations between the average structure and the HU solution structure were found in the beta-ribbon arms, as expected. A 4 A deviation is found at residue 15 of TF1 which is in a loop connecting two helical segments; it has been reported that substitution of Glu15 by Gly increases the thermostability of TF1. The homology between TF1 and other proteins of this family leads us to anticipate similar tertiary structures.

Solution structure of a lipid transfer protein extracted from rice seeds. Comparison with homologous proteins.

PubMed

Poznanski, J; Sodano, P; Suh, S W; Lee, J Y; Ptak, M; Vovelle, F

1999-02-01

Nuclear magnetic resonance (NMR) spectroscopy was used to determine the three dimensional structure of rice nonspecific lipid transfer protein (ns-LTP), a 91 amino acid residue protein belonging to the broad family of plant ns-LTP. Sequence specific assignment was obtained for all but three HN backbone 1H resonances and for more than 95% of the 1H side-chain resonances using a combination of 1H 2D NOESY; TOCSY and COSY experiments at 293 K. The structure was calculated on the basis of four disulfide bridge restraints, 1259 distance constraints derived from 1H-1H Overhauser effects, 72 phi angle restraints and 32 hydrogen-bond restraints. The final solution structure involves four helices (H1: Cys3-Arg18, H2: Ala25-Ala37, H3: Thr41-Ala54 and H4: Ala66-Cys73) followed by a long C-terminal tail (T) with no observable regular structure. N-capping residues (Thr2, Ser24, Thr40), whose side-chain oxygen atoms are involved in hydrogen bonds with i + 3 amide proton additionally stabilize the N termini of the first three helices. The fourth helix involving Pro residues display a mixture of alpha and 3(10) conformation. The rms deviation of 14 final structures with respect to the average structure is 1.14 +/- 0.16 A for all heavy atoms (C, N, O and S) and 0.72 +/- 0.01 A for the backbone atoms. The global fold of rice ns-LTP is close to the previously published structures of wheat, barley and maize ns-LTPs exhibiting nearly identical pattern of the numerous sequence specific interactions. As reported previously for different four-helix topology proteins, hydrophobic, hydrogen bonding and electrostatic mechanisms of fold stabilization were found for the rice ns-LTP. The sequential alignment of 36 ns-LTP primary structures strongly suggests that there is a uniform pattern of specific long-range interactions (in terms of sequence), which stabilize the fold of all plant ns-LTPs.

Dioxaphosphorinane-constrained nucleic Acid dinucleotides as tools for structural tuning of nucleic acids.

PubMed

Catana, Dan-Andrei; Renard, Brice-Loïc; Maturano, Marie; Payrastre, Corinne; Tarrat, Nathalie; Escudier, Jean-Marc

2012-01-01

We describe a rational approach devoted to modulate the sugar-phosphate backbone geometry of nucleic acids. Constraints were generated by connecting one oxygen of the phosphate group to a carbon of the sugar moiety. The so-called dioxaphosphorinane rings were introduced at key positions along the sugar-phosphate backbone allowing the control of the six-torsion angles α to ζ defining the polymer structure. The syntheses of all the members of the D-CNA family are described, and we emphasize the effect on secondary structure stabilization of a couple of diastereoisomers of α,β-D-CNA exhibiting wether B-type canonical values or not.

From protein sequence to dynamics and disorder with DynaMine.

PubMed

Cilia, Elisa; Pancsa, Rita; Tompa, Peter; Lenaerts, Tom; Vranken, Wim F

2013-01-01

Protein function and dynamics are closely related; however, accurate dynamics information is difficult to obtain. Here based on a carefully assembled data set derived from experimental data for proteins in solution, we quantify backbone dynamics properties on the amino-acid level and develop DynaMine--a fast, high-quality predictor of protein backbone dynamics. DynaMine uses only protein sequence information as input and shows great potential in distinguishing regions of different structural organization, such as folded domains, disordered linkers, molten globules and pre-structured binding motifs of different sizes. It also identifies disordered regions within proteins with an accuracy comparable to the most sophisticated existing predictors, without depending on prior disorder knowledge or three-dimensional structural information. DynaMine provides molecular biologists with an important new method that grasps the dynamical characteristics of any protein of interest, as we show here for human p53 and E1A from human adenovirus 5.

Solid state nuclear magnetic resonance investigation of polymer backbone dynamics in poly(ethylene oxide) based lithium and sodium polyether-ester-sulfonate ionomers.

PubMed

Roach, David J; Dou, Shichen; Colby, Ralph H; Mueller, Karl T

2013-05-21

Polymer backbone dynamics of single ion conducting poly(ethylene oxide) (PEO)-based ionomer samples with low glass transition temperatures (T(g)) have been investigated using solid-state nuclear magnetic resonance. Experiments detecting (13)C with (1)H decoupling under magic angle spinning (MAS) conditions identified the different components of the polymer backbone (PEO spacer and isophthalate groups) and their relative mobilities for a suite of lithium- and sodium-containing ionomer samples with varying cation contents. Variable temperature (203-373 K) (1)H-(13)C cross-polarization MAS (CP-MAS) experiments also provided qualitative assessment of the differences in the motions of the polymer backbone components as a function of cation content and identity. Each of the main backbone components exhibit distinct motions, following the trends expected for motional characteristics based on earlier Quasi Elastic Neutron Scattering and (1)H spin-lattice relaxation rate measurements. Previous (1)H and (7)Li spin-lattice relaxation measurements focused on both the polymer backbone and cation motion on the nanosecond timescale. The studies presented here assess the slower timescale motion of the polymer backbone allowing for a more comprehensive understanding of the polymer dynamics. The temperature dependences of (13)C linewidths were used to both qualitatively and quantitatively examine the effects of cation content and identity on PEO spacer mobility. Variable contact time (1)H-(13)C CP-MAS experiments were used to further assess the motions of the polymer backbone on the microsecond timescale. The motion of the PEO spacer, reported via the rate of magnetization transfer from (1)H to (13)C nuclei, becomes similar for T≳1.1 T(g) in all ionic samples, indicating that at similar elevated reduced temperatures the motions of the polymer backbones on the microsecond timescale become insensitive to ion interactions. These results present an improved picture, beyond those of previous findings, for the dependence of backbone dynamics on cation density (and here, cation identity as well) in these amorphous PEO-based ionomer systems.

Residue-Specific α-Helix Propensities from Molecular Simulation

PubMed Central

Best, Robert B.; de Sancho, David; Mittal, Jeetain

2012-01-01

Formation of α-helices is a fundamental process in protein folding and assembly. By studying helix formation in molecular simulations of a series of alanine-based peptides, we obtain the temperature-dependent α-helix propensities of all 20 naturally occurring residues with two recent additive force fields, Amber ff03w and Amber ff99SB∗. Encouragingly, we find that the overall helix propensity of many residues is captured well by both energy functions, with Amber ff99SB∗ being more accurate. Nonetheless, there are some residues that deviate considerably from experiment, which can be attributed to two aspects of the energy function: i), variations of the charge model used to determine the atomic partial charges, with residues whose backbone charges differ most from alanine tending to have the largest error; ii), side-chain torsion potentials, as illustrated by the effect of modifications to the torsion angles of I, L, D, N. We find that constrained refitting of residue charges for charged residues in Amber ff99SB∗ significantly improves their helix propensity. The resulting parameters should more faithfully reproduce helix propensities in simulations of protein folding and disordered proteins. PMID:22455930

Protein Side-Chain Resonance Assignment and NOE Assignment Using RDC-Defined Backbones without TOCSY Data3

PubMed Central

Zeng, Jianyang; Zhou, Pei; Donald, Bruce Randall

2011-01-01

One bottleneck in NMR structure determination lies in the laborious and time-consuming process of side-chain resonance and NOE assignments. Compared to the well-studied backbone resonance assignment problem, automated side-chain resonance and NOE assignments are relatively less explored. Most NOE assignment algorithms require nearly complete side-chain resonance assignments from a series of through-bond experiments such as HCCH-TOCSY or HCCCONH. Unfortunately, these TOCSY experiments perform poorly on large proteins. To overcome this deficiency, we present a novel algorithm, called NASCA (NOE Assignment and Side-Chain Assignment), to automate both side-chain resonance and NOE assignments and to perform high-resolution protein structure determination in the absence of any explicit through-bond experiment to facilitate side-chain resonance assignment, such as HCCH-TOCSY. After casting the assignment problem into a Markov Random Field (MRF), NASCA extends and applies combinatorial protein design algorithms to compute optimal assignments that best interpret the NMR data. The MRF captures the contact map information of the protein derived from NOESY spectra, exploits the backbone structural information determined by RDCs, and considers all possible side-chain rotamers. The complexity of the combinatorial search is reduced by using a dead-end elimination (DEE) algorithm, which prunes side-chain resonance assignments that are provably not part of the optimal solution. Then an A* search algorithm is employed to find a set of optimal side-chain resonance assignments that best fit the NMR data. These side-chain resonance assignments are then used to resolve the NOE assignment ambiguity and compute high-resolution protein structures. Tests on five proteins show that NASCA assigns resonances for more than 90% of side-chain protons, and achieves about 80% correct assignments. The final structures computed using the NOE distance restraints assigned by NASCA have backbone RMSD 0.8 – 1.5 Å from the reference structures determined by traditional NMR approaches. PMID:21706248

Reduced dimensionality tailored HN(C)N experiments for facile backbone resonance assignment of proteins through unambiguous identification of sequential HSQC peaks

NASA Astrophysics Data System (ADS)

Kumar, Dinesh

2013-12-01

Two novel reduced dimensionality (RD) tailored HN(C)N [S.C. Panchal, N.S. Bhavesh, R.V. Hosur, Improved 3D triple resonance experiments, HNN and HN(C)N, for HN and 15N sequential correlations in (13C, 15N) labeled proteins: application to unfolded proteins, J. Biomol. NMR 20 (2001) 135-147] experiments are proposed to facilitate the backbone resonance assignment of proteins both in terms of its accuracy and speed. These experiments - referred here as (4,3)D-hNCOcaNH and (4,3)D-hNcoCANH - exploit the linear combination of backbone 15N and 13C‧/13Cα chemical shifts simultaneously to achieve higher peak dispersion and randomness along their respective F1 dimensions. Simply, this has been achieved by modulating the backbone 15N(i) chemical shifts with that of 13C‧ (i - 1)/13Cα (i - 1) spins following the established reduced dimensionality NMR approach [T. Szyperski, D.C. Yeh, D.K. Sukumaran, H.N. Moseley, G.T. Montelione, Reduced-dimensionality NMR spectroscopy for high-throughput protein resonance assignment, Proc. Natl. Acad. Sci. USA 99 (2002) 8009-8014]. Though the modification is simple it has resulted an ingenious improvement of HN(C)N both in terms of peak dispersion and easiness of establishing the sequential connectivities. The increased dispersion along F1 dimension solves two purposes here: (i) resolves the ambiguities arising because of degenerate 15N chemical shifts and (ii) reduces the signal overlap in F2(15N)-F3(1H) planes (an important requisite in HN(C)N based assignment protocol for facile and unambiguous identification of sequentially connected HSQC peaks). The performance of both these experiments and the assignment protocol has been demonstrated using bovine apo Calbindin-d9k (75 aa) and urea denatured UNC60B (a 152 amino acid ADF/cofilin family protein of Caenorhabditis elegans), as representatives of folded and unfolded protein systems, respectively.

LoopX: A Graphical User Interface-Based Database for Comprehensive Analysis and Comparative Evaluation of Loops from Protein Structures.

PubMed

Kadumuri, Rajashekar Varma; Vadrevu, Ramakrishna

2017-10-01

Due to their crucial role in function, folding, and stability, protein loops are being targeted for grafting/designing to create novel or alter existing functionality and improve stability and foldability. With a view to facilitate a thorough analysis and effectual search options for extracting and comparing loops for sequence and structural compatibility, we developed, LoopX a comprehensively compiled library of sequence and conformational features of ∼700,000 loops from protein structures. The database equipped with a graphical user interface is empowered with diverse query tools and search algorithms, with various rendering options to visualize the sequence- and structural-level information along with hydrogen bonding patterns, backbone φ, ψ dihedral angles of both the target and candidate loops. Two new features (i) conservation of the polar/nonpolar environment and (ii) conservation of sequence and conformation of specific residues within the loops have also been incorporated in the search and retrieval of compatible loops for a chosen target loop. Thus, the LoopX server not only serves as a database and visualization tool for sequence and structural analysis of protein loops but also aids in extracting and comparing candidate loops for a given target loop based on user-defined search options.

Populations of the Minor α-Conformation in AcGXGNH2 and the α-Helical Nucleation Propensities

NASA Astrophysics Data System (ADS)

Zhou, Yanjun; He, Liu; Zhang, Wenwen; Hu, Jingjing; Shi, Zhengshuang

2016-06-01

Intrinsic backbone conformational preferences of different amino acids are important for understanding the local structure of unfolded protein chains. Recent evidence suggests α-structure is relatively minor among three major backbone conformations for unfolded proteins. The α-helices are the dominant structures in many proteins. For these proteins, how could the α-structures occur from the least in unfolded to the most in folded states? Populations of the minor α-conformation in model peptides provide vital information. Reliable determination of populations of the α-conformers in these peptides that exist in multiple equilibriums of different conformations remains a challenge. Combined analyses on data from AcGXPNH2 and AcGXGNH2 peptides allow us to derive the populations of PII, β and α in AcGXGNH2. Our results show that on average residue X in AcGXGNH2 adopt PII, β, and α 44.7%, 44.5% and 10.8% of time, respectively. The contents of α-conformations for different amino acids define an α-helix nucleation propensity scale. With derived PII, β and α-contents, we can construct a free energy-conformation diagram on each AcGXGNH2 in aqueous solution for the three major backbone conformations. Our results would have broad implications on early-stage events of protein folding.

Are Long-Range Structural Correlations Behind the Aggregration Phenomena of Polyglutamine Diseases?

PubMed Central

Moradi, Mahmoud; Babin, Volodymyr; Roland, Christopher; Sagui, Celeste

2012-01-01

We have characterized the conformational ensembles of polyglutamine peptides of various lengths (ranging from to ), both with and without the presence of a C-terminal polyproline hexapeptide. For this, we used state-of-the-art molecular dynamics simulations combined with a novel statistical analysis to characterize the various properties of the backbone dihedral angles and secondary structural motifs of the glutamine residues. For (i.e., just above the pathological length for Huntington's disease), the equilibrium conformations of the monomer consist primarily of disordered, compact structures with non-negligible -helical and turn content. We also observed a relatively small population of extended structures suitable for forming aggregates including - and -strands, and - and -hairpins. Most importantly, for we find that there exists a long-range correlation (ranging for at least residues) among the backbone dihedral angles of the Q residues. For polyglutamine peptides below the pathological length, the population of the extended strands and hairpins is considerably smaller, and the correlations are short-range (at most residues apart). Adding a C-terminal hexaproline to suppresses both the population of these rare motifs and the long-range correlation of the dihedral angles. We argue that the long-range correlation of the polyglutamine homopeptide, along with the presence of these rare motifs, could be responsible for its aggregation phenomena. PMID:22577357

Characterization of a backbone cleavage product of BMS-196854 (Oncostatin M), a recombinant anti-inflammatory cytokine.

PubMed

Zhao, F; Stein, D J; Paborji, M; Cash, P W; Root, B J; Wei, Z; Knupp, C J

2001-01-01

BMS-196843 (Oncostatin M) is a therapeutic recombinant protein in development. Scale-up process changes led to unexpected instability of the bulk drug substance solution during storage. A product with an apparent higher MW than the parent protein was observed by the size-exclusion chromatography (SEC). This study was aimed to fully characterize the product and to identify a solution to stabilize the protein. SEC, SDS-PAGE, tryptic mapping, and N-terminal sequencing were performed to characterize the unknown product. The effect of pH, temperature, bulk concentration, and immobilized trypsin inhibitor on the degradation rate was studied to elucidate the mechanism and to identify stabilization strategies. Despite the apparent high MW indicated initially by SEC, the unknown was characterized to be a degradation product resulted from a backbone cleavage between residues Arg145-Gly146. The resulting fragments from the backbone cleavage were, however, still linked through an intramolecular disulfide bond. Thus, the final product had a more open structure with an increased hydrodynamic radius compared to the parent protein, which explains the initial SEC results. The site-specific backbone cleavage was suspected to be catalyzed by trypsin-like protease impurities in the bulk solution. The bulk drug substance solution was subsequently treated with immobilized soybean trypsin inhibitor, and the degradation rate was significantly reduced. Furthermore, increasing the solution pH from 5 to 8 led to an increase in the degradation rate, which was consistent with the expected pH dependency of trypsin activity. In addition, the effect of bulk concentration also supported the involvement of protease impurities rather than a spontaneous peptide bond hydrolysis reaction. Trace trypsin-like protease impurities led to an unusual site-specific backbone cleavage of BMS-196854. The proteolytic degradation can be minimized by treating the bulk solution with immobilized soybean trypsin inhibitor and/or controlling the solution pH and storage temperature.

Luminescent conjugated oligothiophenes for sensitive fluorescent assignment of protein inclusion bodies.

PubMed

Klingstedt, Therése; Blechschmidt, Cristiane; Nogalska, Anna; Prokop, Stefan; Häggqvist, Bo; Danielsson, Olof; Engel, W King; Askanas, Valerie; Heppner, Frank L; Nilsson, K Peter R

2013-03-18

Small hydrophobic ligands identifying intracellular protein deposits are of great interest, as protein inclusion bodies are the pathological hallmark of several degenerative diseases. Here we report that fluorescent amyloid ligands, termed luminescent conjugated oligothiophenes (LCOs), rapidly and with high sensitivity detect protein inclusion bodies in skeletal muscle tissue from patients with sporadic inclusion body myositis (s-IBM). LCOs having a conjugated backbone of at least five thiophene units emitted strong fluorescence upon binding, and showed co-localization with proteins reported to accumulate in s-IBM protein inclusion bodies. Compared with conventional amyloid ligands, LCOs identified a larger fraction of immunopositive inclusion bodies. When the conjugated thiophene backbone was extended with terminal carboxyl groups, the LCO revealed striking spectral differences between distinct protein inclusion bodies. We conclude that 1) LCOs are sensitive, rapid and powerful tools for identifying protein inclusion bodies and 2) LCOs identify a wider range of protein inclusion bodies than conventional amyloid ligands. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Luminescent Conjugated Oligothiophenes for Sensitive Fluorescent Assignment of Protein Inclusion Bodies

PubMed Central

Klingstedt, Therése; Blechschmidt, Cristiane; Nogalska, Anna; Prokop, Stefan; Häggqvist, Bo; Danielsson, Olof; Engel, W King; Askanas, Valerie; Heppner, Frank L; Nilsson, K Peter R

2013-01-01

Small hydrophobic ligands identifying intracellular protein deposits are of great interest, as protein inclusion bodies are the pathological hallmark of several degenerative diseases. Here we report that fluorescent amyloid ligands, termed luminescent conjugated oligothiophenes (LCOs), rapidly and with high sensitivity detect protein inclusion bodies in skeletal muscle tissue from patients with sporadic inclusion body myositis (s-IBM). LCOs having a conjugated backbone of at least five thiophene units emitted strong fluorescence upon binding, and showed co-localization with proteins reported to accumulate in s-IBM protein inclusion bodies. Compared with conventional amyloid ligands, LCOs identified a larger fraction of immunopositive inclusion bodies. When the conjugated thiophene backbone was extended with terminal carboxyl groups, the LCO revealed striking spectral differences between distinct protein inclusion bodies. We conclude that 1) LCOs are sensitive, rapid and powerful tools for identifying protein inclusion bodies and 2) LCOs identify a wider range of protein inclusion bodies than conventional amyloid ligands. PMID:23450708

On the influence of the mixture of denaturants on protein structure stability: A molecular dynamics study

NASA Astrophysics Data System (ADS)

Shao, Qiang; Wang, Jinan; Zhu, Weiliang

2014-09-01

Mixtures of osmolytes and/or inorganic salts are present in the cell. Therefore, the understanding of the interplay of mixed osmolyte molecules and inorganic salts and their combined effects on protein structure is of fundamental importance. A novel test is presented to investigate the combined effects of urea and a chaotropic inorganic salt, potassium iodide (KI), on protein structure by using molecular dynamics simulation. It is found that the coexistence of KI and urea does not affect their respective distribution in solution. The solvation of KI salt in urea solution makes the electrostatic interactions of urea more favorable, promoting the hydrogen bonding between urea (and water) to protein backbone. The interactions from K+ and hydrogen bonding from urea and water to protein backbone work as the driving force for protein denaturation. The collaborative behavior of urea and KI salt thus enhances the denaturing ability of urea and KI mixed solution.

Temperature dependence of fast carbonyl backbone dynamics in chicken villin headpiece subdomain

PubMed Central

Vugmeyster, Liliya; Ostrovsky, Dmitry

2012-01-01

Temperature-dependence of protein dynamics can provide information on details of the free energy landscape by probing the characteristics of the potential responsible for the fluctuations. We have investigated the temperature-dependence of picosecond to nanosecond backbone dynamics at carbonyl carbon sites in chicken villin headpiece subdomain protein using a combination of three NMR relaxation rates: 13C′ longitudinal rate, and two cross-correlated rates involving dipolar and chemical shift anisotropy (CSA) relaxation mechanisms, 13C′/13C′−13Cα CSA/dipolar and 13C′/13C′−15N CSA/dipolar. Order parameters have been extracted using the Lipari-Szabo model-free approach assuming a separation of the time scales of internal and molecular motions in the 2–16°C temperature range. There is a gradual deviation from this assumption from lower to higher temperatures, such that above 16°C the separation of the time scales is inconsistent with the experimental data and, thus, the Lipari-Szabo formalism can not be applied. While there are variations among the residues, on the average the order parameters indicate a markedly steeper temperature dependence at backbone carbonyl carbons compared to that probed at amide nitrogens in an earlier study. This strongly advocates for probing sites other than amide nitrogen for accurate characterization of the potential and other thermodynamics characteristics of protein backbone. PMID:21416162

How many hydrogen-bonded α-turns are possible?

PubMed

Schreiber, Anette; Schramm, Peter; Hofmann, Hans-Jörg

2011-06-01

The formation of α-turns is a possibility to reverse the direction of peptide sequences via five amino acids. In this paper, a systematic conformational analysis was performed to find the possible isolated α-turns with a hydrogen bond between the first and fifth amino acid employing the methods of ab initio MO theory in vacuum (HF/6-31G*, B3LYP/6-311 + G*) and in solution (CPCM/HF/6-31G*). Only few α-turn structures with glycine and alanine backbones fulfill the geometry criteria for the i←(i + 4) hydrogen bond satisfactorily. The most stable representatives agree with structures found in the Protein Data Bank. There is a general tendency to form additional hydrogen bonds for smaller pseudocycles corresponding to β- and γ-turns with better hydrogen bond geometries. Sometimes, this competition weakens or even destroys the i←(i + 4) hydrogen bond leading to very stable double β-turn structures. This is also the reason why an "ideal" α-turn with three central amino acids having the perfect backbone angle values of an α-helix could not be localized. There are numerous hints for stable α-turns with a distance between the C(α)-atoms of the first and fifth amino acid smaller than 6-7 Å, but without an i←(i + 4) hydrogen bond.

Conformation-dependent restraints for polynucleotides: I. Clustering of the geometry of the phosphodiester group

PubMed Central

Kowiel, Marcin; Brzezinski, Dariusz; Jaskolski, Mariusz

2016-01-01

The refinement of macromolecular structures is usually aided by prior stereochemical knowledge in the form of geometrical restraints. Such restraints are also used for the flexible sugar-phosphate backbones of nucleic acids. However, recent highly accurate structural studies of DNA suggest that the phosphate bond angles may have inadequate description in the existing stereochemical dictionaries. In this paper, we analyze the bonding deformations of the phosphodiester groups in the Cambridge Structural Database, cluster the studied fragments into six conformation-related categories and propose a revised set of restraints for the O-P-O bond angles and distances. The proposed restraints have been positively validated against data from the Nucleic Acid Database and an ultrahigh-resolution Z-DNA structure in the Protein Data Bank. Additionally, the manual classification of PO4 geometry is compared with geometrical clusters automatically discovered by machine learning methods. The machine learning cluster analysis provides useful insights and a practical example for general applications of clustering algorithms for automatic discovery of hidden patterns of molecular geometry. Finally, we describe the implementation and application of a public-domain web server for automatic generation of the proposed restraints. PMID:27521371

Elastin-like Polypeptide (ELP) Charge Influences Self-Assembly of ELP-mCherry Fusion Proteins.

PubMed

Mills, Carolyn E; Michaud, Zachary; Olsen, Bradley D

2018-05-23

Self-assembly of protein-polymer bioconjugates presents an elegant strategy for controlling nanostructure and orientation of globular proteins in functional materials. Recent work has shown that genetic fusion of globular protein mCherry to an elastin-like polypeptide (ELP) yields similar self-assembly behavior to these protein-polymer bioconjugates. In the context of studying protein-polymer bioconjugate self-assembly, the mutability of the ELP sequence allows several different properties of the ELP block to be tuned orthogonally while maintaining consistent polypeptide backbone chemistry. This work uses this ELP sequence tunability in combination with the precise control offered by genetic engineering of an amino acid sequence to generate a library of four novel ELP sequences that are used to study the combined effect of charge and hydrophobicity on ELP-mCherry fusion protein self-assembly. Concentrated solution self-assembly is studied by small-angle X-ray scattering (SAXS) and depolarized light scattering (DPLS). These experiments show that fusions containing a negatively charged ELP block do not assemble at all, and fusions with a charge balanced ELP block exhibit a weak propensity for assembly. By comparison, the fusion containing an uncharged ELP block starts to order at 40 wt % in solution and at all concentrations measured has sharper, more intense SAXS peaks than other fusion proteins. These experiments show that charge character of the ELP block is a stronger predictor of self-assembly behavior than the hydrophobicity of the ELP block. Dilute solution small-angle neutron scattering (SANS) on the ELPs alone suggests that all ELPs used in this study (including the uncharged ELP) adopt dilute solution conformations similar to those of traditional polymers, including polyampholytes and polyelectrolytes. Finally, dynamic light scattering studies on ELP-mCherry blends shows that there is no significant complexation between the charged ELPs and mCherry. Therefore, it is proposed that the superior self-assembly of fusion proteins containing uncharged ELP block is due to effective repulsions between charged and uncharged blocks due to local charge correlation effects and, in the case of anionic ELPs, repulsion between like charges within the ELP block.

Compact representation of continuous energy surfaces for more efficient protein design

PubMed Central

Hallen, Mark A.; Gainza, Pablo; Donald, Bruce R.

2015-01-01

In macromolecular design, conformational energies are sensitive to small changes in atom coordinates, so modeling the small, continuous motions of atoms around low-energy wells confers a substantial advantage in structural accuracy; however, modeling these motions comes at the cost of a very large number of energy function calls, which form the bottleneck in the design calculation. In this work, we remove this bottleneck by consolidating all conformational energy evaluations into the precomputation of a local polynomial expansion of the energy about the “ideal” conformation for each low-energy, “rotameric” state of each residue pair. This expansion is called Energy as Polynomials in Internal Coordinates (EPIC), where the internal coordinates can be sidechain dihedrals, backrub angles, and/or any other continuous degrees of freedom of a macromolecule, and any energy function can be used without adding any asymptotic complexity to the design. We demonstrate that EPIC efficiently represents the energy surface for both molecular-mechanics and quantum-mechanical energy functions, and apply it specifically to protein design to model both sidechain and backbone degrees of freedom. PMID:26089744

Molecular dynamics simulations and CD spectroscopy reveal hydration-induced unfolding of the intrinsically disordered LEA proteins COR15A and COR15B from Arabidopsis thaliana.

PubMed

Navarro-Retamal, Carlos; Bremer, Anne; Alzate-Morales, Jans; Caballero, Julio; Hincha, Dirk K; González, Wendy; Thalhammer, Anja

2016-10-07

The LEA (late embryogenesis abundant) proteins COR15A and COR15B from Arabidopsis thaliana are intrinsically disordered under fully hydrated conditions, but obtain α-helical structure during dehydration, which is reversible upon rehydration. To understand this unusual structural transition, both proteins were investigated by circular dichroism (CD) and molecular dynamics (MD) approaches. MD simulations showed unfolding of the proteins in water, in agreement with CD data obtained with both HIS-tagged and untagged recombinant proteins. Mainly intramolecular hydrogen bonds (H-bonds) formed by the protein backbone were replaced by H-bonds with water molecules. As COR15 proteins function in vivo as protectants in leaves partially dehydrated by freezing, unfolding was further assessed under crowded conditions. Glycerol reduced (40%) or prevented (100%) unfolding during MD simulations, in agreement with CD spectroscopy results. H-bonding analysis indicated that preferential exclusion of glycerol from the protein backbone increased stability of the folded state.

PONDEROSA, an automated 3D-NOESY peak picking program, enables automated protein structure determination.

PubMed

Lee, Woonghee; Kim, Jin Hae; Westler, William M; Markley, John L

2011-06-15

PONDEROSA (Peak-picking Of Noe Data Enabled by Restriction of Shift Assignments) accepts input information consisting of a protein sequence, backbone and sidechain NMR resonance assignments, and 3D-NOESY ((13)C-edited and/or (15)N-edited) spectra, and returns assignments of NOESY crosspeaks, distance and angle constraints, and a reliable NMR structure represented by a family of conformers. PONDEROSA incorporates and integrates external software packages (TALOS+, STRIDE and CYANA) to carry out different steps in the structure determination. PONDEROSA implements internal functions that identify and validate NOESY peak assignments and assess the quality of the calculated three-dimensional structure of the protein. The robustness of the analysis results from PONDEROSA's hierarchical processing steps that involve iterative interaction among the internal and external modules. PONDEROSA supports a variety of input formats: SPARKY assignment table (.shifts) and spectrum file formats (.ucsf), XEASY proton file format (.prot), and NMR-STAR format (.star). To demonstrate the utility of PONDEROSA, we used the package to determine 3D structures of two proteins: human ubiquitin and Escherichia coli iron-sulfur scaffold protein variant IscU(D39A). The automatically generated structural constraints and ensembles of conformers were as good as or better than those determined previously by much less automated means. The program, in the form of binary code along with tutorials and reference manuals, is available at http://ponderosa.nmrfam.wisc.edu/.

Relevance of Internal Friction and Structural Constraints for the Dynamics of Denatured Bovine Serum Albumin.

PubMed

Ameseder, Felix; Radulescu, Aurel; Holderer, Olaf; Falus, Peter; Richter, Dieter; Stadler, Andreas M

2018-05-17

A general property of disordered proteins is their structural expansion that results in a high molecular flexibility. The structure and dynamics of bovine serum albumin (BSA) denatured by guanidinium hydrochloride (GndCl) were investigated using small-angle neutron scattering (SANS) and neutron spin-echo spectroscopy (NSE). SANS experiments demonstrated the relevance of intrachain interactions for structural expansion. Using NSE experiments, we observed a high internal flexibility of denatured BSA in addition to center-of-mass diffusion detected by dynamic light scattering. Internal motions measured by NSE were described using concepts based on polymer theory. The contribution of residue-solvent friction was accounted for using the Zimm model including internal friction (ZIF). Disulfide bonds forming loops of amino acids of the peptide backbone have a major impact on internal dynamics that can be interpreted with a reduced set of Zimm modes.

What induces pocket openings on protein surface patches involved in protein-protein interactions?

PubMed

Eyrisch, Susanne; Helms, Volkhard

2009-02-01

We previously showed for the proteins BCL-X(L), IL-2, and MDM2 that transient pockets at their protein-protein binding interfaces can be identified by applying the PASS algorithm to molecular dynamics (MD) snapshots. We now investigated which aspects of the natural conformational dynamics of proteins induce the formation of such pockets. The pocket detection protocol was applied to three different conformational ensembles for the same proteins that were extracted from MD simulations of the inhibitor bound crystal conformation in water and the free crystal/NMR structure in water and in methanol. Additional MD simulations studied the impact of backbone mobility. The more efficient CONCOORD or normal mode analysis (NMA) techniques gave significantly smaller pockets than MD simulations, whereas tCONCOORD generated pockets comparable to those observed in MD simulations for two of the three systems. Our findings emphasize the influence of solvent polarity and backbone rearrangements on the formation of pockets on protein surfaces and should be helpful in future generation of transient pockets as putative ligand binding sites at protein-protein interfaces.

What induces pocket openings on protein surface patches involved in protein-protein interactions?

NASA Astrophysics Data System (ADS)

Eyrisch, Susanne; Helms, Volkhard

2009-02-01

We previously showed for the proteins BCL-XL, IL-2, and MDM2 that transient pockets at their protein-protein binding interfaces can be identified by applying the PASS algorithm to molecular dynamics (MD) snapshots. We now investigated which aspects of the natural conformational dynamics of proteins induce the formation of such pockets. The pocket detection protocol was applied to three different conformational ensembles for the same proteins that were extracted from MD simulations of the inhibitor bound crystal conformation in water and the free crystal/NMR structure in water and in methanol. Additional MD simulations studied the impact of backbone mobility. The more efficient CONCOORD or normal mode analysis (NMA) techniques gave significantly smaller pockets than MD simulations, whereas tCONCOORD generated pockets comparable to those observed in MD simulations for two of the three systems. Our findings emphasize the influence of solvent polarity and backbone rearrangements on the formation of pockets on protein surfaces and should be helpful in future generation of transient pockets as putative ligand binding sites at protein-protein interfaces.

Using NMR chemical shifts to calculate the propensity for structural order and disorder in proteins.

PubMed

Tamiola, Kamil; Mulder, Frans A A

2012-10-01

NMR spectroscopy offers the unique possibility to relate the structural propensities of disordered proteins and loop segments of folded peptides to biological function and aggregation behaviour. Backbone chemical shifts are ideally suited for this task, provided that appropriate reference data are available and idiosyncratic sensitivity of backbone chemical shifts to structural information is treated in a sensible manner. In the present paper, we describe methods to detect structural protein changes from chemical shifts, and present an online tool [ncSPC (neighbour-corrected Structural Propensity Calculator)], which unites aspects of several current approaches. Examples of structural propensity calculations are given for two well-characterized systems, namely the binding of α-synuclein to micelles and light activation of photoactive yellow protein. These examples spotlight the great power of NMR chemical shift analysis for the quantitative assessment of protein disorder at the atomic level, and further our understanding of biologically important problems.

Recognition of coarse-grained protein tertiary structure.

PubMed

Lezon, Timothy; Banavar, Jayanth R; Maritan, Amos

2004-05-15

A model of the protein backbone is considered in which each residue is characterized by the location of its C(alpha) atom and one of a discrete set of conformal (phi, psi) states. We investigate the key differences between a description that offers a locally precise fit to known backbone structures and one that provides a globally accurate fit to protein structures. Using a statistical scoring scheme and threading, a protein's local best-fit conformation is highly recognizable, but its global structure cannot be directly determined from an amino acid sequence. The incorporation of information about the conformal states of neighboring residues along the chain allows one to accurately translate the local structure into a global structure. We present a two-step algorithm, which recognizes up to 95% of the tested protein native-state structures to within a 2.5 A root mean square deviation. Copyright 2004 Wiley-Liss, Inc.

A role for molecular compression in the post-translational formation of the Green Fluorescent Protein chromophore

NASA Astrophysics Data System (ADS)

Terranova, U.; Nifosı`, R.

2010-05-01

Spontaneous chromophore formation is probably the key feature for the remarkable success of GFPs (Green Fluorescent Proteins) and related proteins in fluorescence microscopy. Though a quantitative analysis of the involved energetics still remains elusive, substantial progress has been made in identifying the steps of chromophore biosynthesis and the contribution of individual residues and surrounding protein matrix. The latter clearly enforces a peculiar configuration of the pre-cyclized chromophore-forming tripeptide. However, it is debated whether a mechanical compression is also at play in triggering backbone cyclization. Here, by molecular dynamics and potential of mean force calculations, we estimate the contribution of the protein scaffold in promoting the proximity of reacting atoms- and hence backbone cyclization - by a sort of compression mechanism. Comparing several mutants we highlight the role of some surrounding residues. Finally, we analyze the case of HAL (Histidine Ammonia-Lyase) active site, which undergoes an analogous cyclization reaction.

Solution Structure and Backbone Dynamics of Human Liver Fatty Acid Binding Protein: Fatty Acid Binding Revisited

PubMed Central

Cai, Jun; Lücke, Christian; Chen, Zhongjing; Qiao, Ye; Klimtchuk, Elena; Hamilton, James A.

2012-01-01

Liver fatty acid binding protein (L-FABP), a cytosolic protein most abundant in liver, is associated with intracellular transport of fatty acids, nuclear signaling, and regulation of intracellular lipolysis. Among the members of the intracellular lipid binding protein family, L-FABP is of particular interest as it can i), bind two fatty acid molecules simultaneously and ii), accommodate a variety of bulkier physiological ligands such as bilirubin and fatty acyl CoA. To better understand the promiscuous binding and transport properties of L-FABP, we investigated structure and dynamics of human L-FABP with and without bound ligands by means of heteronuclear NMR. The overall conformation of human L-FABP shows the typical β-clam motif. Binding of two oleic acid (OA) molecules does not alter the protein conformation substantially, but perturbs the chemical shift of certain backbone and side-chain protons that are involved in OA binding according to the structure of the human L-FABP/OA complex. Comparison of the human apo and holo L-FABP structures revealed no evidence for an “open-cap” conformation or a “swivel-back” mechanism of the K90 side chain upon ligand binding, as proposed for rat L-FABP. Instead, we postulate that the lipid binding process in L-FABP is associated with backbone dynamics. PMID:22713574

Vanishing amplitude of backbone dynamics causes a true protein dynamical transition: H2 NMR studies on perdeuterated C-phycocyanin

NASA Astrophysics Data System (ADS)

Kämpf, Kerstin; Kremmling, Beke; Vogel, Michael

2014-03-01

Using a combination of H2 nuclear magnetic resonance (NMR) methods, we study internal rotational dynamics of the perdeuterated protein C-phycocyanin (CPC) in dry and hydrated states over broad temperature and dynamic ranges with high angular resolution. Separating H2 NMR signals from methyl deuterons, we show that basically all backbone deuterons exhibit highly restricted motion occurring on time scales faster than microseconds. The amplitude of this motion increases when a hydration shell exists, while it decreases upon cooling and vanishes near 175 K. We conclude that the vanishing of the highly restricted motion marks a dynamical transition, which is independent of the time window and of a fundamental importance. This conclusion is supported by results from experimental and computational studies of the proteins myoglobin and elastin. In particular, we argue based on findings in molecular dynamics simulations that the behavior of the highly restricted motion of proteins at the dynamical transition resembles that of a characteristic secondary relaxation of liquids at the glass transition, namely the nearly constant loss. Furthermore, H2 NMR studies on perdeuterated CPC reveal that, in addition to highly restricted motion, small fractions of backbone segments exhibit weakly restricted dynamics when temperature and hydration are sufficiently high.

Combining Rosetta with molecular dynamics (MD): A benchmark of the MD-based ensemble protein design.

PubMed

Ludwiczak, Jan; Jarmula, Adam; Dunin-Horkawicz, Stanislaw

2018-07-01

Computational protein design is a set of procedures for computing amino acid sequences that will fold into a specified structure. Rosetta Design, a commonly used software for protein design, allows for the effective identification of sequences compatible with a given backbone structure, while molecular dynamics (MD) simulations can thoroughly sample near-native conformations. We benchmarked a procedure in which Rosetta design is started on MD-derived structural ensembles and showed that such a combined approach generates 20-30% more diverse sequences than currently available methods with only a slight increase in computation time. Importantly, the increase in diversity is achieved without a loss in the quality of the designed sequences assessed by their resemblance to natural sequences. We demonstrate that the MD-based procedure is also applicable to de novo design tasks started from backbone structures without any sequence information. In addition, we implemented a protocol that can be used to assess the stability of designed models and to select the best candidates for experimental validation. In sum our results demonstrate that the MD ensemble-based flexible backbone design can be a viable method for protein design, especially for tasks that require a large pool of diverse sequences. Copyright © 2018 Elsevier Inc. All rights reserved.

Correlating the Effects of Antimicrobial Preservatives on Conformational Stability, Aggregation Propensity, and Backbone Flexibility of an IgG1 mAb.

PubMed

Arora, Jayant; Joshi, Sangeeta B; Middaugh, C Russell; Weis, David D; Volkin, David B

2017-06-01

Multidose formulations of biotherapeutics, which offer better dosage management and reduced production costs, require the addition of antimicrobial preservatives (APs). APs have been shown, however, to decrease protein stability in solution and cause protein aggregation. In this report, the effect of 4 APs, m-cresol, phenol, phenoxyethanol, and benzyl alcohol on conformational stability, aggregation propensity, and backbone flexibility of an IgG1 mAb, mAb-4, is investigated. Compared with no preservative control, each of the APs decreased the conformational stability of mAb-4 as measured by differential scanning calorimetry and extrinsic fluorescence spectroscopy. The addition of APs resulted in increased monomer loss and aggregate accumulation at 50°C over 28 days, as monitored by size-exclusion chromatography. The extent of conformational destabilization and protein aggregation of mAb-4 induced by APs followed their calculated octanol-water partition coefficients. Increases in backbone flexibility, as measured by hydrogen exchange, of a region located in the C H 2 domain of the mAb (heavy chain 237-254) in the presence of APs also correlated with hydrophobicity. Based on these results, the destabilizing effect of APs on mAb-4 correlates with the increased hydrophobicity of the APs and their ability to enhance the local backbone flexibility of an aggregation hot spot within the C H 2 domain of the mAb. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Approach to characterization of the higher order structure of disulfide-containing proteins using hydrogen/deuterium exchange and top-down mass spectrometry.

PubMed

Wang, Guanbo; Kaltashov, Igor A

2014-08-05

Top-down hydrogen/deuterium exchange (HDX) with mass spectrometric (MS) detection has recently matured to become a potent biophysical tool capable of providing valuable information on higher order structure and conformational dynamics of proteins at an unprecedented level of structural detail. However, the scope of the proteins amenable to the analysis by top-down HDX MS still remains limited, with the protein size and the presence of disulfide bonds being the two most important limiting factors. While the limitations imposed by the physical size of the proteins gradually become more relaxed as the sensitivity, resolution and dynamic range of modern MS instrumentation continue to improve at an ever accelerating pace, the presence of the disulfide linkages remains a much less forgiving limitation even for the proteins of relatively modest size. To circumvent this problem, we introduce an online chemical reduction step following completion and quenching of the HDX reactions and prior to the top-down MS measurements of deuterium occupancy of individual backbone amides. Application of the new methodology to the top-down HDX MS characterization of a small (99 residue long) disulfide-containing protein β2-microglobulin allowed the backbone amide protection to be probed with nearly a single-residue resolution across the entire sequence. The high-resolution backbone protection pattern deduced from the top-down HDX MS measurements carried out under native conditions is in excellent agreement with the crystal structure of the protein and high-resolution NMR data, suggesting that introduction of the chemical reduction step to the top-down routine does not trigger hydrogen scrambling either during the electrospray ionization process or in the gas phase prior to the protein ion dissociation.

Improved protein surface comparison and application to low-resolution protein structure data.

PubMed

Sael, Lee; Kihara, Daisuke

2010-12-14

Recent advancements of experimental techniques for determining protein tertiary structures raise significant challenges for protein bioinformatics. With the number of known structures of unknown function expanding at a rapid pace, an urgent task is to provide reliable clues to their biological function on a large scale. Conventional approaches for structure comparison are not suitable for a real-time database search due to their slow speed. Moreover, a new challenge has arisen from recent techniques such as electron microscopy (EM), which provide low-resolution structure data. Previously, we have introduced a method for protein surface shape representation using the 3D Zernike descriptors (3DZDs). The 3DZD enables fast structure database searches, taking advantage of its rotation invariance and compact representation. The search results of protein surface represented with the 3DZD has showngood agreement with the existing structure classifications, but some discrepancies were also observed. The three new surface representations of backbone atoms, originally devised all-atom-surface representation, and the combination of all-atom surface with the backbone representation are examined. All representations are encoded with the 3DZD. Also, we have investigated the applicability of the 3DZD for searching protein EM density maps of varying resolutions. The surface representations are evaluated on structure retrieval using two existing classifications, SCOP and the CE-based classification. Overall, the 3DZDs representing backbone atoms show better retrieval performance than the original all-atom surface representation. The performance further improved when the two representations are combined. Moreover, we observed that the 3DZD is also powerful in comparing low-resolution structures obtained by electron microscopy.

Structural insights into the backbone-circularized granulocyte colony-stimulating factor containing a short connector.

PubMed

Miyafusa, Takamitsu; Shibuya, Risa; Honda, Shinya

2018-06-02

Backbone circularization is a powerful approach for enhancing the structural stability of polypeptides. Herein, we present the crystal structure of the circularized variant of the granulocyte colony-stimulating factor (G-CSF) in which the terminal helical region was circularized using a short, two-amino acid connector. The structure revealed that the N- and C-termini were indeed connected by a peptide bond. The local structure of the C-terminal region transited from an α helix to 3 10 helix with a bend close to the N-terminal region, indicating that the structural change offset the insufficient length of the connector. This is the first-ever report of a crystal structure of the backbone of a circularized protein. It will facilitate the development of backbone circularization methodology. Copyright © 2018 Elsevier Inc. All rights reserved.

Computational Design of Thermostabilizing d-Amino Acid Substitutions

PubMed Central

Rodriguez-Granillo, Agustina; Annavarapu, Srinivas; Zhang, Lei; Koder, Ronald L.; Nanda, Vikas

2012-01-01

Judicious incorporation of d-amino acids in engineered proteins confer many advantages such as preventing degradation by endogenous proteases, and designing novel structures and functions not accessible to homochiral polypeptides. Glycine to d-alanine substitutions at the carboxy-termini can stabilize α-helices by reducing conformational entropy. Beyond alanine, we propose additional side chain effects on the degree of stabilization conferred by d-amino acid substitutions. A detailed, molecular understanding of backbone and side chain interactions is important for developing rational, broadly applicable strategies in using d-amino acids to increase protein thermostability. Insight from structural bioinformatics combined with computational protein design can successfully guide the selection of stabilizing d-amino acid mutations. Substituting a key glycine in the Trp-Cage mini-protein with d-Gln dramatically stabilizes the fold without altering the protein backbone. Stabilities of individual substitutions can be understood in terms of the balance of intramolecular forces at both the α-helix C-terminus and throughout the protein. PMID:21978298

Protein Structure Determination from Pseudocontact Shifts Using ROSETTA

PubMed Central

Schmitz, Christophe; Vernon, Robert; Otting, Gottfried; Baker, David; Huber, Thomas

2013-01-01

Paramagnetic metal ions generate pseudocontact shifts (PCSs) in nuclear magnetic resonance spectra that are manifested as easily measurable changes in chemical shifts. Metals can be incorporated into proteins through metal binding tags, and PCS data constitute powerful long-range restraints on the positions of nuclear spins relative to the coordinate system of the magnetic susceptibility anisotropy tensor (Δχ-tensor) of the metal ion. We show that three-dimensional structures of proteins can reliably be determined using PCS data from a single metal binding site combined with backbone chemical shifts. The program PCS-ROSETTA automatically determines the Δχ-tensor and metal position from the PCS data during the structure calculations, without any prior knowledge of the protein structure. The program can determine structures accurately for proteins of up to 150 residues, offering a powerful new approach to protein structure determination that relies exclusively on readily measurable backbone chemical shifts and easily discriminates between correctly and incorrectly folded conformations. PMID:22285518

Factors affecting the use of 13Cα chemical shifts to determine, refine, and validate protein structures

PubMed Central

Vila, Jorge A.; Scheraga, Harold A.

2008-01-01

Interest centers here on the analysis of two different, but related, phenomena that affect side-chain conformations and consequently 13Cα chemical shifts and their applications to determine, refine, and validate protein structures. The first is whether 13Cα chemical shifts, computed at the DFT level of approximation with charged residues is a better approximation of observed 13Cα chemical shifts than those computed with neutral residues for proteins in solution. Accurate computation of 13Cα chemical shifts requires a proper representation of the charges, which might not take on integral values. For this analysis, the charges for 139 conformations of the protein ubiquitin were determined by explicit consideration of protein binding equilibria, at a given pH, that is, by exploring the 2ξ possible ionization states of the whole molecule, with ξ being the number of ionizable groups. The results of this analysis, as revealed by the shielding/deshield-ing of the 13Cα nucleus, indicated that: (i) there is a significant difference in the computed 13Cα chemical shifts, between basic and acidic groups, as a function of the degree of charge of the side chain; (ii) this difference is attributed to the distance between the ionizable groups and the 13Cα nucleus, which is shorter for the acidic Asp and Glu groups as compared with that for the basic Lys and Arg groups; and (iii) the use of neutral, rather than charged, basic and acidic groups is a better approximation of the observed 13Cα chemical shifts of a protein in solution. The second is how side-chain flexibility influences computed 13Cα chemical shifts in an additional set of ubiquitin conformations, in which the side chains are generated from an NMR-derived structure with the backbone conformation assumed to be fixed. The 13Cα chemical shift of a given amino acid residue in a protein is determined, mainly, by its own backbone and side-chain torsional angles, independent of the neighboring residues; the conformation of a given residue itself, however, depends on the environment of this residue and, hence, on the whole protein structure. As a consequence, this analysis reveals the role and impact of an accurate side-chain computation in the determination and refinement of protein conformation. The results of this analysis are: (i) a lower error between computed and observed 13Cα chemical shifts (by up to 3.7 ppm), was found for ~68% and ~63% of all ionizable residues and all non-Ala/Pro/Gly residues, respectively, in the additional set of conformations, compared with results for the model from which the set was derived; and (ii) all the additional conformations exhibit a lower root-mean-square-deviation (1.97 ppm ≤ rmsd ≤ 2.13 ppm), between computed and observed 13Cα chemical shifts, than the rmsd (2.32 ppm) computed for the starting conformation from which this additional set was derived. As a validation test, an analysis of the additional set of ubiquitin conformations, comparing computed and observed values of both 13Cα chemical shifts and χ1 torsional angles (given by the vicinal coupling constants, 3JN–Cγ and 3JC′–Cγ, is discussed. PMID:17975838

A protein block based fold recognition method for the annotation of twilight zone sequences.

PubMed

Suresh, V; Ganesan, K; Parthasarathy, S

2013-03-01

The description of protein backbone was recently improved with a group of structural fragments called Structural Alphabets instead of the regular three states (Helix, Sheet and Coil) secondary structure description. Protein Blocks is one of the Structural Alphabets used to describe each and every region of protein backbone including the coil. According to de Brevern (2000) the Protein Blocks has 16 structural fragments and each one has 5 residues in length. Protein Blocks fragments are highly informative among the available Structural Alphabets and it has been used for many applications. Here, we present a protein fold recognition method based on Protein Blocks for the annotation of twilight zone sequences. In our method, we align the predicted Protein Blocks of a query amino acid sequence with a library of assigned Protein Blocks of 953 known folds using the local pair-wise alignment. The alignment results with z-value ≥ 2.5 and P-value ≤ 0.08 are predicted as possible folds. Our method is able to recognize the possible folds for nearly 35.5% of the twilight zone sequences with their predicted Protein Block sequence obtained by pb_prediction, which is available at Protein Block Export server.

Assignment by Negative-Ion Electrospray Tandem Mass Spectrometry of the Tetrasaccharide Backbones of Monosialylated Glycans Released from Bovine Brain Gangliosides

NASA Astrophysics Data System (ADS)

Chai, Wengang; Zhang, Yibing; Mauri, Laura; Ciampa, Maria G.; Mulloy, Barbara; Sonnino, Sandro; Feizi, Ten

2018-05-01

Gangliosides, as plasma membrane-associated sialylated glycolipids, are antigenic structures and they serve as ligands for adhesion proteins of pathogens, for toxins of bacteria, and for endogenous proteins of the host. The detectability by carbohydrate-binding proteins of glycan antigens and ligands on glycolipids can be influenced by the differing lipid moieties. To investigate glycan sequences of gangliosides as recognition structures, we have underway a program of work to develop a "gangliome" microarray consisting of isolated natural gangliosides and neoglycolipids (NGLs) derived from glycans released from them, and each linked to the same lipid molecule for arraying and comparative microarray binding analyses. Here, in the first phase of our studies, we describe a strategy for high-sensitivity assignment of the tetrasaccharide backbones and application to identification of eight of monosialylated glycans released from bovine brain gangliosides. This approach is based on negative-ion electrospray mass spectrometry with collision-induced dissociation (ESI-CID-MS/MS) of the desialylated glycans. Using this strategy, we have the data on backbone regions of four minor components among the monosialo-ganglioside-derived glycans; these are of the ganglio-, lacto-, and neolacto-series.

Chemical crosslinking and mass spectrometry studies of the structure and dynamics of membrane proteins and receptors.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Haskins, William E.; Leavell, Michael D.; Lane, Pamela

2005-03-01

Membrane proteins make up a diverse and important subset of proteins for which structural information is limited. In this study, chemical cross-linking and mass spectrometry were used to explore the structure of the G-protein-coupled photoreceptor bovine rhodopsin in the dark-state conformation. All experiments were performed in rod outer segment membranes using amino acid 'handles' in the native protein sequence and thus minimizing perturbations to the native protein structure. Cysteine and lysine residues were covalently cross-linked using commercially available reagents with a range of linker arm lengths. Following chemical digestion of cross-linked protein, cross-linked peptides were identified by accurate mass measurementmore » using liquid chromatography-fourier transform mass spectrometry and an automated data analysis pipeline. Assignments were confirmed and, if necessary, resolved, by tandem MS. The relative reactivity of lysine residues participating in cross-links was evaluated by labeling with NHS-esters. A distinct pattern of cross-link formation within the C-terminal domain, and between loop I and the C-terminal domain, emerged. Theoretical distances based on cross-linking were compared to inter-atomic distances determined from the energy-minimized X-ray crystal structure and Monte Carlo conformational search procedures. In general, the observed cross-links can be explained by re-positioning participating side-chains without significantly altering backbone structure. One exception, between C3 16 and K325, requires backbone motion to bring the reactive atoms into sufficient proximity for cross-linking. Evidence from other studies suggests that residues around K325 for a region of high backbone mobility. These findings show that cross-linking studies can provide insight into the structural dynamics of membrane proteins in their native environment.« less

Flexible Xxx–Asp/Asn and Gly–Xxx Residues of Equine Cytochrome c in Matrix-Assisted Laser Desorption/Ionization In-Source Decay Mass Spectrometry

PubMed Central

Takayama, Mitsuo

2012-01-01

The backbone flexibility of a protein has been studied from the standpoint of the susceptibility of amino acid residues to in-source decay (ISD) in matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Residues more susceptible to MALDI-ISD, namely Xxx–Asp/Asn and Gly–Xxx, were identified from the discontinuous intense peak of c′-ions originating from specific cleavage at N–Cα bonds of the backbone of equine cytochrome c. The identity of the residues susceptible to ISD was consistent with the known flexible backbone amides as estimated by hydrogen/deuterium exchange (HDX) experiments. The identity of these flexible amino acid residues (Asp, Asn, and Gly) is consistent with the fact that these residues are preferred in flexible secondary structure free from intramolecular hydrogen-bonded structures such as α-helix and β-sheet. The MALDI-ISD spectrum of equine cytochrome c gave not only intense N-terminal side c′-ions originating from N–Cα bond cleavage at Xxx–Asp/Asn and Gly–Xxx residues, but also C-terminal side complement z′-ions originating from the same cleavage sites. The present study implies that MALDI-ISD can give information about backbone flexibility of proteins, comparable with the protection factors estimated by HDX. PMID:24349908

Flexible xxx-asp/asn and gly-xxx residues of equine cytochrome C in matrix-assisted laser desorption/ionization in-source decay mass spectrometry.

PubMed

Takayama, Mitsuo

2012-01-01

The backbone flexibility of a protein has been studied from the standpoint of the susceptibility of amino acid residues to in-source decay (ISD) in matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Residues more susceptible to MALDI-ISD, namely Xxx-Asp/Asn and Gly-Xxx, were identified from the discontinuous intense peak of c'-ions originating from specific cleavage at N-Cα bonds of the backbone of equine cytochrome c. The identity of the residues susceptible to ISD was consistent with the known flexible backbone amides as estimated by hydrogen/deuterium exchange (HDX) experiments. The identity of these flexible amino acid residues (Asp, Asn, and Gly) is consistent with the fact that these residues are preferred in flexible secondary structure free from intramolecular hydrogen-bonded structures such as α-helix and β-sheet. The MALDI-ISD spectrum of equine cytochrome c gave not only intense N-terminal side c'-ions originating from N-Cα bond cleavage at Xxx-Asp/Asn and Gly-Xxx residues, but also C-terminal side complement z'-ions originating from the same cleavage sites. The present study implies that MALDI-ISD can give information about backbone flexibility of proteins, comparable with the protection factors estimated by HDX.

Mutation of charged residues to neutral ones accelerates urea denaturation of HP-35.

PubMed

Wei, Haiyan; Yang, Lijiang; Gao, Yi Qin

2010-09-16

Following the studies of urea denaturation of β-hairpins using molecular dynamics, in this paper, molecular dynamics simulations of two peptides, a 35 residue three helix bundle villin headpiece protein HP-35 and its doubly norleucine-substituent mutant (Lys24Nle/Lys29Nle) HP-35 NleNle, were undertaken in urea solutions to understand the molecular mechanism of urea denaturation of α-helices. The mutant HP-35 NleNle was found to denature more easily than the wild type. During the expansion of the small hydrophobic core, water penetration occurs first, followed by that of urea molecules. It was also found that the initial hydration of the peptide backbone is achieved through water hydrogen bonding with the backbone CO groups during the denaturation of both polypeptides. The mutation of the two charged lysine residues to apolar norleucine enhances the accumulation of urea near the hydrophobic core and facilitates the denaturation process. Urea also interacts directly with the peptide backbone as well as side chains, thereby stabilizing nonnative conformations. The mechanism revealed here is consistent with the previous study on secondary structure of β-hairpin polypeptide, GB1, PEPTIDE 1, and TRPZIP4, suggesting that there is a general mechanism in the denaturation of protein backbone hydrogen bonds by urea.

Quantitative comparison of structure and dynamics of elastin following three isolation schemes by 13C solid state NMR and MALDI mass spectrometry.

PubMed

Papaioannou, A; Louis, M; Dhital, B; Ho, H P; Chang, E J; Boutis, G S

2015-05-01

Methods for isolating elastin from fat, collagen, and muscle, commonly used in the design of artificial elastin based biomaterials, rely on exposing tissue to harsh pH levels and temperatures that usually denature many proteins. At present, a quantitative measurement of the modifications to elastin following isolation from other extracellular matrix constituents has not been reported. Using magic angle spinning (13)C NMR spectroscopy and relaxation methodologies, we have measured the modification in structure and dynamics following three known purification protocols. Our experimental data reveal that the (13)C spectra of the hydrated samples appear remarkably similar across the various purification methods. Subtle differences in the half maximum widths were observed in the backbone carbonyl suggesting possible structural heterogeneity across the different methods of purification. Additionally, small differences in the relative signal intensities were observed between purified samples. Lyophilizing the samples results in a reduction of backbone motion and reveals additional differences across the purification methods studied. These differences were most notable in the alanine motifs indicating possible changes in cross-linking or structural rigidity. The measured correlation times of glycine and proline moieties are observed to also vary considerably across the different purification methods, which may be related to peptide bond cleavage. Lastly, the relative concentration of desmosine cross-links in the samples quantified by MALDI mass spectrometry is reported. Copyright © 2015 Elsevier B.V. All rights reserved.

Deciphering the shape and deformation of secondary structures through local conformation analysis

PubMed Central

2011-01-01

Background Protein deformation has been extensively analysed through global methods based on RMSD, torsion angles and Principal Components Analysis calculations. Here we use a local approach, able to distinguish among the different backbone conformations within loops, α-helices and β-strands, to address the question of secondary structures' shape variation within proteins and deformation at interface upon complexation. Results Using a structural alphabet, we translated the 3 D structures of large sets of protein-protein complexes into sequences of structural letters. The shape of the secondary structures can be assessed by the structural letters that modeled them in the structural sequences. The distribution analysis of the structural letters in the three protein compartments (surface, core and interface) reveals that secondary structures tend to adopt preferential conformations that differ among the compartments. The local description of secondary structures highlights that curved conformations are preferred on the surface while straight ones are preferred in the core. Interfaces display a mixture of local conformations either preferred in core or surface. The analysis of the structural letters transition occurring between protein-bound and unbound conformations shows that the deformation of secondary structure is tightly linked to the compartment preference of the local conformations. Conclusion The conformation of secondary structures can be further analysed and detailed thanks to a structural alphabet which allows a better description of protein surface, core and interface in terms of secondary structures' shape and deformation. Induced-fit modification tendencies described here should be valuable information to identify and characterize regions under strong structural constraints for functional reasons. PMID:21284872

Deciphering the shape and deformation of secondary structures through local conformation analysis.

PubMed

Baussand, Julie; Camproux, Anne-Claude

2011-02-01

Protein deformation has been extensively analysed through global methods based on RMSD, torsion angles and Principal Components Analysis calculations. Here we use a local approach, able to distinguish among the different backbone conformations within loops, α-helices and β-strands, to address the question of secondary structures' shape variation within proteins and deformation at interface upon complexation. Using a structural alphabet, we translated the 3 D structures of large sets of protein-protein complexes into sequences of structural letters. The shape of the secondary structures can be assessed by the structural letters that modeled them in the structural sequences. The distribution analysis of the structural letters in the three protein compartments (surface, core and interface) reveals that secondary structures tend to adopt preferential conformations that differ among the compartments. The local description of secondary structures highlights that curved conformations are preferred on the surface while straight ones are preferred in the core. Interfaces display a mixture of local conformations either preferred in core or surface. The analysis of the structural letters transition occurring between protein-bound and unbound conformations shows that the deformation of secondary structure is tightly linked to the compartment preference of the local conformations. The conformation of secondary structures can be further analysed and detailed thanks to a structural alphabet which allows a better description of protein surface, core and interface in terms of secondary structures' shape and deformation. Induced-fit modification tendencies described here should be valuable information to identify and characterize regions under strong structural constraints for functional reasons.

Frequency Response of a Protein to Local Conformational Perturbations

PubMed Central

Eren, Dilek; Alakent, Burak

2013-01-01

Signals created by local perturbations are known to propagate long distances through proteins via backbone connectivity and nonbonded interactions. In the current study, signal propagation from the flexible ligand binding loop to the rest of Protein Tyrosine Phosphatase 1B (PTP1B) was investigated using frequency response techniques. Using restrained Targeted Molecular Dynamics (TMD) potential on WPD and R loops, PTP1B was driven between its crystal structure conformations at different frequencies. Propagation of the local perturbation signal was manifested via peaks at the fundamental frequency and upper harmonics of 1/f distributed spectral density of atomic variables, such as Cα atoms, dihedral angles, or polar interaction distances. Frequency of perturbation was adjusted high enough (simulation length >∼10×period of a perturbation cycle) not to be clouded by random diffusional fluctuations, and low enough (<∼0.8 ns−1) not to attenuate the propagating signal and enhance the contribution of the side-chains to the dissipation of the signals. Employing Discrete Fourier Transform (DFT) to TMD simulation trajectories of 16 cycles of conformational transitions at periods of 1.2 to 5 ns yielded Cα displacements consistent with those obtained from crystal structures. Identification of the perturbed atomic variables by statistical t-tests on log-log scale spectral densities revealed the extent of signal propagation in PTP1B, while phase angles of the filtered trajectories at the fundamental frequency were used to cluster collectively fluctuating elements. Hydrophobic interactions were found to have a higher contribution to signal transduction between side-chains compared to the role of polar interactions. Most of in-phase fluctuating residues on the signaling pathway were found to have high identity among PTP domains, and located over a wide region of PTP1B including the allosteric site. Due to its simplicity and efficiency, the suggested technique may find wide applications in identification of signaling pathways of different proteins. PMID:24086121

Building proteins from C alpha coordinates using the dihedral probability grid Monte Carlo method.

PubMed Central

Mathiowetz, A. M.; Goddard, W. A.

1995-01-01

Dihedral probability grid Monte Carlo (DPG-MC) is a general-purpose method of conformational sampling that can be applied to many problems in peptide and protein modeling. Here we present the DPG-MC method and apply it to predicting complete protein structures from C alpha coordinates. This is useful in such endeavors as homology modeling, protein structure prediction from lattice simulations, or fitting protein structures to X-ray crystallographic data. It also serves as an example of how DPG-MC can be applied to systems with geometric constraints. The conformational propensities for individual residues are used to guide conformational searches as the protein is built from the amino-terminus to the carboxyl-terminus. Results for a number of proteins show that both the backbone and side chain can be accurately modeled using DPG-MC. Backbone atoms are generally predicted with RMS errors of about 0.5 A (compared to X-ray crystal structure coordinates) and all atoms are predicted to an RMS error of 1.7 A or better. PMID:7549885

OPAL: prediction of MoRF regions in intrinsically disordered protein sequences.

PubMed

Sharma, Ronesh; Raicar, Gaurav; Tsunoda, Tatsuhiko; Patil, Ashwini; Sharma, Alok

2018-06-01

Intrinsically disordered proteins lack stable 3-dimensional structure and play a crucial role in performing various biological functions. Key to their biological function are the molecular recognition features (MoRFs) located within long disordered regions. Computationally identifying these MoRFs from disordered protein sequences is a challenging task. In this study, we present a new MoRF predictor, OPAL, to identify MoRFs in disordered protein sequences. OPAL utilizes two independent sources of information computed using different component predictors. The scores are processed and combined using common averaging method. The first score is computed using a component MoRF predictor which utilizes composition and sequence similarity of MoRF and non-MoRF regions to detect MoRFs. The second score is calculated using half-sphere exposure (HSE), solvent accessible surface area (ASA) and backbone angle information of the disordered protein sequence, using information from the amino acid properties of flanks surrounding the MoRFs to distinguish MoRF and non-MoRF residues. OPAL is evaluated using test sets that were previously used to evaluate MoRF predictors, MoRFpred, MoRFchibi and MoRFchibi-web. The results demonstrate that OPAL outperforms all the available MoRF predictors and is the most accurate predictor available for MoRF prediction. It is available at http://www.alok-ai-lab.com/tools/opal/. ashwini@hgc.jp or alok.sharma@griffith.edu.au. Supplementary data are available at Bioinformatics online.

Dynamical role of phosphorylation on serine/threonine-proline Pin1 substrates from constant force molecular dynamics simulations.

PubMed

Velazquez, Hector A; Hamelberg, Donald

2015-02-21

Cis-trans isomerization of peptidyl-prolyl bonds of the protein backbone plays an important role in numerous biological processes. Cis-trans isomerization can be the rate-limiting step due its extremely slow dynamics, compared to the millisecond time scale of many processes, and is catalyzed by a widely studied family of peptidyl-prolyl cis-trans isomerase enzymes. Also, mechanical forces along the peptide chain can speed up the rate of isomerization, resulting in "mechanical catalysis," and have been used to study peptidyl-prolyl cis-trans isomerization and other mechanical properties of proteins. Here, we use constant force molecular dynamics simulations to study the dynamical effects of phosphorylation on serine/threonine-proline protein motifs that are involved in the function of many proteins and have been implicated in many aberrant biological processes. We show that the rate of cis-trans isomerization is slowed down by phosphorylation, in excellent agreement with experiments. We use a well-grounded theory to describe the force dependent rate of isomerization. The calculated rates at zero force are also in excellent agreement with experimentally measured rates, providing additional validation of the models and force field parameters. Our results suggest that the slowdown in the rate upon phosphorylation is mainly due to an increase in the friction along the peptidyl-prolyl bond angle during isomerization. Our results provide a microscopic description of the dynamical effects of post-translational phosphorylation on cis-trans isomerization and insights into the properties of proteins under tension.

Dynamical role of phosphorylation on serine/threonine-proline Pin1 substrates from constant force molecular dynamics simulations

NASA Astrophysics Data System (ADS)

Velazquez, Hector A.; Hamelberg, Donald

2015-02-01

Cis-trans isomerization of peptidyl-prolyl bonds of the protein backbone plays an important role in numerous biological processes. Cis-trans isomerization can be the rate-limiting step due its extremely slow dynamics, compared to the millisecond time scale of many processes, and is catalyzed by a widely studied family of peptidyl-prolyl cis-trans isomerase enzymes. Also, mechanical forces along the peptide chain can speed up the rate of isomerization, resulting in "mechanical catalysis," and have been used to study peptidyl-prolyl cis-trans isomerization and other mechanical properties of proteins. Here, we use constant force molecular dynamics simulations to study the dynamical effects of phosphorylation on serine/threonine-proline protein motifs that are involved in the function of many proteins and have been implicated in many aberrant biological processes. We show that the rate of cis-trans isomerization is slowed down by phosphorylation, in excellent agreement with experiments. We use a well-grounded theory to describe the force dependent rate of isomerization. The calculated rates at zero force are also in excellent agreement with experimentally measured rates, providing additional validation of the models and force field parameters. Our results suggest that the slowdown in the rate upon phosphorylation is mainly due to an increase in the friction along the peptidyl-prolyl bond angle during isomerization. Our results provide a microscopic description of the dynamical effects of post-translational phosphorylation on cis-trans isomerization and insights into the properties of proteins under tension.

Arginine side chain stacking with peptide plane stabilizes the protein helix conformation in a cooperative way.

PubMed

Wang, Jia; Chen, Jingfei; Li, Jingwen; An, Liaoyuan; Wang, Yefei; Huang, Qingshan; Yao, Lishan

2018-06-01

A combined experimental and computational study is performed for arginine side chain stacking with the protein α-helix. Theremostability measurements of Aristaless homeodomain, a helical protein, suggest that mutating the arginine residue R106, R137 or R141, which has the guanidino side chain stacking with the peptide plane, to alanine, destabilizes the protein. The R-PP stacking has an energy of ∼0.2-0.4 kcal/mol. This stacking interaction mainly comes from dispersion and electrostatics, based on MP2 calculations with the energy decomposition analysis. The calculations also suggest that the stacking stabilizes 2 backbone-backbone h-bonds (i→i-4 and i-3→i-7) in a cooperative way. Desolvation and electrostatic polarization are responsible for cooperativity with the i→i-4 and i-3→i-7 h-bonds, respectively. This cooperativity is supported by a protein α-helices h-bond survey in the pdb databank where stacking shortens the corresponding h-bond distances. © 2018 Wiley Periodicals, Inc.

An energetic scale for equilibrium H/D fractionation factors illuminates hydrogen bond free energies in proteins

PubMed Central

Cao, Zheng; Bowie, James U

2014-01-01

Equilibrium H/D fractionation factors have been extensively employed to qualitatively assess hydrogen bond strengths in protein structure, enzyme active sites, and DNA. It remains unclear how fractionation factors correlate with hydrogen bond free energies, however. Here we develop an empirical relationship between fractionation factors and free energy, allowing for the simple and quantitative measurement of hydrogen bond free energies. Applying our empirical relationship to prior fractionation factor studies in proteins, we find: [1] Within the folded state, backbone hydrogen bonds are only marginally stronger on average in α-helices compared to β-sheets by ∼0.2 kcal/mol. [2] Charge-stabilized hydrogen bonds are stronger than neutral hydrogen bonds by ∼2 kcal/mol on average, and can be as strong as –7 kcal/mol. [3] Changes in a few hydrogen bonds during an enzyme catalytic cycle can stabilize an intermediate state by –4.2 kcal/mol. [4] Backbone hydrogen bonds can make a large overall contribution to the energetics of conformational changes, possibly playing an important role in directing conformational changes. [5] Backbone hydrogen bonding becomes more uniform overall upon ligand binding, which may facilitate participation of the entire protein structure in events at the active site. Our energetic scale provides a simple method for further exploration of hydrogen bond free energies. PMID:24501090

Semiempirical prediction of protein folds

NASA Astrophysics Data System (ADS)

Fernández, Ariel; Colubri, Andrés; Appignanesi, Gustavo

2001-08-01

We introduce a semiempirical approach to predict ab initio expeditious pathways and native backbone geometries of proteins that fold under in vitro renaturation conditions. The algorithm is engineered to incorporate a discrete codification of local steric hindrances that constrain the movements of the peptide backbone throughout the folding process. Thus, the torsional state of the chain is assumed to be conditioned by the fact that hopping from one basin of attraction to another in the Ramachandran map (local potential energy surface) of each residue is energetically more costly than the search for a specific (Φ, Ψ) torsional state within a single basin. A combinatorial procedure is introduced to evaluate coarsely defined torsional states of the chain defined ``modulo basins'' and translate them into meaningful patterns of long range interactions. Thus, an algorithm for structure prediction is designed based on the fact that local contributions to the potential energy may be subsumed into time-evolving conformational constraints defining sets of restricted backbone geometries whereupon the patterns of nonbonded interactions are constructed. The predictive power of the algorithm is assessed by (a) computing ab initio folding pathways for mammalian ubiquitin that ultimately yield a stable structural pattern reproducing all of its native features, (b) determining the nucleating event that triggers the hydrophobic collapse of the chain, and (c) comparing coarse predictions of the stable folds of moderately large proteins (N~100) with structural information extracted from the protein data bank.

Contact- and distance-based principal component analysis of protein dynamics.

PubMed

Ernst, Matthias; Sittel, Florian; Stock, Gerhard

2015-12-28

To interpret molecular dynamics simulations of complex systems, systematic dimensionality reduction methods such as principal component analysis (PCA) represent a well-established and popular approach. Apart from Cartesian coordinates, internal coordinates, e.g., backbone dihedral angles or various kinds of distances, may be used as input data in a PCA. Adopting two well-known model problems, folding of villin headpiece and the functional dynamics of BPTI, a systematic study of PCA using distance-based measures is presented which employs distances between Cα-atoms as well as distances between inter-residue contacts including side chains. While this approach seems prohibitive for larger systems due to the quadratic scaling of the number of distances with the size of the molecule, it is shown that it is sufficient (and sometimes even better) to include only relatively few selected distances in the analysis. The quality of the PCA is assessed by considering the resolution of the resulting free energy landscape (to identify metastable conformational states and barriers) and the decay behavior of the corresponding autocorrelation functions (to test the time scale separation of the PCA). By comparing results obtained with distance-based, dihedral angle, and Cartesian coordinates, the study shows that the choice of input variables may drastically influence the outcome of a PCA.

Contact- and distance-based principal component analysis of protein dynamics

NASA Astrophysics Data System (ADS)

Ernst, Matthias; Sittel, Florian; Stock, Gerhard

2015-12-01

To interpret molecular dynamics simulations of complex systems, systematic dimensionality reduction methods such as principal component analysis (PCA) represent a well-established and popular approach. Apart from Cartesian coordinates, internal coordinates, e.g., backbone dihedral angles or various kinds of distances, may be used as input data in a PCA. Adopting two well-known model problems, folding of villin headpiece and the functional dynamics of BPTI, a systematic study of PCA using distance-based measures is presented which employs distances between Cα-atoms as well as distances between inter-residue contacts including side chains. While this approach seems prohibitive for larger systems due to the quadratic scaling of the number of distances with the size of the molecule, it is shown that it is sufficient (and sometimes even better) to include only relatively few selected distances in the analysis. The quality of the PCA is assessed by considering the resolution of the resulting free energy landscape (to identify metastable conformational states and barriers) and the decay behavior of the corresponding autocorrelation functions (to test the time scale separation of the PCA). By comparing results obtained with distance-based, dihedral angle, and Cartesian coordinates, the study shows that the choice of input variables may drastically influence the outcome of a PCA.

Structural and dynamic characterization of eukaryotic gene regulatory protein domains in solution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Andrew Loyd

Solution NMR was primarily used to characterize structure and dynamics in two different eukaryotic protein systems: the δ-Al-ε activation domain from c-jun and the Drosophila RNA-binding protein Sex-lethal. The second system is the Drosophila Sex-lethal (Sxl) protein, an RNA-binding protein which is the ``master switch`` in sex determination. Sxl contains two adjacent RNA-binding domains (RBDs) of the RNP consensus-type. The NMR spectrum of the second RBD (Sxl-RBD2) was assigned using multidimensional heteronuclear NMR, and an intermediate-resolution family of structures was calculated from primarily NOE distance restraints. The overall fold was determined to be similar to other RBDs: a βαβ-βαβ patternmore » of secondary structure, with the two helices packed against a 4-stranded anti-parallel β-sheet. In addition 15N T 1, T 2, and 15N/ 1H NOE relaxation measurements were carried out to characterize the backbone dynamics of Sxl-RBD2 in solution. RNA corresponding to the polypyrimidine tract of transformer pre-mRNA was generated and titrated into 3 different Sxl-RBD protein constructs. Combining Sxl-RBD1+2 (bht RBDs) with this RNA formed a specific, high affinity protein/RNA complex that is amenable to further NMR characterization. The backbone 1H, 13C, and 15N resonances of Sxl-RBD1+2 were assigned using a triple-resonance approach, and 15N relaxation experiments were carried out to characterize the backbone dynamics of this complex. The changes in chemical shift in Sxl-RBD1+2 upon binding RNA are observed using Sxl-RBD2 as a substitute for unbound Sxl-RBD1+2. This allowed the binding interface to be qualitatively mapped for the second domain.« less

Improved protein surface comparison and application to low-resolution protein structure data

PubMed Central

2010-01-01

Background Recent advancements of experimental techniques for determining protein tertiary structures raise significant challenges for protein bioinformatics. With the number of known structures of unknown function expanding at a rapid pace, an urgent task is to provide reliable clues to their biological function on a large scale. Conventional approaches for structure comparison are not suitable for a real-time database search due to their slow speed. Moreover, a new challenge has arisen from recent techniques such as electron microscopy (EM), which provide low-resolution structure data. Previously, we have introduced a method for protein surface shape representation using the 3D Zernike descriptors (3DZDs). The 3DZD enables fast structure database searches, taking advantage of its rotation invariance and compact representation. The search results of protein surface represented with the 3DZD has showngood agreement with the existing structure classifications, but some discrepancies were also observed. Results The three new surface representations of backbone atoms, originally devised all-atom-surface representation, and the combination of all-atom surface with the backbone representation are examined. All representations are encoded with the 3DZD. Also, we have investigated the applicability of the 3DZD for searching protein EM density maps of varying resolutions. The surface representations are evaluated on structure retrieval using two existing classifications, SCOP and the CE-based classification. Conclusions Overall, the 3DZDs representing backbone atoms show better retrieval performance than the original all-atom surface representation. The performance further improved when the two representations are combined. Moreover, we observed that the 3DZD is also powerful in comparing low-resolution structures obtained by electron microscopy. PMID:21172052

Strike a Balance: Optimization of Backbone Torsion Parameters of AMBER Polarizable Force Field for Simulations of Proteins and Peptides

PubMed Central

WANG, ZHI-XIANG; ZHANG, WEI; WU, CHUN; LEI, HONGXING; CIEPLAK, PIOTR; DUAN, YONG

2014-01-01

Based on the AMBER polarizable model (ff02), we have reoptimized the parameters related to the main-chain (Φ, Ψ) torsion angles by fitting to the Boltzmann-weighted average quantum mechanical (QM) energies of the important regions (i.e., β, PII, αR, and αL regions). Following the naming convention of the AMBER force field series, this release will be called ff02pol.rl The force field has been assessed both by energetic comparison against the QM data and by the replica exchange molecular dynamics simulations of short alanine peptides in water. For Ace-Ala-Nme, the simulated populations in the β, PII and αR regions were approximately 30, 43, and 26%, respectively. For Ace-(Ala)7-Nme, the populations in these three regions were approximately 24, 49, and 26%. Both were in qualitative agreement with the NMR and CD experimental conclusions. In comparison with the previous force field, ff02pol.rl demonstrated good balance among these three important regions. The optimized torsion parameters, together with those in ff02, allow us to carry out simulations on proteins and peptides with the consideration of polarization. PMID:16526038

Computational Study of Environmental Effects on Torsional Free Energy Surface of N-Acetyl-N'-methyl-L-alanylamide Dipeptide

ERIC Educational Resources Information Center

Carlotto, Silvia; Zerbetto, Mirco

2014-01-01

We propose an articulated computational experiment in which both quantum mechanics (QM) and molecular mechanics (MM) methods are employed to investigate environment effects on the free energy surface for the backbone dihedral angles rotation of the small dipeptide N-Acetyl-N'-methyl-L-alanylamide. This computation exercise is appropriate for an…

Methods in Enzymology: “Flexible backbone sampling methods to model and design protein alternative conformations”

PubMed Central

Ollikainen, Noah; Smith, Colin A.; Fraser, James S.; Kortemme, Tanja

2013-01-01

Sampling alternative conformations is key to understanding how proteins work and engineering them for new functions. However, accurately characterizing and modeling protein conformational ensembles remains experimentally and computationally challenging. These challenges must be met before protein conformational heterogeneity can be exploited in protein engineering and design. Here, as a stepping stone, we describe methods to detect alternative conformations in proteins and strategies to model these near-native conformational changes based on backrub-type Monte Carlo moves in Rosetta. We illustrate how Rosetta simulations that apply backrub moves improve modeling of point mutant side chain conformations, native side chain conformational heterogeneity, functional conformational changes, tolerated sequence space, protein interaction specificity, and amino acid co-variation across protein-protein interfaces. We include relevant Rosetta command lines and RosettaScripts to encourage the application of these types of simulations to other systems. Our work highlights that critical scoring and sampling improvements will be necessary to approximate conformational landscapes. Challenges for the future development of these methods include modeling conformational changes that propagate away from designed mutation sites and modulating backbone flexibility to predictively design functionally important conformational heterogeneity. PMID:23422426

Density functional theory study of the conformational space of an infinitely long polypeptide chain

NASA Astrophysics Data System (ADS)

Ireta, Joel; Scheffler, Matthias

2009-08-01

The backbone conformational space of infinitely long polyalanine is investigated with density-functional theory and mapping the potential energy surface in terms of (L, θ) cylindrical coordinates. A comparison of the obtained (L, θ) Ramachandran-like plot with results from an extended set of protein structures shows excellent conformity, with the exception of the polyproline II region. It is demonstrated the usefulness of infinitely long polypeptide models for investigating the influence of hydrogen bonding and its cooperative effect on the backbone conformations. The results imply that hydrogen bonding together with long-range electrostatics is the main actuator for most of the structures assumed by protein residues.

Molecular Dynamics Simulation of Membranes and a Transmembrane Helix

NASA Astrophysics Data System (ADS)

Duong, Tap Ha; Mehler, Ernest L.; Weinstein, Harel

1999-05-01

Three molecular dynamics (MD) simulations of 1.5-ns length were carried out on fully hydrated patches of dimyristoyl phosphatidylcholine (DMPC) bilayers in the liquid-crystalline phase. The simulations were performed using different ensembles and electrostatic conditions: a microcanonical ensemble or constant pressure-temperature ensemble, with or without truncated electrostatic interactions. Calculated properties of the membrane patches from the three different protocols were compared to available data from experiments. These data include the resulting overall geometrical dimensions, the order characteristics of the lipid hydrocarbon chains, as well as various measures of the conformations of the polar head groups. The comparisons indicate that the simulation carried out within the microcanonical ensemble with truncated electrostatic interactions yielded results closest to the experimental data, provided that the initial equilibration phase preceding the production run was sufficiently long. The effects of embedding a non-ideal helical protein domain in the membrane patch were studied with the same MD protocols. This simulation was carried out for 2.5 ns. The protein domain corresponds to the seventh transmembrane segment (TMS7) of the human serotonin 5HT 2Areceptor. The peptide is composed of two α-helical segments linked by a hinge domain around a perturbing Asn-Pro motif that produces at the end of the simulation a kink angle of nearly 80° between the two helices. Several aspects of the TMS7 structure, such as the bending angle, backbone Φ and Ψ torsion angles, the intramolecular hydrogen bonds, and the overall conformation, were found to be very similar to those determined by NMR for the corresponding transmembrane segment of the tachykinin NK-1 receptor. In general, the simulations were found to yield structural and dynamic characteristics that are in good agreement with experiment. These findings support the application of simulation methods to the study of the complex biomolecular systems at the membrane interface of cells.

Solution structure of murine macrophage inflammatory protein-2.

PubMed

Shao, W; Jerva, L F; West, J; Lolis, E; Schweitzer, B I

1998-06-09

The solution structure of murine macrophage inflammatory protein-2 (MIP-2), a heparin-binding chemokine that is secreted in response to inflammatory stimuli, has been determined using two-dimensional homonuclear and heteronuclear NMR spectroscopy. Structure calculations were carried out by means of torsion-angle molecular dynamics using the program X-PLOR. The structure is based on a total of 2390 experimental restraints, comprising 2246 NOE-derived distance restraints, 44 distance restraints for 22 hydrogen bonds, and 100 torsion angle restraints. The structure is well-defined, with the backbone (N, Calpha, C) and heavy atom atomic rms distribution about the mean coordinates for residues 9-69 of the dimer being 0.57 +/- 0.16 A and 0.96 +/- 0.12 A, respectively. The N- and C-terminal residues (1-8 and 70-73, respectively) are disordered. The overall structure of the MIP-2 dimer is similar to that reported previously for the NMR structures of MGSA and IL-8 and consists of a six-stranded antiparallel beta-sheet (residue 25-29, 39-44, and 48-52) packed against two C-terminal antiparallel alpha-helices. A best fit superposition of the NMR structure of MIP-2 on the structures of MGSA, NAP-2, and the NMR and X-ray structures of IL-8 are 1.11, 1.02, 1.27, and 1.19 A, respectively, for the monomers, and 1.28, 1.10, 1.55, and 1.36 A, respectively, for the dimers (IL-8 residues 7-14 and 16-67, NAP-2 residues 25-84). At the tertiary level, the main differences between the MIP-2 solution structure and the IL-8, MGSA, and NAP-2 structures involve the N-terminal loop between residues 9-23 and the loops formed by residues 30-38 and residues 53-58. At the quaternary level, the difference between MIP-2 and IL-8, MGSA, or NAP-2 results from differing interhelical angles and separations.

Solid-state NMR spectroscopy of 18.5 kDa myelin basic protein reconstituted with lipid vesicles: spectroscopic characterisation and spectral assignments of solvent-exposed protein fragments.

PubMed

Zhong, Ligang; Bamm, Vladimir V; Ahmed, Mumdooh A M; Harauz, George; Ladizhansky, Vladimir

2007-12-01

Myelin basic protein (MBP, 18.5 kDa isoform) is a peripheral membrane protein that is essential for maintaining the structural integrity of the multilamellar myelin sheath of the central nervous system. Reconstitution of the most abundant 18.5 kDa MBP isoform with lipid vesicles yields an aggregated assembly mimicking the protein's natural environment, but which is not amenable to standard solution NMR spectroscopy. On the other hand, the mobility of MBP in such a system is variable, depends on the local strength of the protein-lipid interaction, and in general is of such a time scale that the dipolar interactions are averaged out. Here, we used a combination of solution and solid-state NMR (ssNMR) approaches: J-coupling-driven polarization transfers were combined with magic angle spinning and high-power decoupling to yield high-resolution spectra of the mobile fragments of 18.5 kDa murine MBP in membrane-associated form. To partially circumvent the problem of short transverse relaxation, we implemented three-dimensional constant-time correlation experiments (NCOCX, NCACX, CONCACX, and CAN(CO)CX) that were able to provide interresidue and intraresidue backbone correlations. These experiments resulted in partial spectral assignments for mobile fragments of the protein. Additional nuclear Overhauser effect spectroscopy (NOESY)-based experiments revealed that the mobile fragments were exposed to solvent and were likely located outside the lipid bilayer, or in its hydrophilic portion. Chemical shift index analysis showed that the fragments were largely disordered under these conditions. These combined approaches are applicable to ssNMR investigations of other peripheral membrane proteins reconstituted with lipids.

Local Structural Differences in Homologous Proteins: Specificities in Different SCOP Classes

PubMed Central

Joseph, Agnel Praveen; Valadié, Hélène; Srinivasan, Narayanaswamy; de Brevern, Alexandre G.

2012-01-01

The constant increase in the number of solved protein structures is of great help in understanding the basic principles behind protein folding and evolution. 3-D structural knowledge is valuable in designing and developing methods for comparison, modelling and prediction of protein structures. These approaches for structure analysis can be directly implicated in studying protein function and for drug design. The backbone of a protein structure favours certain local conformations which include α-helices, β-strands and turns. Libraries of limited number of local conformations (Structural Alphabets) were developed in the past to obtain a useful categorization of backbone conformation. Protein Block (PB) is one such Structural Alphabet that gave a reasonable structure approximation of 0.42 Å. In this study, we use PB description of local structures to analyse conformations that are preferred sites for structural variations and insertions, among group of related folds. This knowledge can be utilized in improving tools for structure comparison that work by analysing local structure similarities. Conformational differences between homologous proteins are known to occur often in the regions comprising turns and loops. Interestingly, these differences are found to have specific preferences depending upon the structural classes of proteins. Such class-specific preferences are mainly seen in the all-β class with changes involving short helical conformations and hairpin turns. A test carried out on a benchmark dataset also indicates that the use of knowledge on the class specific variations can improve the performance of a PB based structure comparison approach. The preference for the indel sites also seem to be confined to a few backbone conformations involving β-turns and helix C-caps. These are mainly associated with short loops joining the regular secondary structures that mediate a reversal in the chain direction. Rare β-turns of type I’ and II’ are also identified as preferred sites for insertions. PMID:22745680

Validating a Coarse-Grained Potential Energy Function through Protein Loop Modelling

PubMed Central

MacDonald, James T.; Kelley, Lawrence A.; Freemont, Paul S.

2013-01-01

Coarse-grained (CG) methods for sampling protein conformational space have the potential to increase computational efficiency by reducing the degrees of freedom. The gain in computational efficiency of CG methods often comes at the expense of non-protein like local conformational features. This could cause problems when transitioning to full atom models in a hierarchical framework. Here, a CG potential energy function was validated by applying it to the problem of loop prediction. A novel method to sample the conformational space of backbone atoms was benchmarked using a standard test set consisting of 351 distinct loops. This method used a sequence-independent CG potential energy function representing the protein using -carbon positions only and sampling conformations with a Monte Carlo simulated annealing based protocol. Backbone atoms were added using a method previously described and then gradient minimised in the Rosetta force field. Despite the CG potential energy function being sequence-independent, the method performed similarly to methods that explicitly use either fragments of known protein backbones with similar sequences or residue-specific /-maps to restrict the search space. The method was also able to predict with sub-Angstrom accuracy two out of seven loops from recently solved crystal structures of proteins with low sequence and structure similarity to previously deposited structures in the PDB. The ability to sample realistic loop conformations directly from a potential energy function enables the incorporation of additional geometric restraints and the use of more advanced sampling methods in a way that is not possible to do easily with fragment replacement methods and also enable multi-scale simulations for protein design and protein structure prediction. These restraints could be derived from experimental data or could be design restraints in the case of computational protein design. C++ source code is available for download from http://www.sbg.bio.ic.ac.uk/phyre2/PD2/. PMID:23824634

General order parameter based correlation analysis of protein backbone motions between experimental NMR relaxation measurements and molecular dynamics simulations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Qing; Shi, Chaowei; Yu, Lu

Internal backbone dynamic motions are essential for different protein functions and occur on a wide range of time scales, from femtoseconds to seconds. Molecular dynamic (MD) simulations and nuclear magnetic resonance (NMR) spin relaxation measurements are valuable tools to gain access to fast (nanosecond) internal motions. However, there exist few reports on correlation analysis between MD and NMR relaxation data. Here, backbone relaxation measurements of {sup 15}N-labeled SH3 (Src homology 3) domain proteins in aqueous buffer were used to generate general order parameters (S{sup 2}) using a model-free approach. Simultaneously, 80 ns MD simulations of SH3 domain proteins in amore » defined hydrated box at neutral pH were conducted and the general order parameters (S{sup 2}) were derived from the MD trajectory. Correlation analysis using the Gromos force field indicated that S{sup 2} values from NMR relaxation measurements and MD simulations were significantly different. MD simulations were performed on models with different charge states for three histidine residues, and with different water models, which were SPC (simple point charge) water model and SPC/E (extended simple point charge) water model. S{sup 2} parameters from MD simulations with charges for all three histidines and with the SPC/E water model correlated well with S{sup 2} calculated from the experimental NMR relaxation measurements, in a site-specific manner. - Highlights: • Correlation analysis between NMR relaxation measurements and MD simulations. • General order parameter (S{sup 2}) as common reference between the two methods. • Different protein dynamics with different Histidine charge states in neutral pH. • Different protein dynamics with different water models.« less

Charge Neutralization Drives the Shape Reconfiguration of DNA Nanotubes.

PubMed

Liu, Pi; Zhao, Yan; Liu, Xiaoguo; Sun, Jixue; Xu, Dede; Li, Yang; Li, Qian; Wang, Lihua; Yang, Sichun; Fan, Chunhai; Lin, Jianping

2018-05-04

Reconfiguration of membrane protein channels for gated transport is highly regulated under physiological conditions. However, a mechanistic understanding of such channels remains challenging owing to the difficulty in probing subtle gating-associated structural changes. Herein, we show that charge neutralization can drive the shape reconfiguration of a biomimetic 6-helix bundle DNA nanotube (6HB). Specifically, 6HB adopts a compact state when its charge is neutralized by Mg 2+ ; whereas Na + switches it to the expanded state, as revealed by MD simulations, small-angle X-ray scattering (SAXS), and FRET characterization. Furthermore, partial neutralization of the DNA backbone charges by chemical modification renders 6HB compact and insensitive to ions, suggesting an interplay between electrostatic and hydrophobic forces in the channels. This system provides a platform for understanding the structure-function relationship of biological channels and designing rules for the shape control of DNA nanostructures in biomedical applications. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Side-chain mobility in the folded state of Myoglobin

NASA Astrophysics Data System (ADS)

Lammert, Heiko; Onuchic, Jose

We study the accessibility of alternative side-chain rotamer configurations in the native state of Myoglobin, using an all-atom structure-based model. From long, unbiased simulation trajectories we determine occupancies of rotameric states and also estimate configurational and vibrational entropies. Direct sampling of the full native-state dynamics, enabled by the simple model, reveals facilitation of side-chain motions by backbone dynamics. Correlations between different dihedral angles are quantified and prove to be weak. We confirm global trends in the mobilities of side-chains, following burial and also the chemical character of residues. Surface residues loose little configurational entropy upon folding; side-chains contribute significantly to the entropy of the folded state. Mobilities of buried side-chains vary strongly with temperature. At ambient temperature, individual side-chains in the core of the protein gain substantial access to alternative rotamers, with occupancies that are likely observable experimentally. Finally, the dynamics of buried side-chains may be linked to the internal pockets, available to ligand gas molecules in Myoglobin.

Which similarity measure is better for analyzing protein structures in a molecular dynamics trajectory?

PubMed

Cossio, Pilar; Laio, Alessandro; Pietrucci, Fabio

2011-06-14

An important step in the computer simulation of the dynamics of biomolecules is the comparison of structures in a trajectory by exploiting a measure of distance. This allows distinguishing structures which are geometrically similar from those which are different. By analyzing microseconds-long all-atom molecular dynamics simulations of a polypeptide, we find that a distance based on backbone dihedral angles performs very well in distinguishing structures that are kinetically correlated from those that are not, while the widely used C(α) root mean square distance performs more poorly. The root mean square difference between contact matrices turns out instead to be the metric providing the highest clustering coefficient, namely, according to this similarity measure, the neighbors of a structure are also, on average, neighbors among themselves. We also propose a combined distance measure which, for the system considered here, performs well both for distinguishing structures which are distant in time and for giving a consistent cluster analysis. This journal is © the Owner Societies 2011

¹⁵N, ¹³C and ¹H resonance assignments and secondary structure determination of a variable heavy domain of a heavy chain antibody.

PubMed

Prosser, Christine E; Waters, Lorna C; Muskett, Frederick W; Veverka, Vaclav; Addis, Philip W; Griffin, Laura M; Baker, Terry S; Lawson, Alastair D G; Wernery, Ulrich; Kinne, Jorg; Henry, Alistair J; Taylor, Richard J; Carr, Mark D

2014-04-01

Heavy chain antibodies differ in structure to conventional antibodies lacking both the light chain and the first heavy chain constant domain (CH1). Characteristics of the antigen-binding variable heavy domain of the heavy chain antibody (VHH) including the smaller size, high solubility and stability make them an attractive alternative to more traditional antibody fragments for detailed NMR-based structural analysis. Here we report essentially complete backbone and side chain (15)N, (13)C and (1)H assignments for a free VHH. Analysis of the backbone chemical shift data obtained indicates that the VHH is comprised predominantly of β-sheets corresponding to nearly 60% of the protein backbone.

NMR structural and dynamic characterization of the acid-unfolded state of apomyoglobin provides insights into the early events in protein folding.

PubMed

Yao, J; Chung, J; Eliezer, D; Wright, P E; Dyson, H J

2001-03-27

Apomyoglobin forms a denatured state under low-salt conditions at pH 2.3. The conformational propensities and polypeptide backbone dynamics of this state have been characterized by NMR. Nearly complete backbone and some side chain resonance assignments have been obtained, using a triple-resonance assignment strategy tailored to low protein concentration (0.2 mM) and poor chemical shift dispersion. An estimate of the population and location of residual secondary structure has been made by examining deviations of (13)C(alpha), (13)CO, and (1)H(alpha) chemical shifts from random coil values, scalar (3)J(HN,H)(alpha) coupling constants and (1)H-(1)H NOEs. Chemical shifts constitute a highly reliable indicator of secondary structural preferences, provided the appropriate random coil chemical shift references are used, but in the case of acid-unfolded apomyoglobin, (3)J(HN,H)(alpha) coupling constants are poor diagnostics of secondary structure formation. Substantial populations of helical structure, in dynamic equilibrium with unfolded states, are formed in regions corresponding to the A and H helices of the folded protein. In addition, the deviation of the chemical shifts from random coil values indicates the presence of helical structure encompassing the D helix and extending into the first turn of the E helix. The polypeptide backbone dynamics of acid-unfolded apomyoglobin have been investigated using reduced spectral density function analysis of (15)N relaxation data. The spectral density J(omega(N)) is particularly sensitive to variations in backbone fluctuations on the picosecond to nanosecond time scale. The central region of the polypeptide spanning the C-terminal half of the E helix, the EF turn, and the F helix behaves as a free-flight random coil chain, but there is evidence from J(omega(N)) of restricted motions on the picosecond to nanosecond time scale in the A and H helix regions where there is a propensity to populate helical secondary structure in the acid-unfolded state. Backbone fluctuations are also restricted in parts of the B and G helices due to formation of local hydrophobic clusters. Regions of restricted backbone flexibility are generally associated with large buried surface area. A significant increase in J(0) is observed for the NH resonances of some residues located in the A and G helices of the folded protein and is associated with fluctuations on a microsecond to millisecond time scale that probably arise from transient contacts between these distant regions of the polypeptide chain. Our results indicate that the equilibrium unfolded state of apomyoglobin formed at pH 2.3 is an excellent model for the events that are expected to occur in the earliest stages of protein folding, providing insights into the regions of the polypeptide that spontaneously undergo local hydrophobic collapse and sample nativelike secondary structure.

Consequences of non-uniformity in the stoichiometry of component fractions within one and two loops models of alpha-helical peptides

USDA-ARS?s Scientific Manuscript database

Atoms in biomolecular structures like alpha helices contain an array of distances and angles which include abundant multiple patterns of redundancies. Thus all peptides backbones contain the three atom sequence N-C*C, whereas the repeating set of a four atom sequences (N-C*C-N, C*-C-N-C*, and C-N-C...

Packing of sidechains in low-resolution models for proteins.

PubMed

Keskin, O; Bahar, I

1998-01-01

Atomic level rotamer libraries for sidechains in proteins have been proposed by several groups. Conformations of side groups in coarse-grained models, on the other hand, have not yet been analyzed, although low resolution approaches are the only efficient way to explore global structural features. A residue-specific backbone-dependent library for sidechain isomers, compatible with a coarse-grained model, is proposed. The isomeric states are utilized in packing sidechains of known backbone structures. Sidechain positions are predicted with a root-mean-square deviation (r.m.s.d.) of 2.40 A with respect to crystal structure for 50 test proteins. The rmsd for core residues is 1.60 A and decreases to 1.35 A when conformational correlations and directional effects in inter-residue couplings are considered. An automated method for assigning sidechain positions in coarse-grained model proteins is proposed and made available on the internet; the method accounts satisfactorily for sidechain packing, particularly in the core.

The Dominant Folding Route Minimizes Backbone Distortion in SH3

PubMed Central

Lammert, Heiko; Noel, Jeffrey K.; Onuchic, José N.

2012-01-01

Energetic frustration in protein folding is minimized by evolution to create a smooth and robust energy landscape. As a result the geometry of the native structure provides key constraints that shape protein folding mechanisms. Chain connectivity in particular has been identified as an essential component for realistic behavior of protein folding models. We study the quantitative balance of energetic and geometrical influences on the folding of SH3 in a structure-based model with minimal energetic frustration. A decomposition of the two-dimensional free energy landscape for the folding reaction into relevant energy and entropy contributions reveals that the entropy of the chain is not responsible for the folding mechanism. Instead the preferred folding route through the transition state arises from a cooperative energetic effect. Off-pathway structures are penalized by excess distortion in local backbone configurations and contact pair distances. This energy cost is a new ingredient in the malleable balance of interactions that controls the choice of routes during protein folding. PMID:23166485

Chemical synthesis of membrane proteins by the removable backbone modification method.

PubMed

Tang, Shan; Zuo, Chao; Huang, Dong-Liang; Cai, Xiao-Ying; Zhang, Long-Hua; Tian, Chang-Lin; Zheng, Ji-Shen; Liu, Lei

2017-12-01

Chemical synthesis can produce membrane proteins bearing specifically designed modifications (e.g., phosphorylation, isotope labeling) that are difficult to obtain through recombinant protein expression approaches. The resulting homogeneously modified synthetic membrane proteins are valuable tools for many advanced biochemical and biophysical studies. This protocol describes the chemical synthesis of membrane proteins by condensation of transmembrane peptide segments through native chemical ligation. To avoid common problems encountered due to the poor solubility of transmembrane peptides in almost any solvent, we describe an effective procedure for the chemical synthesis of membrane proteins through the removable-backbone modification (RBM) strategy. Two key steps of this protocol are: (i) installation of solubilizing Arg4-tagged RBM groups into the transmembrane peptides at any primary amino acid through Fmoc (9-fluorenylmethyloxycarbonyl) solid-phase peptide synthesis and (ii) native ligation of the full-length sequence, followed by removal of the RBM tags by TFA (trifluoroacetic acid) cocktails to afford the native protein. The installation of RBM groups is achieved by using 4-methoxy-5-nitrosalicyladehyde by reduction amination to incorporate an activated O-to-N acyl transfer auxiliary. The Arg4-tag-modified membrane-spanning peptide segments behave like water-soluble peptides to facilitate their purification, ligation and mass characterization.

Salting effects on protein components in aqueous NaCl and urea solutions: toward understanding of urea-induced protein denaturation.

PubMed

Li, Weifeng; Zhou, Ruhong; Mu, Yuguang

2012-02-02

The mechanism of urea-induced protein denaturation is explored through studying the salting effect of urea on 14 amino acid side chain analogues, and N-methylacetamide (NMA) which mimics the protein backbone. The solvation free energies of the 15 molecules were calculated in pure water, aqueous urea, and NaCl solutions. Our results show that NaCl displays strong capability to salt out all 15 molecules, while urea facilitates the solvation (salting-in) of all the 15 molecules on the other hand. The salting effect is found to be largely enthalpy-driven for both NaCl and urea. Our observations can explain the higher stability of protein's secondary and tertiary structures in typical salt solutions than that in pure water. Meanwhile, urea's capability to better solvate protein backbone and side-chain components can be extrapolated to explain protein's denaturation in aqueous urea solution. Urea salts in molecules through direct binding to solute surface, and the strength is linearly dependent on the number of heavy atoms of solute molecules. The van der Waals interactions are found to be the dominant force, which challenges a hydrogen-bonding-driven mechanism proposed previously.

Antibody side chain conformations are position-dependent.

PubMed

Leem, Jinwoo; Georges, Guy; Shi, Jiye; Deane, Charlotte M

2018-04-01

Side chain prediction is an integral component of computational antibody design and structure prediction. Current antibody modelling tools use backbone-dependent rotamer libraries with conformations taken from general proteins. Here we present our antibody-specific rotamer library, where rotamers are binned according to their immunogenetics (IMGT) position, rather than their local backbone geometry. We find that for some amino acid types at certain positions, only a restricted number of side chain conformations are ever observed. Using this information, we are able to reduce the breadth of the rotamer sampling space. Based on our rotamer library, we built a side chain predictor, position-dependent antibody rotamer swapper (PEARS). On a blind test set of 95 antibody model structures, PEARS had the highest average χ 1 and χ1+2 accuracy (78.7% and 64.8%) compared to three leading backbone-dependent side chain predictors. Our use of IMGT position, rather than backbone ϕ/ψ, meant that PEARS was more robust to errors in the backbone of the model structure. PEARS also achieved the lowest number of side chain-side chain clashes. PEARS is freely available as a web application at http://opig.stats.ox.ac.uk/webapps/pears. © 2018 Wiley Periodicals, Inc.

Discrete RNA libraries from pseudo-torsional space

PubMed Central

Humphris-Narayanan, Elisabeth

2012-01-01

The discovery that RNA molecules can fold into complex structures and carry out diverse cellular roles has led to interest in developing tools for modeling RNA tertiary structure. While significant progress has been made in establishing that the RNA backbone is rotameric, few libraries of discrete conformations specifically for use in RNA modeling have been validated. Here, we present six libraries of discrete RNA conformations based on a simplified pseudo-torsional notation of the RNA backbone, comparable to phi and psi in the protein backbone. We evaluate the ability of each library to represent single nucleotide backbone conformations and we show how individual library fragments can be assembled into dinucleotides that are consistent with established RNA backbone descriptors spanning from sugar to sugar. We then use each library to build all-atom models of 20 test folds and we show how the composition of a fragment library can limit model quality. Despite the limitations inherent in using discretized libraries, we find that several hundred discrete fragments can rebuild RNA folds up to 174 nucleotides in length with atomic-level accuracy (<1.5Å RMSD). We anticipate the libraries presented here could easily be incorporated into RNA structural modeling, analysis, or refinement tools. PMID:22425640

TRX-LOGOS - a graphical tool to demonstrate DNA information content dependent upon backbone dynamics in addition to base sequence.

PubMed

Fortin, Connor H; Schulze, Katharina V; Babbitt, Gregory A

2015-01-01

It is now widely-accepted that DNA sequences defining DNA-protein interactions functionally depend upon local biophysical features of DNA backbone that are important in defining sites of binding interaction in the genome (e.g. DNA shape, charge and intrinsic dynamics). However, these physical features of DNA polymer are not directly apparent when analyzing and viewing Shannon information content calculated at single nucleobases in a traditional sequence logo plot. Thus, sequence logos plots are severely limited in that they convey no explicit information regarding the structural dynamics of DNA backbone, a feature often critical to binding specificity. We present TRX-LOGOS, an R software package and Perl wrapper code that interfaces the JASPAR database for computational regulatory genomics. TRX-LOGOS extends the traditional sequence logo plot to include Shannon information content calculated with regard to the dinucleotide-based BI-BII conformation shifts in phosphate linkages on the DNA backbone, thereby adding a visual measure of intrinsic DNA flexibility that can be critical for many DNA-protein interactions. TRX-LOGOS is available as an R graphics module offered at both SourceForge and as a download supplement at this journal. To demonstrate the general utility of TRX logo plots, we first calculated the information content for 416 Saccharomyces cerevisiae transcription factor binding sites functionally confirmed in the Yeastract database and matched to previously published yeast genomic alignments. We discovered that flanking regions contain significantly elevated information content at phosphate linkages than can be observed at nucleobases. We also examined broader transcription factor classifications defined by the JASPAR database, and discovered that many general signatures of transcription factor binding are locally more information rich at the level of DNA backbone dynamics than nucleobase sequence. We used TRX-logos in combination with MEGA 6.0 software for molecular evolutionary genetics analysis to visually compare the human Forkhead box/FOX protein evolution to its binding site evolution. We also compared the DNA binding signatures of human TP53 tumor suppressor determined by two different laboratory methods (SELEX and ChIP-seq). Further analysis of the entire yeast genome, center aligned at the start codon, also revealed a distinct sequence-independent 3 bp periodic pattern in information content, present only in coding region, and perhaps indicative of the non-random organization of the genetic code. TRX-LOGOS is useful in any situation in which important information content in DNA can be better visualized at the positions of phosphate linkages (i.e. dinucleotides) where the dynamic properties of the DNA backbone functions to facilitate DNA-protein interaction.

Beta-Strand Interfaces of Non-Dimeric Protein Oligomers Are Characterized by Scattered Charged Residue Patterns

PubMed Central

Feverati, Giovanni; Achoch, Mounia; Zrimi, Jihad; Vuillon, Laurent; Lesieur, Claire

2012-01-01

Protein oligomers are formed either permanently, transiently or even by default. The protein chains are associated through intermolecular interactions constituting the protein interface. The protein interfaces of 40 soluble protein oligomers of stœchiometries above two are investigated using a quantitative and qualitative methodology, which analyzes the x-ray structures of the protein oligomers and considers their interfaces as interaction networks. The protein oligomers of the dataset share the same geometry of interface, made by the association of two individual β-strands (β-interfaces), but are otherwise unrelated. The results show that the β-interfaces are made of two interdigitated interaction networks. One of them involves interactions between main chain atoms (backbone network) while the other involves interactions between side chain and backbone atoms or between only side chain atoms (side chain network). Each one has its own characteristics which can be associated to a distinct role. The secondary structure of the β-interfaces is implemented through the backbone networks which are enriched with the hydrophobic amino acids favored in intramolecular β-sheets (MCWIV). The intermolecular specificity is provided by the side chain networks via positioning different types of charged residues at the extremities (arginine) and in the middle (glutamic acid and histidine) of the interface. Such charge distribution helps discriminating between sequences of intermolecular β-strands, of intramolecular β-strands and of β-strands forming β-amyloid fibers. This might open new venues for drug designs and predictive tool developments. Moreover, the β-strands of the cholera toxin B subunit interface, when produced individually as synthetic peptides, are capable of inhibiting the assembly of the toxin into pentamers. Thus, their sequences contain the features necessary for a β-interface formation. Such β-strands could be considered as ‘assemblons’, independent associating units, by homology to the foldons (independent folding unit). Such property would be extremely valuable in term of assembly inhibitory drug development. PMID:22496732

Backbone resonance assignments of the PRYSPRY domain of TRIM25.

PubMed

Kong, Chen; Penumutchu, Srinivasa R; Hung, Kuo-Wei; Huang, Huiying; Lin, Tianwei; Yu, Chin

2015-10-01

TRIM25 is a member of the tripartite motif (TRIM) family and has been implicated in the regulation of innate immune signaling via the RIG-I (retinoic acid-inducible gene-I) pathway for antiviral defense. As the essential first step towards the structural and functional characterization of the TRIM25/RIG-I interaction, the backbone resonance of the PRYSPRY domain of TRIM25 is assigned here based on triple-resonance experiments using uniformly [(2)H, (13)C, (15)N]-labeled protein.

Conformational Plasticity of the Influenza A M2 Transmembrane Helix in Lipid Bilayers Under Varying pH, Drug Binding and Membrane Thickness

PubMed Central

Hu, Fanghao; Luo, Wenbin; Cady, Sarah D.; Hong, Mei

2010-01-01

Membrane proteins change their conformations to respond to environmental cues, thus conformational plasticity is important for function. The influenza A M2 protein forms an acid-activated proton channel important for the virus lifecycle. Here we have used solid-state NMR spectroscopy to examine the conformational plasticity of membrane-bound transmembrane domain of M2 (M2TM). 13C and 15N chemical shifts indicate coupled conformational changes of several pore-facing residues due to changes in bilayer thickness, drug binding and pH. The structural changes are attributed to the formation of a well-defined helical kink at G34 in the drug-bound state and in thick lipid bilayers, non-ideal backbone conformation of the secondary-gate residue V27 in the presence of drug, and non-ideal conformation of the proton-sensing residue H37 at high pH. The chemical shifts constrained the (ϕ, ψ) torsion angles for three basis states, the equilibrium among which explains the multiple resonances per site in the NMR spectra under different combinations of bilayer thickness, drug binding and pH conditions. Thus, conformational plasticity is important for the proton conduction and inhibition of M2TM. The study illustrates the utility of NMR chemical shifts for probing the structural plasticity and folding of membrane proteins. PMID:20883664

Backbone-only restraints for fast determination of the protein fold: The role of paramagnetism-based restraints. Cytochrome b562 as an example

NASA Astrophysics Data System (ADS)

Banci, Lucia; Bertini, Ivano; Felli, Isabella C.; Sarrou, Josephine

2005-02-01

CH α residual dipolar couplings (Δ rdc's) were measured for the oxidized cytochrome b562 from Escherichia coli as a result of its partial self-orientation in high magnetic fields due to the anisotropy of the overall magnetic susceptibility tensor. Both the low spin iron (III) heme and the four-helix bundle fold contribute to the magnetic anisotropy tensor. CH α Δ rdc's, which span a larger range than the analogous NH values (already available in the literature) sample large space variations at variance with NH Δ rdc's, which are largely isooriented within α helices. The whole structure is now significantly refined with the chemical shift index and CH α Δ rdc's. The latter are particularly useful also in defining the molecular magnetic anisotropy parameters. It is shown here that the backbone folding can be conveniently and accurately determined using backbone restraints only, which include NOEs, hydrogen bonds, residual dipolar couplings, pseudocontact shifts, and chemical shift index. All these restraints are easily and quickly determined from the backbone assignment. The calculated backbone structure is comparable to that obtained by using also side chain restraint. Furthermore, the structure obtained with backbone only restraints is, in its whole, very similar to that obtained with the complete set of restraints. The paramagnetism based restraints are shown to be absolutely relevant, especially for Δ rdc's.

Solution structure of the phosphoryl transfer complex between the cytoplasmic A domain of the mannitol transporter IIMannitol and HPr of the Escherichia coli phosphotransferase system.

PubMed

Cornilescu, Gabriel; Lee, Byeong Ryong; Cornilescu, Claudia C; Wang, Guangshun; Peterkofsky, Alan; Clore, G Marius

2002-11-01

The solution structure of the complex between the cytoplasmic A domain (IIA(Mtl)) of the mannitol transporter II(Mannitol) and the histidine-containing phosphocarrier protein (HPr) of the Escherichia coli phosphotransferase system has been solved by NMR, including the use of conjoined rigid body/torsion angle dynamics, and residual dipolar couplings, coupled with cross-validation, to permit accurate orientation of the two proteins. A convex surface on HPr, formed by helices 1 and 2, interacts with a complementary concave depression on the surface of IIA(Mtl) formed by helix 3, portions of helices 2 and 4, and beta-strands 2 and 3. The majority of intermolecular contacts are hydrophobic, with a small number of electrostatic interactions at the periphery of the interface. The active site histidines, His-15 of HPr and His-65 of IIA(Mtl), are in close spatial proximity, and a pentacoordinate phosphoryl transition state can be readily accommodated with no change in protein-protein orientation and only minimal perturbations of the backbone immediately adjacent to the histidines. Comparison with two previously solved structures of complexes of HPr with partner proteins of the phosphotransferase system, the N-terminal domain of enzyme I (EIN) and enzyme IIA(Glucose) (IIA(Glc)), reveals a number of common features despite the fact that EIN, IIA(Glc), and IIA(Mtl) bear no structural resemblance to one another. Thus, entirely different underlying structural elements can form binding surfaces for HPr that are similar in terms of both shape and residue composition. These structural comparisons illustrate the roles of surface and residue complementarity, redundancy, incremental build-up of specificity and conformational side chain plasticity in the formation of transient specific protein-protein complexes in signal transduction pathways.

Influence of proline upon the folding and geometry of the WALP19 transmembrane peptide.

PubMed

Thomas, Rachel; Vostrikov, Vitaly V; Greathouse, Denise V; Koeppe, Roger E

2009-12-22

The orientations, geometries, and lipid interactions of designed transmembrane (TM) peptides have attracted significant experimental and theoretical interest. Because the amino acid proline will introduce a known discontinuity into an alpha helix, we have sought to measure the extent of helix kinking caused by a single proline within the isolated TM helical domain of WALP19. For this purpose, we synthesized acetyl-GWWLALALAP(10)ALALALWWA-ethanolamide and included pairs of deuterated alanines by using 60-100% Fmoc-l-Ala-d(4) at selected sequence positions. Solid-state deuterium ((2)H) magnetic resonance spectra from oriented, hydrated samples (1/40, peptide/lipid; using several lipids) reveal signals from many of the alanine backbone C(alpha) deuterons as well as the alanine side-chain C(beta) methyl groups, whereas signals from C(alpha) deuterons generally have not been observed for similar peptides without proline. It is conceivable that altered peptide dynamics may be responsible for the apparent "unmasking" of the backbone resonances in the presence of the proline. Data analysis using the geometric analysis of labeled alanines (GALA) method reveals that the peptide helix is distorted due to the presence of the proline. To provide additional data points for evaluating the segmental tilt angles of the two halves of the peptide, we substituted selected leucines with l-Ala-d(4). Using this approach, we were able to deduce that the apparent average tilt of the C-terminal increases from approximately 4 degrees to approximately 12 degrees when Pro(10) is introduced. The segment N-terminal to proline is more complex and possibly is more dynamically flexible; Leu to Ala mutations within the N-terminal segment alter the average orientations of alanines in both segments. Nevertheless, in DOPC, we could estimate an apparent kink angle of approximately 19 degrees . Together, the results suggest that the central proline influences not only the geometry but also the dynamics of the membrane-spanning peptide. The results make up an important basis for understanding the functional role of proline in several families of membrane proteins.

PONDEROSA, an automated 3D-NOESY peak picking program, enables automated protein structure determination

PubMed Central

Lee, Woonghee; Kim, Jin Hae; Westler, William M.; Markley, John L.

2011-01-01

Summary: PONDEROSA (Peak-picking Of Noe Data Enabled by Restriction of Shift Assignments) accepts input information consisting of a protein sequence, backbone and sidechain NMR resonance assignments, and 3D-NOESY (13C-edited and/or 15N-edited) spectra, and returns assignments of NOESY crosspeaks, distance and angle constraints, and a reliable NMR structure represented by a family of conformers. PONDEROSA incorporates and integrates external software packages (TALOS+, STRIDE and CYANA) to carry out different steps in the structure determination. PONDEROSA implements internal functions that identify and validate NOESY peak assignments and assess the quality of the calculated three-dimensional structure of the protein. The robustness of the analysis results from PONDEROSA's hierarchical processing steps that involve iterative interaction among the internal and external modules. PONDEROSA supports a variety of input formats: SPARKY assignment table (.shifts) and spectrum file formats (.ucsf), XEASY proton file format (.prot), and NMR-STAR format (.star). To demonstrate the utility of PONDEROSA, we used the package to determine 3D structures of two proteins: human ubiquitin and Escherichia coli iron-sulfur scaffold protein variant IscU(D39A). The automatically generated structural constraints and ensembles of conformers were as good as or better than those determined previously by much less automated means. Availability: The program, in the form of binary code along with tutorials and reference manuals, is available at http://ponderosa.nmrfam.wisc.edu/. Contact: whlee@nmrfam.wisc.edu; markley@nmrfam.wisc.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:21511715

Progress in protein-protein docking: atomic resolution predictions in the CAPRI experiment using RosettaDock with an improved treatment of side-chain flexibility.

PubMed

Schueler-Furman, Ora; Wang, Chu; Baker, David

2005-08-01

RosettaDock uses real-space Monte Carlo minimization (MCM) on both rigid-body and side-chain degrees of freedom to identify the lowest free energy docked arrangement of 2 protein structures. An improved version of the method that uses gradient-based minimization for off-rotamer side-chain optimization and includes information from unbound structures was used to create predictions for Rounds 4 and 5 of CAPRI. First, large numbers of independent MCM trajectories were carried out and the lowest free energy docked configurations identified. Second, new trajectories were started from these lowest energy structures to thoroughly sample the surrounding conformation space, and the lowest energy configurations were submitted as predictions. For all cases in which there were no significant backbone conformational changes, a small number of very low-energy configurations were identified in the first, global search and subsequently found to be close to the center of the basin of attraction in the free energy landscape in the second, local search. Following the release of the experimental coordinates, it was found that the centers of these free energy minima were remarkably close to the native structures in not only the rigid-body orientation but also the detailed conformations of the side-chains. Out of 8 targets, the lowest energy models had interface root-mean-square deviations (RMSDs) less than 1.1 A from the correct structures for 6 targets, and interface RMSDs less than 0.4 A for 3 targets. The predictions were top submissions to CAPRI for Targets 11, 12, 14, 15, and 19. The close correspondence of the lowest free energy structures found in our searches to the experimental structures suggests that our free energy function is a reasonable representation of the physical chemistry, and that the real space search with full side-chain flexibility to some extent solves the protein-protein docking problem in the absence of significant backbone conformational changes. On the other hand, the approach fails when there are significant backbone conformational changes as the steric complementarity of the 2 proteins cannot be modeled without incorporating backbone flexibility, and this is the major goal of our current work.

Structure and self-assembly properties of a new chitosan-based amphiphile.

PubMed

Huang, Yuping; Yu, Hailong; Guo, Liang; Huang, Qingrong

2010-06-17

A new chitosan-based amphiphile, octanoyl-chitosan-polyethylene glycol monomethyl ether (acylChitoMPEG), has been prepared using both hydrophobic octanoyl and hydrophilic polyethylene glycol monomethyl ether (MPEG) substitutions. The success of synthesis was confirmed by Fourier transform infrared (FT-IR) and (1)H NMR spectroscopy. The synthesized acylChitoMPEG exhibited good solubility in either aqueous solution or common organic solvents such as ethanol, acetone, and CHCl(3). The self-aggregation behavior of acylChitoMPEG in solutions was studied by a combination of pyrene fluorescence technique, dynamic light scattering, atomic force microscopy, and small-angle X-ray scattering (SAXS). The critical aggregation concentration (CAC) and hydrodynamic diameter were found to be 0.066 mg/mL and 24.4 nm, respectively. SAXS results suggested a coiled structure of the triple helical acylChitoMPEG backbone with the hydrophobic moieties hiding in the center of the backbone, and the hydrophilic MPEG chains surrounding the acylChitoMPEG backbone in a random Gaussian chain conformation. Cytotoxicity results showed that acylChitoMPEG exhibited negligible cytotoxicity even at concentrations as high as 1.0 mg/mL. All results implied that acylChitoMPEG has the potential to be used for biological or medical applications.

Synthetic beta-solenoid proteins with the fragment-free computational design of a beta-hairpin extension

PubMed Central

MacDonald, James T.; Kabasakal, Burak V.; Godding, David; Kraatz, Sebastian; Henderson, Louie; Barber, James; Freemont, Paul S.; Murray, James W.

2016-01-01

The ability to design and construct structures with atomic level precision is one of the key goals of nanotechnology. Proteins offer an attractive target for atomic design because they can be synthesized chemically or biologically and can self-assemble. However, the generalized protein folding and design problem is unsolved. One approach to simplifying the problem is to use a repetitive protein as a scaffold. Repeat proteins are intrinsically modular, and their folding and structures are better understood than large globular domains. Here, we have developed a class of synthetic repeat proteins based on the pentapeptide repeat family of beta-solenoid proteins. We have constructed length variants of the basic scaffold and computationally designed de novo loops projecting from the scaffold core. The experimentally solved 3.56-Å resolution crystal structure of one designed loop matches closely the designed hairpin structure, showing the computational design of a backbone extension onto a synthetic protein core without the use of backbone fragments from known structures. Two other loop designs were not clearly resolved in the crystal structures, and one loop appeared to be in an incorrect conformation. We have also shown that the repeat unit can accommodate whole-domain insertions by inserting a domain into one of the designed loops. PMID:27573845

Ser/Thr Motifs in Transmembrane Proteins: Conservation Patterns and Effects on Local Protein Structure and Dynamics

PubMed Central

del Val, Coral; White, Stephen H.

2014-01-01

We combined systematic bioinformatics analyses and molecular dynamics simulations to assess the conservation patterns of Ser and Thr motifs in membrane proteins, and the effect of such motifs on the structure and dynamics of α-helical transmembrane (TM) segments. We find that Ser/Thr motifs are often present in β-barrel TM proteins. At least one Ser/Thr motif is present in almost half of the sequences of α-helical proteins analyzed here. The extensive bioinformatics analyses and inspection of protein structures led to the identification of molecular transporters with noticeable numbers of Ser/Thr motifs within the TM region. Given the energetic penalty for burying multiple Ser/Thr groups in the membrane hydrophobic core, the observation of transporters with multiple membrane-embedded Ser/Thr is intriguing and raises the question of how the presence of multiple Ser/Thr affects protein local structure and dynamics. Molecular dynamics simulations of four different Ser-containing model TM peptides indicate that backbone hydrogen bonding of membrane-buried Ser/Thr hydroxyl groups can significantly change the local structure and dynamics of the helix. Ser groups located close to the membrane interface can hydrogen bond to solvent water instead of protein backbone, leading to an enhanced local solvation of the peptide. PMID:22836667

Hidden Markov model approach for identifying the modular framework of the protein backbone.

PubMed

Camproux, A C; Tuffery, P; Chevrolat, J P; Boisvieux, J F; Hazout, S

1999-12-01

The hidden Markov model (HMM) was used to identify recurrent short 3D structural building blocks (SBBs) describing protein backbones, independently of any a priori knowledge. Polypeptide chains are decomposed into a series of short segments defined by their inter-alpha-carbon distances. Basically, the model takes into account the sequentiality of the observed segments and assumes that each one corresponds to one of several possible SBBs. Fitting the model to a database of non-redundant proteins allowed us to decode proteins in terms of 12 distinct SBBs with different roles in protein structure. Some SBBs correspond to classical regular secondary structures. Others correspond to a significant subdivision of their bounding regions previously considered to be a single pattern. The major contribution of the HMM is that this model implicitly takes into account the sequential connections between SBBs and thus describes the most probable pathways by which the blocks are connected to form the framework of the protein structures. Validation of the SBBs code was performed by extracting SBB series repeated in recoding proteins and examining their structural similarities. Preliminary results on the sequence specificity of SBBs suggest promising perspectives for the prediction of SBBs or series of SBBs from the protein sequences.

Improving Antibody-Based Cancer Therapeutics Through Glycan Engineering.

PubMed

Yu, Xiaojie; Marshall, Michael J E; Cragg, Mark S; Crispin, Max

2017-06-01

Antibody-based therapeutics has emerged as a major tool in cancer treatment. Guided by the superb specificity of the antibody variable domain, it allows the precise targeting of tumour markers. Recently, eliciting cellular effector functions, mediated by the Fc domain, has gained traction as a means by which to generate more potent antibody therapeutics. Extensive mutagenesis studies of the Fc protein backbone has enabled the generation of Fc variants that more optimally engage the Fcγ receptors known to mediate cellular effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and cellular phagocytosis. In addition to the protein backbone, the homodimeric Fc domain contains two opposing N-linked glycans, which represent a further point of potential immunomodulation, independent of the Fc protein backbone. For example, a lack of core fucose usually attached to the IgG Fc glycan leads to enhanced ADCC activity, whereas a high level of terminal sialylation is associated with reduced inflammation. Significant growth in knowledge of Fc glycosylation over the last decade, combined with advancement in genetic engineering, has empowered glyco-engineering to fine-tune antibody therapeutics. This has culminated in the approval of two glyco-engineered antibodies for cancer therapy: the anti-CCR4 mogamulizumab approved in 2012 and the anti-CD20 obinutuzumab in 2013. We discuss here the technological platforms for antibody glyco-engineering and review the current clinical landscape of glyco-engineered antibodies.

Computational protein design: validation and possible relevance as a tool for homology searching and fold recognition.

PubMed

Schmidt Am Busch, Marcel; Sedano, Audrey; Simonson, Thomas

2010-05-05

Protein fold recognition usually relies on a statistical model of each fold; each model is constructed from an ensemble of natural sequences belonging to that fold. A complementary strategy may be to employ sequence ensembles produced by computational protein design. Designed sequences can be more diverse than natural sequences, possibly avoiding some limitations of experimental databases. WE EXPLORE THIS STRATEGY FOR FOUR SCOP FAMILIES: Small Kunitz-type inhibitors (SKIs), Interleukin-8 chemokines, PDZ domains, and large Caspase catalytic subunits, represented by 43 structures. An automated procedure is used to redesign the 43 proteins. We use the experimental backbones as fixed templates in the folded state and a molecular mechanics model to compute the interaction energies between sidechain and backbone groups. Calculations are done with the Proteins@Home volunteer computing platform. A heuristic algorithm is used to scan the sequence and conformational space, yielding 200,000-300,000 sequences per backbone template. The results confirm and generalize our earlier study of SH2 and SH3 domains. The designed sequences ressemble moderately-distant, natural homologues of the initial templates; e.g., the SUPERFAMILY, profile Hidden-Markov Model library recognizes 85% of the low-energy sequences as native-like. Conversely, Position Specific Scoring Matrices derived from the sequences can be used to detect natural homologues within the SwissProt database: 60% of known PDZ domains are detected and around 90% of known SKIs and chemokines. Energy components and inter-residue correlations are analyzed and ways to improve the method are discussed. For some families, designed sequences can be a useful complement to experimental ones for homologue searching. However, improved tools are needed to extract more information from the designed profiles before the method can be of general use.

Characterization of beta-turn and Asx-turns mimicry in a model peptide: stabilization via C--H . . . O interaction.

PubMed

Thakur, A K; Kishore, R

2006-04-15

The chemical synthesis and single-crystal X-ray diffraction analysis of a model peptide, Boc-Thr-Thr-NH2 (1) comprised of proteinogenic residues bearing an amphiphilic Cbeta -stereogenic center, has been described. Interestingly, the analysis of its molecular structure revealed the existence of a distinct conformation that mimics a typical beta-turn and Asx-turns, i.e., the two Thr residues occupy the left- and right-corner positions. The main-chain torsion angles of the N- and C-terminal residues i.e., semiextended: phi = -68.9 degrees , psi = 128.6 degrees ; semifolded: phi = -138.1 degrees , psi = 2.5 degrees conformations, respectively, in conjunction with a gauche- disposition of the obligatory C-terminus Thr CgammaH3 group, characterize the occurrence of the newly described beta-turn- and Asx-turns-like topology. The preferred molecular structure is suggested to be stabilized by an effective nonconventional main-chain to side-chain Ci=O . . . H--Cgamma(i+2)-type intraturn hydrogen bond. Noteworthy, the observed topology of the resulting 10-membered hydrogen-bonded ring is essentially similar to the one perceived for a classical beta-turn and the Asx-turns, stabilized by a conventional intraturn hydrogen bond. Considering the signs as well as magnitudes of the backbone torsion angles and the orientation of the central peptide bond, the overall mimicked topology resembles the type II beta-turn or type II Asx-turns. An analysis of Xaa-Thr sequences in high-resolution X-ray elucidated protein structures revealed the novel topology prevalence in functional proteins (unpublished). In view of indubitable structural as well as functional importance of nonconventional interactions in bioorganic and biomacromolecules, we intend to highlight the participation of Thr CgammaH in the creation of a short-range C=O . . . H--Cgamma -type interaction in peptides and proteins. Copyright 2006 Wiley Periodicals, Inc.

Targeting Common but Complex Proteoglycans on Breast Cancer Cells and Stem Cells Using Evolutionary Refined Malaria Proteins

DTIC Science & Technology

2014-09-01

protein VAR2CSA. We have extensive data demonstrating that this protein specifically targets sulfated chondroitin sulfate A proteoglycans present on all... chondroitin sulfate A on circulating tumor cells using a evolutionary refined malaria protein B) National Annual PhD meeting in Oncology, March 26-27...malaria protein VAR2CSA when sulfated on carbon 4 of the CS backbone. We have identified CSPG4 as a major protein in breast cancer cells, but also a

Solution structure and backbone dynamics of the N-terminal region of the calcium regulatory domain from soybean calcium-dependent protein kinase alpha.

PubMed

Weljie, Aalim M; Gagné, Stéphane M; Vogel, Hans J

2004-12-07

Ca(2+)-dependent protein kinases (CDPKs) are vital Ca(2+)-signaling proteins in plants and protists which have both a kinase domain and a self-contained calcium regulatory calmodulin-like domain (CLD). Despite being very similar to CaM (>40% identity) and sharing the same fold, recent biochemical and structural evidence suggests that the behavior of CLD is distinct from its namesake, calmodulin. In this study, NMR spectroscopy is employed to examine the structure and backbone dynamics of a 168 amino acid Ca(2+)-saturated construct of the CLD (NtH-CLD) in which almost the entire C-terminal domain is exchange broadened and not visible in the NMR spectra. Structural characterization of the N-terminal domain indicates that the first Ca(2+)-binding loop is significantly more open than in a recently reported structure of the CLD complexed with a putative intramolecular binding region (JD) in the CDPK. Backbone dynamics suggest that parts of the third helix exhibit unusually high mobility, and significant exchange, consistent with previous findings that this helix interacts with the C-terminal domain. Dynamics data also show that the "tether" region, consisting of the first 11 amino acids of CLD, is highly mobile and these residues exhibit distinctive beta-type secondary structure, which may help to position the JD and CLD. Finally, the unusual global dynamic behavior of the protein is rationalized on the basis of possible interdomain rearrangements and the highly variable environments of the C- and N-terminal domains.

Hierarchical Protein Free Energy Landscapes from Variationally Enhanced Sampling.

PubMed

Shaffer, Patrick; Valsson, Omar; Parrinello, Michele

2016-12-13

In recent work, we demonstrated that it is possible to obtain approximate representations of high-dimensional free energy surfaces with variationally enhanced sampling ( Shaffer, P.; Valsson, O.; Parrinello, M. Proc. Natl. Acad. Sci. , 2016 , 113 , 17 ). The high-dimensional spaces considered in that work were the set of backbone dihedral angles of a small peptide, Chignolin, and the high-dimensional free energy surface was approximated as the sum of many two-dimensional terms plus an additional term which represents an initial estimate. In this paper, we build on that work and demonstrate that we can calculate high-dimensional free energy surfaces of very high accuracy by incorporating additional terms. The additional terms apply to a set of collective variables which are more coarse than the base set of collective variables. In this way, it is possible to build hierarchical free energy surfaces, which are composed of terms that act on different length scales. We test the accuracy of these free energy landscapes for the proteins Chignolin and Trp-cage by constructing simple coarse-grained models and comparing results from the coarse-grained model to results from atomistic simulations. The approach described in this paper is ideally suited for problems in which the free energy surface has important features on different length scales or in which there is some natural hierarchy.

Improvements to robotics-inspired conformational sampling in rosetta.

PubMed

Stein, Amelie; Kortemme, Tanja

2013-01-01

To accurately predict protein conformations in atomic detail, a computational method must be capable of sampling models sufficiently close to the native structure. All-atom sampling is difficult because of the vast number of possible conformations and extremely rugged energy landscapes. Here, we test three sampling strategies to address these difficulties: conformational diversification, intensification of torsion and omega-angle sampling and parameter annealing. We evaluate these strategies in the context of the robotics-based kinematic closure (KIC) method for local conformational sampling in Rosetta on an established benchmark set of 45 12-residue protein segments without regular secondary structure. We quantify performance as the fraction of sub-Angstrom models generated. While improvements with individual strategies are only modest, the combination of intensification and annealing strategies into a new "next-generation KIC" method yields a four-fold increase over standard KIC in the median percentage of sub-Angstrom models across the dataset. Such improvements enable progress on more difficult problems, as demonstrated on longer segments, several of which could not be accurately remodeled with previous methods. Given its improved sampling capability, next-generation KIC should allow advances in other applications such as local conformational remodeling of multiple segments simultaneously, flexible backbone sequence design, and development of more accurate energy functions.

Improvements to Robotics-Inspired Conformational Sampling in Rosetta

PubMed Central

Stein, Amelie; Kortemme, Tanja

2013-01-01

To accurately predict protein conformations in atomic detail, a computational method must be capable of sampling models sufficiently close to the native structure. All-atom sampling is difficult because of the vast number of possible conformations and extremely rugged energy landscapes. Here, we test three sampling strategies to address these difficulties: conformational diversification, intensification of torsion and omega-angle sampling and parameter annealing. We evaluate these strategies in the context of the robotics-based kinematic closure (KIC) method for local conformational sampling in Rosetta on an established benchmark set of 45 12-residue protein segments without regular secondary structure. We quantify performance as the fraction of sub-Angstrom models generated. While improvements with individual strategies are only modest, the combination of intensification and annealing strategies into a new “next-generation KIC” method yields a four-fold increase over standard KIC in the median percentage of sub-Angstrom models across the dataset. Such improvements enable progress on more difficult problems, as demonstrated on longer segments, several of which could not be accurately remodeled with previous methods. Given its improved sampling capability, next-generation KIC should allow advances in other applications such as local conformational remodeling of multiple segments simultaneously, flexible backbone sequence design, and development of more accurate energy functions. PMID:23704889

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bolton, Justin; Rzayev, Javid

Polystyrene–poly(methyl methacrylate)–polylactide (PS–PMMA–PLA) triblock bottlebrush copolymer with nearly symmetric volume fractions was synthesized by grafting from a symmetrical triblock backbone and the resulting melt was characterized by scanning electron microscopy and small-angle X-ray scattering. The copolymer backbone was prepared by sequential reversible addition–fragmentation chain transfer (RAFT) polymerization of solketal methacrylate (SM), 2-(bromoisobutyryl)ethyl methacrylate (BIEM), and 5-(trimethylsilyl)-4-pentyn-1-ol methacrylate (TPYM). PMMA branches were grafted by atom transfer radical polymerization from the poly(BIEM) segment, PS branches were grafted by RAFT polymerization from the poly(TPYM) block after installment of the RAFT agents, while PLA side chains were grafted from the deprotected poly(SM) block. Themore » resulting copolymer was found to exhibit a lamellae morphology with a domain spacing of 79 nm. Differential scanning calorimetry analysis indicated that PMMA was preferentially mixing with PS while phase separating from PLA domains.« less

Improved Force Fields for Peptide Nucleic Acids with Optimized Backbone Torsion Parameters.

PubMed

Jasiński, Maciej; Feig, Michael; Trylska, Joanna

2018-06-06

Peptide nucleic acids are promising nucleic acid analogs for antisense therapies as they can form stable duplex and triplex structures with DNA and RNA. Computational studies of PNA-containing duplexes and triplexes are an important component for guiding their design, yet existing force fields have not been well validated and parametrized with modern computational capabilities. We present updated CHARMM and Amber force fields for PNA that greatly improve the stability of simulated PNA-containing duplexes and triplexes in comparison with experimental structures and allow such systems to be studied on microsecond time scales. The force field modifications focus on reparametrized PNA backbone torsion angles to match high-level quantum mechanics reference energies for a model compound. The microsecond simulations of PNA-PNA, PNA-DNA, PNA-RNA, and PNA-DNA-PNA complexes also allowed a comprehensive analysis of hydration and ion interactions with such systems.

Top-Down Hydrogen-Deuterium Exchange Analysis of Protein Structures Using Ultraviolet Photodissociation.

PubMed

Brodie, Nicholas I; Huguet, Romain; Zhang, Terry; Viner, Rosa; Zabrouskov, Vlad; Pan, Jingxi; Petrotchenko, Evgeniy V; Borchers, Christoph H

2018-03-06

Top-down hydrogen-deuterium exchange (HDX) analysis using electron capture or transfer dissociation Fourier transform mass spectrometry (FTMS) is a powerful method for the analysis of secondary structure of proteins in solution. The resolution of the method is a function of the degree of fragmentation of backbone bonds in the proteins. While fragmentation is usually extensive near the N- and C-termini, electron capture (ECD) or electron transfer dissociation (ETD) fragmentation methods sometimes lack good coverage of certain regions of the protein, most often in the middle of the sequence. Ultraviolet photodissociation (UVPD) is a recently developed fast-fragmentation technique, which provides extensive backbone fragmentation that can be complementary in sequence coverage to the aforementioned electron-based fragmentation techniques. Here, we explore the application of electrospray ionization (ESI)-UVPD FTMS on an Orbitrap Fusion Lumos Tribrid mass spectrometer to top-down HDX analysis of proteins. We have incorporated UVPD-specific fragment-ion types and fragment-ion mixtures into our isotopic envelope fitting software (HDX Match) for the top-down HDX analysis. We have shown that UVPD data is complementary to ETD, thus improving the overall resolution when used as a combined approach.

AUTOBA: automation of backbone assignment from HN(C)N suite of experiments.

PubMed

Borkar, Aditi; Kumar, Dinesh; Hosur, Ramakrishna V

2011-07-01

Development of efficient strategies and automation represent important milestones of progress in rapid structure determination efforts in proteomics research. In this context, we present here an efficient algorithm named as AUTOBA (Automatic Backbone Assignment) designed to automate the assignment protocol based on HN(C)N suite of experiments. Depending upon the spectral dispersion, the user can record 2D or 3D versions of the experiments for assignment. The algorithm uses as inputs: (i) protein primary sequence and (ii) peak-lists from user defined HN(C)N suite of experiments. In the end, one gets H(N), (15)N, C(α) and C' assignments (in common BMRB format) for the individual residues along the polypeptide chain. The success of the algorithm has been demonstrated, not only with experimental spectra recorded on two small globular proteins: ubiquitin (76 aa) and M-crystallin (85 aa), but also with simulated spectra of 27 other proteins using assignment data from the BMRB.

Conformational analysis investigation into the influence of nano-porosity of ultra-permeable ultra-selective polyimides on its diffusivity as potential membranes for use in the "green" separation of natural gases

NASA Astrophysics Data System (ADS)

Madkour, Tarek M.

2013-08-01

Nano-porous polymers of intrinsic microporosity, PIM, have exhibited excellent permeability and selectivity characteristics that could be utilized in an environmentally friendly gas separation process. A full understanding of the mechanism through which these membranes effectively and selectively allow for the permeation of specific gases will lead to further development of these membranes. Three factors obviously influenced the conformational behavior of these polymers, which are the presence of electronegative atoms, the presence of non-linearity in the polymeric backbones (backbone kinks) and the presence of bulky side groups on the polymeric chains. The dipole moment increased sharply with the presence of backbone kinks more than any other factor. Replacing the fluorine atoms with bulky alkyl groups didn't influence the dipole moment greatly indicating that the size of the side chains had much less dramatic influence on the dipole moment than having a bent backbone. Similarly, the presence of the backbone kinks in the polymeric chains influenced the polymeric chains to assume less extended configuration causing the torsional angles around the interconnecting bonds unable to cross the high potential energy barriers. The presence of the bulky side groups also caused the energy barriers of the cis-configurations to increase dramatically, which prevented the polymeric segments from experiencing full rotation about the connecting bonds. For these polymers, it was clear that the fully extended configurations are the preferred configurations in the absence of strong electronegative atoms, backbones kinks or bulky side groups. The addition of any of these factors to the polymeric structures resulted in the polymeric chains being forced to assume less extended configurations. Rather interestingly, the length or bulkiness of the side groups didn't affect the end-to-end distance distribution to a great deal since the presence of quite large bulky side chain such as the pentyl group has caused the polymeric chains to revert back to the fully extended configurations possibly due to the quite high potential energy barriers that the chains have to cross to reach the less extended configurational states.

Sulfation and cation effects on the conformational properties of the glycan backbone of chondroitin sulfate disaccharides.

PubMed

Faller, Christina E; Guvench, Olgun

2015-05-21

Chondroitin sulfate (CS) is one of several glycosaminoglycans that are major components of proteoglycans. A linear polymer consisting of repeats of the disaccharide -4GlcAβ1-3GalNAcβ1-, CS undergoes differential sulfation resulting in five unique sulfation patterns. Because of the dimer repeat, the CS glycosidic "backbone" has two distinct sets of conformational degrees of freedom defined by pairs of dihedral angles: (ϕ1, ψ1) about the β1-3 glycosidic linkage and (ϕ2, ψ2) about the β1-4 glycosidic linkage. Differential sulfation and the possibility of cation binding, combined with the conformational flexibility and biological diversity of CS, complicate experimental efforts to understand CS three-dimensional structures at atomic resolution. Therefore, all-atom explicit-solvent molecular dynamics simulations with Adaptive Biasing Force sampling of the CS backbone were applied to obtain high-resolution, high-precision free energies of CS disaccharides as a function of all possible backbone geometries. All 10 disaccharides (β1-3 vs β1-4 linkage × five different sulfation patterns) were studied; additionally, ion effects were investigated by considering each disaccharide in the presence of either neutralizing sodium or calcium cations. GlcAβ1-3GalNAc disaccharides have a single, broad, thermodynamically important free-energy minimum, whereas GalNAcβ1-4GlcA disaccharides have two such minima. Calcium cations but not sodium cations bind to the disaccharides, and binding is primarily to the GlcA -COO(-) moiety as opposed to sulfate groups. This binding alters the glycan backbone thermodynamics in instances where a calcium cation bound to -COO(-) can act to bridge and stabilize an interaction with an adjacent sulfate group, whereas, in the absence of this cation, the proximity of a sulfate group to -COO(-) results in two like charges being both desolvated and placed adjacent to each other and is found to be destabilizing. In addition to providing information on sulfation and cation effects, the present results can be applied to building models of CS polymers and as a point of comparison in studies of CS polymer backbone dynamics and thermodynamics.

Conformational changes induced by a single amino acid substitution in the trans-membrane domain of Vpu: implications for HIV-1 susceptibility to channel blocking drugs.

PubMed

Park, Sang Ho; Opella, Stanley J

2007-10-01

The channel-forming trans-membrane domain of Vpu (Vpu TM) from HIV-1 is known to enhance virion release from the infected cells and is a potential target for ion-channel blockers. The substitution of alanine at position 18 by a histidine (A18H) has been shown to render HIV-1 infections susceptible to rimantadine, a channel blocker of M2 protein from the influenza virus. In order to describe the influence of the mutation on the structure and rimantadine susceptibility of Vpu, we determined the structure of A18H Vpu TM, and compared it to those of wild-type Vpu TM and M2 TM. Both isotropic and orientationally dependent NMR frequencies of the backbone amide resonance of His18 were perturbed by rimantadine, and those of Ile15 and Trp22 were also affected, suggesting that His18 is the key residue for rimantadine binding and that residues located on the same face of the TM helix are also involved. A18H Vpu TM has an ideal, straight alpha-helix spanning residues 6-27 with an average tilt angle of 41 degrees in C14 phospholipid bicelles, indicating that the tilt angle is increased by 11 degrees compared to that of wild-type Vpu TM. The longer helix formed by the A18H mutation has a larger tilt angle to compensate for the hydrophobic mismatch with the length of the phospholipids in the bilayer. These results demonstrate that the local change of the primary structure plays an important role in secondary and tertiary structures of Vpu TM in lipid bilayers and affects its ability to interact with channel blockers.

Experimental conformational energy maps of proteins and peptides.

PubMed

Balaji, Govardhan A; Nagendra, H G; Balaji, Vitukudi N; Rao, Shashidhar N

2017-06-01

We have presented an extensive analysis of the peptide backbone dihedral angles in the PDB structures and computed experimental Ramachandran plots for their distributions seen under a various constraints on X-ray resolution, representativeness at different sequence identity percentages, and hydrogen bonding distances. These experimental distributions have been converted into isoenergy contour plots using the approach employed previously by F. M. Pohl. This has led to the identification of energetically favored minima in the Ramachandran (ϕ, ψ) plots in which global minima are predominantly observed either in the right-handed α-helical or the polyproline II regions. Further, we have identified low energy pathways for transitions between various minima in the (ϕ,ψ) plots. We have compared and presented the experimental plots with published theoretical plots obtained from both molecular mechanics and quantum mechanical approaches. In addition, we have developed and employed a root mean square deviation (RMSD) metric for isoenergy contours in various ranges, as a measure (in kcal.mol -1 ) to compare any two plots and determine the extent of correlation and similarity between their isoenergy contours. In general, we observe a greater degree of compatibility with experimental plots for energy maps obtained from molecular mechanics methods compared to most quantum mechanical methods. The experimental energy plots we have investigated could be helpful in refining protein structures obtained from X-ray, NMR, and electron microscopy and in refining force field parameters to enable simulations of peptide and protein structures that have higher degree of consistency with experiments. Proteins 2017; 85:979-1001. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Error assessment in molecular dynamics trajectories using computed NMR chemical shifts.

PubMed

Koes, David R; Vries, John K

2017-01-01

Accurate chemical shifts for the atoms in molecular mechanics (MD) trajectories can be obtained from quantum mechanical (QM) calculations that depend solely on the coordinates of the atoms in the localized regions surrounding atoms of interest. If these coordinates are correct and the sample size is adequate, the ensemble average of these chemical shifts should be equal to the chemical shifts obtained from NMR spectroscopy. If this is not the case, the coordinates must be incorrect. We have utilized this fact to quantify the errors associated with the backbone atoms in MD simulations of proteins. A library of regional conformers containing 169,499 members was constructed from 6 model proteins. The chemical shifts associated with the backbone atoms in each of these conformers was obtained from QM calculations using density functional theory at the B3LYP level with a 6-311+G(2d,p) basis set. Chemical shifts were assigned to each backbone atom in each MD simulation frame using a template matching approach. The ensemble average of these chemical shifts was compared to chemical shifts from NMR spectroscopy. A large systematic error was identified that affected the 1 H atoms of the peptide bonds involved in hydrogen bonding with water molecules or peptide backbone atoms. This error was highly sensitive to changes in electrostatic parameters. Smaller errors affecting the 13 C a and 15 N atoms were also detected. We believe these errors could be useful as metrics for comparing the force-fields and parameter sets used in MD simulation because they are directly tied to errors in atomic coordinates.

Targeting allosteric disulphide bonds in cancer.

PubMed

Hogg, Philip J

2013-06-01

Protein action in nature is generally controlled by the amount of protein produced and by chemical modification of the protein, and both are often perturbed in cancer. The amino acid side chains and the peptide and disulphide bonds that bind the polypeptide backbone can be post-translationally modified. Post-translational cleavage or the formation of disulphide bonds are now being identified in cancer-related proteins and it is timely to consider how these allosteric bonds could be targeted for new therapies.

Effects of solvent concentration and composition on protein dynamics: 13C MAS NMR studies of elastin in glycerol-water mixtures.

PubMed

Demuth, Dominik; Haase, Nils; Malzacher, Daniel; Vogel, Michael

2015-08-01

We use (13)C CP MAS NMR to investigate the dependence of elastin dynamics on the concentration and composition of the solvent at various temperatures. For elastin in pure glycerol, line-shape analysis shows that larger-scale fluctuations of the protein backbone require a minimum glycerol concentration of ~0.6 g/g at ambient temperature, while smaller-scale fluctuations are activated at lower solvation levels of ~0.2 g/g. Immersing elastin in various glycerol-water mixtures, we observe at room temperature that the protein mobility is higher for lower glycerol fractions in the solvent and, thus, lower solvent viscosity. When decreasing the temperature, the elastin spectra approach the line shape for the rigid protein at 245 K for all studied samples, indicating that the protein ceases to be mobile on the experimental time scale of ~10(-5) s. Our findings yield evidence for a strong coupling between elastin fluctuations and solvent dynamics and, hence, such interaction is not restricted to the case of protein-water mixtures. Spectral resolution of different carbon species reveals that the protein-solvent couplings can, however, be different for side chain and backbone units. We discuss these results against the background of the slaving model for protein dynamics. Copyright © 2015 Elsevier B.V. All rights reserved.

Cleavage of the main carbon chain backbone of high molecular weight polyacrylamide by aerobic and anaerobic biological treatment.

PubMed

Song, Wenzhe; Zhang, Yu; Gao, Yingxin; Chen, Dong; Yang, Min

2017-12-01

High molecular weight partially hydrolyzed polyacrylamide (PAM) can be bio-hydrolyzed on the amide side group, however, solid evidence regarding the biological cleavage of its main carbon chain backbone is limited. In this study, viscometry, flow field-flow fractionation multi-angle light scattering (FFF-MALS), and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) analysis were used to investigate the biodegradability of PAM with a nominal molecular weight of 2 × 10 7  Da (Da) in two suspended aerobic (25 and 40 °C) and two upflow anaerobic blanket reactors (35 and 55 °C) operated for 470 d under a hydraulic residence time (HRT) of 2 d. Both anaerobic and aerobic biological treatment reduced the viscosity from 2.02 cp in the influent to 1.45-1.60 cp, and reduced the molecular weight of PAM using FFF-MALS from 2.17 × 10 7  Da to less than one-third its original size. The removals of both the amide group and carbon chain backbone in the PAM molecule were further supported by the FTIR analysis. In comparison with the other conditions, thermophilic anaerobic treatment exhibited higher efficiency for PAM biodegradation. Batch test excluded the influence of temperature on the molecular weight of PAM over the range 25-55 °C, suggesting that cleavage of the main carbon chain backbone was attributed to biological degradation. Our results suggested that high molecular weight PAM was biodegradable, but mineralization did not occur. Copyright © 2017 Elsevier Ltd. All rights reserved.

Using Molecular Dynamics Simulations as an Aid in the Prediction of Domain Swapping of Computationally Designed Protein Variants.

PubMed

Mou, Yun; Huang, Po-Ssu; Thomas, Leonard M; Mayo, Stephen L

2015-08-14

In standard implementations of computational protein design, a positive-design approach is used to predict sequences that will be stable on a given backbone structure. Possible competing states are typically not considered, primarily because appropriate structural models are not available. One potential competing state, the domain-swapped dimer, is especially compelling because it is often nearly identical with its monomeric counterpart, differing by just a few mutations in a hinge region. Molecular dynamics (MD) simulations provide a computational method to sample different conformational states of a structure. Here, we tested whether MD simulations could be used as a post-design screening tool to identify sequence mutations leading to domain-swapped dimers. We hypothesized that a successful computationally designed sequence would have backbone structure and dynamics characteristics similar to that of the input structure and that, in contrast, domain-swapped dimers would exhibit increased backbone flexibility and/or altered structure in the hinge-loop region to accommodate the large conformational change required for domain swapping. While attempting to engineer a homodimer from a 51-amino-acid fragment of the monomeric protein engrailed homeodomain (ENH), we had instead generated a domain-swapped dimer (ENH_DsD). MD simulations on these proteins showed increased B-factors derived from MD simulation in the hinge loop of the ENH_DsD domain-swapped dimer relative to monomeric ENH. Two point mutants of ENH_DsD designed to recover the monomeric fold were then tested with an MD simulation protocol. The MD simulations suggested that one of these mutants would adopt the target monomeric structure, which was subsequently confirmed by X-ray crystallography. Copyright © 2015. Published by Elsevier Ltd.

Solution structure of the DNA-binding domain of the heat shock transcription factor determined by multidimensional heteronuclear magnetic resonance spectroscopy.

PubMed Central

Damberger, F. F.; Pelton, J. G.; Harrison, C. J.; Nelson, H. C.; Wemmer, D. E.

1994-01-01

The solution structure of the 92-residue DNA-binding domain of the heat shock transcription factor from Kluyveromyces lactis has been determined using multidimensional NMR methods. Three-dimensional (3D) triple resonance, 1H-13C-13C-1H total correlation spectroscopy, and 15N-separated total correlation spectroscopy-heteronuclear multiple quantum correlation experiments were used along with various 2D spectra to make nearly complete assignments for the backbone and side-chain 1H, 15N, and 13C resonances. Five-hundred eighty-three NOE constraints identified in 3D 13C- and 15N-separated NOE spectroscopy (NOESY)-heteronuclear multiple quantum correlation spectra and a 4-dimensional 13C/13C-edited NOESY spectrum, along with 35 phi, 9 chi 1, and 30 hydrogen bond constraints, were used to calculate 30 structures by hybrid distance geometry/stimulated annealing protocol, of which 24 were used for structural comparison. The calculations revealed that a 3-helix bundle packs against a small 4-stranded antiparallel beta-sheet. The backbone RMS deviation (RMSD) for the family of structures was 1.03 +/- 0.19 A with respect to the average structure. The topology is analogous to that of the C-terminal domain of the catabolite gene activator protein and appears to be in the helix-turn-helix family of DNA-binding proteins. The overall fold determined by the NMR data is consistent with recent crystallographic work on this domain (Harrison CJ, Bohm AA, Nelson HCM, 1994, Science 263:224) as evidenced by RMSD between backbone atoms in the NMR and X-ray structures of 1.77 +/- 0.20 A. Several differences were identified some of which may be due to protein-protein interactions in the crystal. PMID:7849597

Analysis of Electrowetting Dynamics with Level Set Method

NASA Astrophysics Data System (ADS)

Park, Jun Kwon; Hong, Jiwoo; Kang, Kwan Hyoung

2009-11-01

Electrowetting is a versatile tool to handle tiny droplets and forms a backbone of digital microfluidics. Numerical analysis is necessary to fully understand the dynamics of electrowetting, especially in designing electrowetting-based liquid lenses and reflective displays. We developed a numerical method to analyze the general contact-line problems, incorporating dynamic contact angle models. The method was applied to the analysis of spreading process of a sessile droplet for step input voltages in electrowetting. The result was compared with experimental data and analytical result which is based on the spectral method. It is shown that contact line friction significantly affects the contact line motion and the oscillation amplitude. The pinning process of contact line was well represented by including the hysteresis effect in the contact angle models.

Solid-Phase Synthesis of RNA Analogs Containing Phosphorodithioate Linkages.

PubMed

Yang, Xianbin

2017-09-18

The oligoribonucleotide phosphorodithioate (PS2-RNA) modification uses two sulfur atoms to replace two non-bridging oxygen atoms at an internucleotide phosphorodiester backbone linkage. Like a natural phosphodiester RNA backbone linkage, a PS2-modified backbone linkage is achiral at phosphorus. PS2-RNAs are highly stable to nucleases and several in vitro assays have demonstrated their biological activity. For example, PS2-RNAs silenced mRNA in vitro and bound to protein targets in the form of PS2-aptamers (thioaptamers). Thus, the interest in and promise of PS2-RNAs has drawn attention to synthesizing, isolating, and characterizing these compounds. RNA-thiophosphoramidite monomers are commercially available from AM Biotechnologies and this unit describes an effective methodology for solid-phase synthesis, deprotection, and purification of RNAs having PS2 internucleotide linkages. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Uranyl mediated photofootprinting reveals strong E. coli RNA polymerase--DNA backbone contacts in the +10 region of the DeoP1 promoter open complex.

PubMed Central

Jeppesen, C; Nielsen, P E

1989-01-01

Employing a newly developed uranyl photofootprinting technique (Nielsen et al. (1988) FEBS Lett. 235, 122), we have analyzed the structure of the E. coli RNA polymerase deoP1 promoter open complex. The results show strong polymerase DNA backbone contacts in the -40, -10, and most notably in the +10 region. These results suggest that unwinding of the -12 to +3 region of the promoter in the open complex is mediated through polymerase DNA backbone contacts on both sides of this region. The pattern of bases that are hyperreactive towards KMnO4 or uranyl within the -12 to +3 region furthermore argues against a model in which this region is simply unwound and/or single stranded. The results indicate specific protein contacts and/or a fixed DNA conformation within the -12 to +3 region. Images PMID:2503811

Biopolymer Chain Elasticity: a novel concept and a least deformation energy principle predicts backbone and overall folding of DNA TTT hairpins in agreement with NMR distances

PubMed Central

Pakleza, Christophe; Cognet, Jean A. H.

2003-01-01

A new molecular modelling methodology is presented and shown to apply to all published solution structures of DNA hairpins with TTT in the loop. It is based on the theory of elasticity of thin rods and on the assumption that single-stranded B-DNA behaves as a continuous, unshearable, unstretchable and flexible thin rod. It requires four construction steps: (i) computation of the tri-dimensional trajectory of the elastic line, (ii) global deformation of single-stranded helical DNA onto the elastic line, (iii) optimisation of the nucleoside rotations about the elastic line, (iv) energy minimisation to restore backbone bond lengths and bond angles. This theoretical approach called ‘Biopolymer Chain Elasticity’ (BCE) is capable of reproducing the tri-dimensional course of the sugar–phosphate chain and, using NMR-derived distances, of reproducing models close to published solution structures. This is shown by computing three different types of distance criteria. The natural description provided by the elastic line and by the new parameter, Ω, which corresponds to the rotation angles of nucleosides about the elastic line, offers a considerable simplification of molecular modelling of hairpin loops. They can be varied independently from each other, since the global shape of the hairpin loop is preserved in all cases. PMID:12560506

Protein oxidation and peroxidation

PubMed Central

Davies, Michael J.

2016-01-01

Proteins are major targets for radicals and two-electron oxidants in biological systems due to their abundance and high rate constants for reaction. With highly reactive radicals damage occurs at multiple side-chain and backbone sites. Less reactive species show greater selectivity with regard to the residues targeted and their spatial location. Modification can result in increased side-chain hydrophilicity, side-chain and backbone fragmentation, aggregation via covalent cross-linking or hydrophobic interactions, protein unfolding and altered conformation, altered interactions with biological partners and modified turnover. In the presence of O2, high yields of peroxyl radicals and peroxides (protein peroxidation) are formed; the latter account for up to 70% of the initial oxidant flux. Protein peroxides can oxidize both proteins and other targets. One-electron reduction results in additional radicals and chain reactions with alcohols and carbonyls as major products; the latter are commonly used markers of protein damage. Direct oxidation of cysteine (and less commonly) methionine residues is a major reaction; this is typically faster than with H2O2, and results in altered protein activity and function. Unlike H2O2, which is rapidly removed by protective enzymes, protein peroxides are only slowly removed, and catabolism is a major fate. Although turnover of modified proteins by proteasomal and lysosomal enzymes, and other proteases (e.g. mitochondrial Lon), can be efficient, protein hydroperoxides inhibit these pathways and this may contribute to the accumulation of modified proteins in cells. Available evidence supports an association between protein oxidation and multiple human pathologies, but whether this link is causal remains to be established. PMID:27026395

An algorithm for converting a virtual-bond chain into a complete polypeptide backbone chain

NASA Technical Reports Server (NTRS)

Luo, N.; Shibata, M.; Rein, R.

1991-01-01

A systematic analysis is presented of the algorithm for converting a virtual-bond chain, defined by the coordinates of the alpha-carbons of a given protein, into a complete polypeptide backbone. An alternative algorithm, based upon the same set of geometric parameters used in the Purisima-Scheraga algorithm but with a different "linkage map" of the algorithmic procedures, is proposed. The global virtual-bond chain geometric constraints are more easily separable from the loal peptide geometric and energetic constraints derived from, for example, the Ramachandran criterion, within the framework of this approach.

Structure-Functional Basis of Ion Transport in Sodium–Calcium Exchanger (NCX) Proteins

PubMed Central

Giladi, Moshe; Shor, Reut; Lisnyansky, Michal; Khananshvili, Daniel

2016-01-01

The membrane-bound sodium–calcium exchanger (NCX) proteins shape Ca2+ homeostasis in many cell types, thus participating in a wide range of physiological and pathological processes. Determination of the crystal structure of an archaeal NCX (NCX_Mj) paved the way for a thorough and systematic investigation of ion transport mechanisms in NCX proteins. Here, we review the data gathered from the X-ray crystallography, molecular dynamics simulations, hydrogen–deuterium exchange mass-spectrometry (HDX-MS), and ion-flux analyses of mutants. Strikingly, the apo NCX_Mj protein exhibits characteristic patterns in the local backbone dynamics at particular helix segments, thereby possessing characteristic HDX profiles, suggesting structure-dynamic preorganization (geometric arrangements of catalytic residues before the transition state) of conserved α1 and α2 repeats at ion-coordinating residues involved in transport activities. Moreover, dynamic preorganization of local structural entities in the apo protein predefines the status of ion-occlusion and transition states, even though Na+ or Ca2+ binding modifies the preceding backbone dynamics nearby functionally important residues. Future challenges include resolving the structural-dynamic determinants governing the ion selectivity, functional asymmetry and ion-induced alternating access. Taking into account the structural similarities of NCX_Mj with the other proteins belonging to the Ca2+/cation exchanger superfamily, the recent findings can significantly improve our understanding of ion transport mechanisms in NCX and similar proteins. PMID:27879668

Structure-Functional Basis of Ion Transport in Sodium-Calcium Exchanger (NCX) Proteins.

PubMed

Giladi, Moshe; Shor, Reut; Lisnyansky, Michal; Khananshvili, Daniel

2016-11-22

The membrane-bound sodium-calcium exchanger (NCX) proteins shape Ca 2+ homeostasis in many cell types, thus participating in a wide range of physiological and pathological processes. Determination of the crystal structure of an archaeal NCX (NCX_Mj) paved the way for a thorough and systematic investigation of ion transport mechanisms in NCX proteins. Here, we review the data gathered from the X-ray crystallography, molecular dynamics simulations, hydrogen-deuterium exchange mass-spectrometry (HDX-MS), and ion-flux analyses of mutants. Strikingly, the apo NCX_Mj protein exhibits characteristic patterns in the local backbone dynamics at particular helix segments, thereby possessing characteristic HDX profiles, suggesting structure-dynamic preorganization (geometric arrangements of catalytic residues before the transition state) of conserved α₁ and α₂ repeats at ion-coordinating residues involved in transport activities. Moreover, dynamic preorganization of local structural entities in the apo protein predefines the status of ion-occlusion and transition states, even though Na⁺ or Ca 2+ binding modifies the preceding backbone dynamics nearby functionally important residues. Future challenges include resolving the structural-dynamic determinants governing the ion selectivity, functional asymmetry and ion-induced alternating access. Taking into account the structural similarities of NCX_Mj with the other proteins belonging to the Ca 2+ /cation exchanger superfamily, the recent findings can significantly improve our understanding of ion transport mechanisms in NCX and similar proteins.

Searching for protein binding sites from Molecular Dynamics simulations and paramagnetic fragment-based NMR studies.

PubMed

Bernini, Andrea; Henrici De Angelis, Lucia; Morandi, Edoardo; Spiga, Ottavia; Santucci, Annalisa; Assfalg, Michael; Molinari, Henriette; Pillozzi, Serena; Arcangeli, Annarosa; Niccolai, Neri

2014-03-01

Hotspot delineation on protein surfaces represents a fundamental step for targeting protein-protein interfaces. Disruptors of protein-protein interactions can be designed provided that the sterical features of binding pockets, including the transient ones, can be defined. Molecular Dynamics, MD, simulations have been used as a reliable framework for identifying transient pocket openings on the protein surface. Accessible surface area and intramolecular H-bond involvement of protein backbone amides are proposed as descriptors for characterizing binding pocket occurrence and evolution along MD trajectories. TEMPOL induced paramagnetic perturbations on (1)H-(15)N HSQC signals of protein backbone amides have been analyzed as a fragment-based search for surface hotspots, in order to validate MD predicted pockets. This procedure has been applied to CXCL12, a small chemokine responsible for tumor progression and proliferation. From combined analysis of MD data and paramagnetic profiles, two CXCL12 sites suitable for the binding of small molecules were identified. One of these sites is the already well characterized CXCL12 region involved in the binding to CXCR4 receptor. The other one is a transient pocket predicted by Molecular Dynamics simulations, which could not be observed from static analysis of CXCL12 PDB structures. The present results indicate how TEMPOL, instrumental in identifying this transient pocket, can be a powerful tool to delineate minor conformations which can be highly relevant in dynamic discovery of antitumoral drugs. Copyright © 2013 Elsevier B.V. All rights reserved.

Unifying the microscopic picture of His-containing turns: from gas phase model peptides to crystallized proteins.

PubMed

Sohn, Woon Yong; Habka, Sana; Gloaguen, Eric; Mons, Michel

2017-07-14

The presence in crystallized proteins of a local anchoring between the side chain of a His residue, located in the central position of a γ- or β-turn, and its local main chain environment, was assessed by the comparison of protein structures with relevant isolated model peptides. Gas phase laser spectroscopy, combined with relevant quantum chemistry methods, was used to characterize the γ- and β-turn structures in these model peptides. A conformer-selective NH stretch infrared study provided evidence for the formation in vacuo of two types of short-range H-bonded motifs, labelled ε-6 δ and δ- δ 7/π H , bridging the His side chain (in its gauche+ rotamer) to the neighbouring NH(i) and CO(i) sites of the backbone; each side chain-backbone motif was found to be specific of the tautomer (ε or δ) adopted by the His side chain in its neutral form. A close comparison between β- and γ-turns, selected from the Protein Data Bank, and the gas phase models demonstrated that a significant proportion of the gauche+ His rotamer distribution of proteins was well described by the corresponding gas phase H-bonded structures. This is consistent with the persistence of local 6 δ and δ 7/π H intramolecular interactions in proteins, emphasizing the relevance of gas phase data to secondary structures that are poorly accessible to solvents, e.g., in the case of a specific compact topology (Xxx-His β-turns). Deviations from the gas phase structures were also observed, mainly in His-Xxx β-turns, and assigned to solvent accessible turn structures. They were well accounted for by theoretical models of microhydrated turns, in which a few solvent molecules take over the gas phase motifs, constituting a water-mediated local anchoring of the His side chain to the backbone. Finally, the present gas phase benchmark models also pinpointed weaknesses in the protein structure determination by X-ray diffraction analysis; in particular, besides the lack of tautomer information, inaccuracies in the description of imidazole ring flip rotamerism were identified.

Design of HIV-1 Protease Inhibitors with Amino-bis-tetrahydrofuran Derivatives as P2-Ligands to Enhance Backbone-Binding Interactions. Synthesis, Biological Evaluation, and Protein-Ligand X-ray Studies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ghosh, Arun K.; Martyr, Cuthbert D.; Osswald, Heather L.

Structure-based design, synthesis, and biological evaluation of a series of very potent HIV-1 protease inhibitors are described. In an effort to improve backbone ligand–binding site interactions, we have incorporated basic-amines at the C4 position of the bis-tetrahydrofuran (bis-THF) ring. We speculated that these substituents would make hydrogen bonding interactions in the flap region of HIV-1 protease. Synthesis of these inhibitors was performed diastereoselectively. A number of inhibitors displayed very potent enzyme inhibitory and antiviral activity. Inhibitors 25f, 25i, and 25j were evaluated against a number of highly-PI-resistant HIV-1 strains, and they exhibited improved antiviral activity over darunavir. Two high resolutionmore » X-ray structures of 25f- and 25g-bound HIV-1 protease revealed unique hydrogen bonding interactions with the backbone carbonyl group of Gly48 as well as with the backbone NH of Gly48 in the flap region of the enzyme active site. These ligand–binding site interactions are possibly responsible for their potent activity.« less

Three-dimensional structure of interleukin 8 in solution.

PubMed

Clore, G M; Appella, E; Yamada, M; Matsushima, K; Gronenborn, A M

1990-02-20

The solution structure of the interleukin 8 (IL-8) dimer has been solved by nuclear magnetic resonance (NMR) spectroscopy and hybrid distance geometry-dynamical simulated annealing calculations. The structure determination is based on a total of 1880 experimental distance restraints (of which 82 are intersubunit) and 362 torsion angle restraints (comprising phi, psi, and chi 1 torsion angles). A total of 30 simulated annealing structures were calculated, and the atomic rms distribution about the mean coordinate positions (excluding residues 1-5 of each subunit) is 0.41 +/- 0.08 A for the backbone atoms and 0.90 +/- 0.08 A for all atoms. The three-dimensional solution structure of the IL-8 dimer reveals a structural motif in which two symmetry-related antiparallel alpha-helices, approximately 24 A long and separated by about 14 A, lie on top of a six-stranded antiparallel beta-sheet platform derived from two three-stranded Greek keys, one from each monomer unit. The general architecture is similar to that of the alpha 1/alpha 2 domains of the human class I histocompatibility antigen HLA-A2. It is suggested that the two alpha-helices form the binding site for the cellular receptor and that the specificity of IL-8, as well as that of a number of related proteins involved in cell-specific chemotaxis, mediation of cell growth, and the inflammatory response, is achieved by the distinct distribution of charged and polar residues at the surface of the helices.

A NMR experiment for simultaneous correlations of valine and leucine/isoleucine methyls with carbonyl chemical shifts in proteins.

PubMed

Tugarinov, Vitali; Venditti, Vincenzo; Marius Clore, G

2014-01-01

A methyl-detected 'out-and-back' NMR experiment for obtaining simultaneous correlations of methyl resonances of valine and isoleucine/leucine residues with backbone carbonyl chemical shifts, SIM-HMCM(CGCBCA)CO, is described. The developed pulse-scheme serves the purpose of convenience in recording a single data set for all Ile(δ1), Leu(δ) and Val(γ) (ILV) methyl positions instead of acquiring two separate spectra selective for valine or leucine/isoleucine residues. The SIM-HMCM(CGCBCA)CO experiment can be used for ILV methyl assignments in moderately sized protein systems (up to ~100 kDa) where the backbone chemical shifts of (13)C(α), (13)Cβ and (13)CO are known from prior NMR studies and where some losses in sensitivity can be tolerated for the sake of an overall reduction in NMR acquisition time.

Crystallization of dienelactone hydrolase in two space groups: structural changes caused by crystal packing

PubMed Central

Porter, Joanne L.; Carr, Paul D.; Collyer, Charles A.; Ollis, David L.

2014-01-01

Dienelactone hydrolase (DLH) is a monomeric protein with a simple α/β-hydrolase fold structure. It readily crystallizes in space group P212121 from either a phosphate or ammonium sulfate precipitation buffer. Here, the structure of DLH at 1.85 Å resolution crystallized in space group C2 with two molecules in the asymmetric unit is reported. When crystallized in space group P212121 DLH has either phosphates or sulfates bound to the protein in crucial locations, one of which is located in the active site, preventing substrate/inhibitor binding. Another is located on the surface of the enzyme coordinated by side chains from two different molecules. Crystallization in space group C2 from a sodium citrate buffer results in new crystallographic protein–protein interfaces. The protein backbone is highly similar, but new crystal contacts cause changes in side-chain orientations and in loop positioning. In regions not involved in crystal contacts, there is little change in backbone or side-chain configuration. The flexibility of surface loops and the adaptability of side chains are important factors enabling DLH to adapt and form different crystal lattices. PMID:25005082

Probing specific molecular processes and intermediates by time-resolved Fourier transform infrared spectroscopy: application to the bacteriorhodopsin photocycle.

PubMed

Lórenz-Fonfría, Víctor A; Kandori, Hideki; Padrós, Esteve

2011-06-23

We present a general approach for probing the kinetics of specific molecular processes in proteins by time-resolved Fourier transform infrared (IR) spectroscopy. Using bacteriorhodopsin (bR) as a model we demonstrate that by appropriately monitoring some selected IR bands it is possible obtaining the kinetics of the most important events occurring in the photocycle, namely changes in the chromophore and the protein backbone conformation, and changes in the protonation state of the key residues implicated in the proton transfers. Besides confirming widely accepted views of the bR photocycle, our analysis also sheds light into some disputed issues: the degree of retinal torsion in the L intermediate to respect the ground state; the possibility of a proton transfer from Asp85 to Asp212; the relationship between the protonation/deprotonation of Asp85 and the proton release complex; and the timing of the protein backbone dynamics. By providing a direct way to estimate the kinetics of photocycle intermediates the present approach opens new prospects for a robust quantitative kinetic analysis of the bR photocycle, which could also benefit the study of other proteins involved in photosynthesis, in phototaxis, or in respiratory chains.

1H, 15N and 13C resonance assignments for free and IEEVD peptide-bound forms of the tetratricopeptide repeat domain from the human E3 ubiquitin ligase CHIP.

PubMed

Zhang, Huaqun; McGlone, Cameron; Mannion, Matthew M; Page, Richard C

2017-04-01

The ubiquitin ligase CHIP catalyzes covalent attachment of ubiquitin to unfolded proteins chaperoned by the heat shock proteins Hsp70/Hsc70 and Hsp90. CHIP interacts with Hsp70/Hsc70 and Hsp90 by binding of a C-terminal IEEVD motif found in Hsp70/Hsc70 and Hsp90 to the tetratricopeptide repeat (TPR) domain of CHIP. Although recruitment of heat shock proteins to CHIP via interaction with the CHIP-TPR domain is well established, alterations in structure and dynamics of CHIP upon binding are not well understood. In particular, the absence of a structure for CHIP-TPR in the free form presents a significant limitation upon studies seeking to rationally design inhibitors that may disrupt interactions between CHIP and heat shock proteins. Here we report the 1 H, 13 C, and 15 N backbone and side chain chemical shift assignments for CHIP-TPR in the free form, and backbone chemical shift assignments for CHIP-TPR in the IEEVD-bound form. The NMR resonance assignments will enable further studies examining the roles of dynamics and structure in regulating interactions between CHIP and the heat shock proteins Hsp70/Hsc70 and Hsp90.

SASSIE: A program to study intrinsically disordered biological molecules and macromolecular ensembles using experimental scattering restraints

NASA Astrophysics Data System (ADS)

Curtis, Joseph E.; Raghunandan, Sindhu; Nanda, Hirsh; Krueger, Susan

2012-02-01

A program to construct ensembles of biomolecular structures that are consistent with experimental scattering data are described. Specifically, we generate an ensemble of biomolecular structures by varying sets of backbone dihedral angles that are then filtered using experimentally determined restraints to rapidly determine structures that have scattering profiles that are consistent with scattering data. We discuss an application of these tools to predict a set of structures for the HIV-1 Gag protein, an intrinsically disordered protein, that are consistent with small-angle neutron scattering experimental data. We have assembled these algorithms into a program called SASSIE for structure generation, visualization, and analysis of intrinsically disordered proteins and other macromolecular ensembles using neutron and X-ray scattering restraints. Program summaryProgram title: SASSIE Catalogue identifier: AEKL_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AEKL_v1_0.html Program obtainable from: CPC Program Library, Queen's University, Belfast, N. Ireland Licensing provisions: GNU General Public License v3 No. of lines in distributed program, including test data, etc.: 3 991 624 No. of bytes in distributed program, including test data, etc.: 826 Distribution format: tar.gz Programming language: Python, C/C++, Fortran Computer: PC/Mac Operating system: 32- and 64-bit Linux (Ubuntu 10.04, Centos 5.6) and Mac OS X (10.6.6) RAM: 1 GB Classification: 3 External routines: Python 2.6.5, numpy 1.4.0, swig 1.3.40, scipy 0.8.0, Gnuplot-py-1.8, Tcl 8.5, Tk 8.5, Mac installation requires aquaterm 1.0 (or X window system) and Xcode 3 development tools. Nature of problem: Open source software to generate structures of disordered biological molecules that subsequently allow for the comparison of computational and experimental results is limiting the use of scattering resources. Solution method: Starting with an all atom model of a protein, for example, users can input regions to vary dihedral angles, ensembles of structures can be generated. Additionally, simple two-body rigid-body rotations are supported with and without disordered regions. Generated structures can then be used to calculate small-angle scattering profiles which can then be filtered against experimentally determined data. Filtered structures can be visualized individually or as an ensemble using density plots. In the modular and expandable program framework the user can easily access our subroutines and structural coordinates can be easily obtained for study using other computational physics methods. Additional comments: The distribution file for this program is over 159 Mbytes and therefore is not delivered directly when download or Email is requested. Instead an html file giving details of how the program can be obtained is sent. Running time: Varies depending on application. Typically 10 minutes to 24 hours depending on the number of generated structures.

Hydrophobic core malleability of a de novo designed three-helix bundle protein.

PubMed

Walsh, S T; Sukharev, V I; Betz, S F; Vekshin, N L; DeGrado, W F

2001-01-12

De novo protein design provides a tool for testing the principles that stabilize the structures of proteins. Recently, we described the design and structure determination of alpha(3)D, a three-helix bundle protein with a well-packed hydrophobic core. Here, we test the malleability and adaptability of this protein's structure by mutating a small, Ala residue (A60) in its core to larger, hydrophobic side-chains, Leu and Ile. Such changes introduce strain into the structures of natural proteins, and therefore generally destabilize the native state. By contrast, these mutations were slightly stabilizing ( approximately 1.5 kcal mol(-1)) to the tertiary structure of alpha(3)D. The value of DeltaC(p) for unfolding of these mutants was not greatly affected relative to wild-type, indicating that the change in solvent accessibility for unfolding was similar. However, two-dimensional heteronuclear single quantum coherence spectra indicate that the protein adjusts to the introduction of steric bulk in different ways. A60L-alpha(3)D showed serious erosion in the dispersion of both the amide backbone as well as the side-chain methyl chemical shifts. By contrast, A60I-alpha(3)D showed excellent dispersion of the backbone resonances, and selective changes in dispersion of the aliphatic side-chains proximal to the site of mutation. Together, these data suggest that alpha(3)D, although folded into a unique three-dimensional structure, is nevertheless more malleable and flexible than most natural, native proteins. Copyright 2001 Academic Press.

Peptoid nanosheets exhibit a new secondary-structure motif.

PubMed

Mannige, Ranjan V; Haxton, Thomas K; Proulx, Caroline; Robertson, Ellen J; Battigelli, Alessia; Butterfoss, Glenn L; Zuckermann, Ronald N; Whitelam, Stephen

2015-10-15

A promising route to the synthesis of protein-mimetic materials that are capable of complex functions, such as molecular recognition and catalysis, is provided by sequence-defined peptoid polymers--structural relatives of biologically occurring polypeptides. Peptoids, which are relatively non-toxic and resistant to degradation, can fold into defined structures through a combination of sequence-dependent interactions. However, the range of possible structures that are accessible to peptoids and other biological mimetics is unknown, and our ability to design protein-like architectures from these polymer classes is limited. Here we use molecular-dynamics simulations, together with scattering and microscopy data, to determine the atomic-resolution structure of the recently discovered peptoid nanosheet, an ordered supramolecular assembly that extends macroscopically in only two dimensions. Our simulations show that nanosheets are structurally and dynamically heterogeneous, can be formed only from peptoids of certain lengths, and are potentially porous to water and ions. Moreover, their formation is enabled by the peptoids' adoption of a secondary structure that is not seen in the natural world. This structure, a zigzag pattern that we call a Σ('sigma')-strand, results from the ability of adjacent backbone monomers to adopt opposed rotational states, thereby allowing the backbone to remain linear and untwisted. Linear backbones tiled in a brick-like way form an extended two-dimensional nanostructure, the Σ-sheet. The binary rotational-state motif of the Σ-strand is not seen in regular protein structures, which are usually built from one type of rotational state. We also show that the concept of building regular structures from multiple rotational states can be generalized beyond the peptoid nanosheet system.

Comparison of the dynamics of substrate access channels in three cytochrome P450s reveals different opening mechanisms and a novel functional role for a buried arginine

PubMed Central

Winn, Peter J.; Lüdemann, Susanna K.; Gauges, Ralph; Lounnas, Valère; Wade, Rebecca C.

2002-01-01

Understanding the mechanism and specificity of substrate binding in the cytochrome P450 (P450) superfamily is an important step toward explaining its key role in drug metabolism, toxicity, xenobiotic degradation, and several biosynthetic pathways. Here we investigate the ligand exit pathways and mechanisms of P450cam (CYP101), P450BM-3 (CYP102), and P450eryF (CYP107A1) by using random expulsion molecular dynamics and classical molecular dynamics simulations. Although several different pathways are found for each protein, one pathway is common to all three. The mechanism of ligand exit along this pathway is, however, quite different in the three different proteins. For P450cam, small backbone conformational changes, in combination with aromatic side chain rotation, allow for the passage of the rather rigid, compact, and hydrophobic substrate, camphor. In P450BM-3, larger transient backbone changes are observed on ligand exit. R47, situated at the entrance to the channel, appears important in guiding negatively charged fatty acid substrates in and out of the active site. In P450eryF, an isolated buried arginine, R185, stabilized by four hydrogen bonds to backbone carbonyl oxygen atoms, is located in the exit channel and is identified as having a particularly unusual functionality, dynamically gating channel opening. The results for these three P450s suggest that the channel opening mechanisms are adjusted to the physico-chemical properties of the substrate and can kinetically modulate protein-substrate specificity. PMID:11959989

Relationship between the structure of SET/TAF-Ibeta/INHAT and its histone chaperone activity.

PubMed

Muto, Shinsuke; Senda, Miki; Akai, Yusuke; Sato, Lui; Suzuki, Toru; Nagai, Ryozo; Senda, Toshiya; Horikoshi, Masami

2007-03-13

Histone chaperones assemble and disassemble nucleosomes in an ATP-independent manner and thus regulate the most fundamental step in the alteration of chromatin structure. The molecular mechanisms underlying histone chaperone activity remain unclear. To gain insights into these mechanisms, we solved the crystal structure of the functional domain of SET/TAF-Ibeta/INHAT at a resolution of 2.3 A. We found that SET/TAF-Ibeta/INHAT formed a dimer that assumed a "headphone"-like structure. Each subunit of the SET/TAF-Ibeta/INHAT dimer consisted of an N terminus, a backbone helix, and an "earmuff" domain. It resembles the structure of the related protein NAP-1. Comparison of the crystal structures of SET/TAF-Ibeta/INHAT and NAP-1 revealed that the two proteins were folded similarly except for an inserted helix. However, their backbone helices were shaped differently, and the relative dispositions of the backbone helix and the earmuff domain between the two proteins differed by approximately 40 degrees . Our biochemical analyses of mutants revealed that the region of SET/TAF-Ibeta/INHAT that is engaged in histone chaperone activity is the bottom surface of the earmuff domain, because this surface bound both core histones and double-stranded DNA. This overlap or closeness of the activity surface and the binding surfaces suggests that the specific association among SET/TAF-Ibeta/INHAT, core histones, and double-stranded DNA is requisite for histone chaperone activity. These findings provide insights into the possible mechanisms by which histone chaperones assemble and disassemble nucleosome structures.

Pilus distribution among lineages of group b streptococcus: an evolutionary and clinical perspective

PubMed Central

2014-01-01

Background Group B Streptococcus (GBS) is an opportunistic pathogen in both humans and bovines. Epidemiological and phylogenetic analyses have found strains belonging to certain phylogenetic lineages to be more frequently associated with invasive newborn disease, asymptomatic maternal colonization, and subclinical bovine mastitis. Pilus structures in GBS facilitate colonization and invasion of host tissues and play a role in biofilm formation, though few large-scale studies have estimated the frequency and diversity of the three pilus islands (PIs) across diverse genotypes. Here, we examined the distribution of pilus islands (PI) 1, 2a and 2b among 295 GBS strains representing 73 multilocus sequence types (STs) belonging to eight clonal complexes. PCR-based RFLP was also used to evaluate variation in the genes encoding pilus backbone proteins of PI-2a and PI-2b. Results All 295 strains harbored one of the PI-2 variants and most human-derived strains contained PI-1. Bovine-derived strains lacked PI-1 and possessed a unique PI-2b backbone protein allele. Neonatal strains more frequently had PI-1 and a PI-2 variant than maternal colonizing strains, and most CC-17 strains had PI-1 and PI-2b with a distinct backbone protein allele. Furthermore, we present evidence for the frequent gain and loss of genes encoding certain pilus types. Conclusions These data suggest that pilus combinations impact host specificity and disease presentation and that diversification often involves the loss or acquisition of PIs. Such findings have implications for the development of GBS vaccines that target the three pilus islands. PMID:24943359

Solvation thermodynamics of amino acid side chains on a short peptide backbone

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hajari, Timir; Vegt, Nico F. A. van der, E-mail: vandervegt@csi.tu-darmstadt.de

The hydration process of side chain analogue molecules differs from that of the actual amino acid side chains in peptides and proteins owing to the effects of the peptide backbone on the aqueous solvent environment. A recent molecular simulation study has provided evidence that all nonpolar side chains, attached to a short peptide backbone, are considerably less hydrophobic than the free side chain analogue molecules. In contrast to this, the hydrophilicity of the polar side chains is hardly affected by the backbone. To analyze the origin of these observations, we here present a molecular simulation study on temperature dependent solvationmore » free energies of nonpolar and polar side chains attached to a short peptide backbone. The estimated solvation entropies and enthalpies of the various amino acid side chains are compared with existing side chain analogue data. The solvation entropies and enthalpies of the polar side chains are negative, but in absolute magnitude smaller compared with the corresponding analogue data. The observed differences are large; however, owing to a nearly perfect enthalpy-entropy compensation, the solvation free energies of polar side chains remain largely unaffected by the peptide backbone. We find that a similar compensation does not apply to the nonpolar side chains; while the backbone greatly reduces the unfavorable solvation entropies, the solvation enthalpies are either more favorable or only marginally affected. This results in a very small unfavorable free energy cost, or even free energy gain, of solvating the nonpolar side chains in strong contrast to solvation of small hydrophobic or nonpolar molecules in bulk water. The solvation free energies of nonpolar side chains have been furthermore decomposed into a repulsive cavity formation contribution and an attractive dispersion free energy contribution. We find that cavity formation next to the peptide backbone is entropically favored over formation of similar sized nonpolar side chain cavities in bulk water, in agreement with earlier work in the literature on analysis of cavity fluctuations at nonpolar molecular surfaces. The cavity and dispersion interaction contributions correlate quite well with the solvent accessible surface area of the nonpolar side chains attached to the backbone. This correlation however is weak for the overall solvation free energies owing to the fact that the cavity and dispersion free energy contributions are almost exactly cancelling each other.« less

Solvation thermodynamics of amino acid side chains on a short peptide backbone

NASA Astrophysics Data System (ADS)

Hajari, Timir; van der Vegt, Nico F. A.

2015-04-01

The hydration process of side chain analogue molecules differs from that of the actual amino acid side chains in peptides and proteins owing to the effects of the peptide backbone on the aqueous solvent environment. A recent molecular simulation study has provided evidence that all nonpolar side chains, attached to a short peptide backbone, are considerably less hydrophobic than the free side chain analogue molecules. In contrast to this, the hydrophilicity of the polar side chains is hardly affected by the backbone. To analyze the origin of these observations, we here present a molecular simulation study on temperature dependent solvation free energies of nonpolar and polar side chains attached to a short peptide backbone. The estimated solvation entropies and enthalpies of the various amino acid side chains are compared with existing side chain analogue data. The solvation entropies and enthalpies of the polar side chains are negative, but in absolute magnitude smaller compared with the corresponding analogue data. The observed differences are large; however, owing to a nearly perfect enthalpy-entropy compensation, the solvation free energies of polar side chains remain largely unaffected by the peptide backbone. We find that a similar compensation does not apply to the nonpolar side chains; while the backbone greatly reduces the unfavorable solvation entropies, the solvation enthalpies are either more favorable or only marginally affected. This results in a very small unfavorable free energy cost, or even free energy gain, of solvating the nonpolar side chains in strong contrast to solvation of small hydrophobic or nonpolar molecules in bulk water. The solvation free energies of nonpolar side chains have been furthermore decomposed into a repulsive cavity formation contribution and an attractive dispersion free energy contribution. We find that cavity formation next to the peptide backbone is entropically favored over formation of similar sized nonpolar side chain cavities in bulk water, in agreement with earlier work in the literature on analysis of cavity fluctuations at nonpolar molecular surfaces. The cavity and dispersion interaction contributions correlate quite well with the solvent accessible surface area of the nonpolar side chains attached to the backbone. This correlation however is weak for the overall solvation free energies owing to the fact that the cavity and dispersion free energy contributions are almost exactly cancelling each other.

Interplay Between Hydrophobic Effect and Dipole Interactions in Peptide Aggregation

NASA Astrophysics Data System (ADS)

Ganesan, Sai; Matysiak, Silvina

In the past decade, the development of various coarse-grained models for proteins have provided key insights into the driving forces in folding and aggregation.We recently developed a low resolution Water Explicit Polarizable PROtein coarse-grained Model by adding oppositely charged dummy particles inside protein backbone beads.With this model,we were able to achieve significant α/ β secondary structure content,without any added bias.We now extend the model to study peptide aggregation at hydrophobic-hydrophilic interface using elastin-like octapeptides (GV)4 as a model system.A condensation-ordering mechanism of aggregation is observed in water.Our results suggest that backbone interpeptide dipolar interactions,not hydrophobicity,plays a more significant role in fibril-like peptide aggregation.We observe a cooperative effect in hydrogen bonding or dipolar interactions, with increase in aggregate size in water and interface.Based on this cooperative effect, we provide a potential explanation for the observed nucleus size in peptide aggregation pathways.Without dipolar particles,peptide aggregation is not observed at the hydrophilic-hydrophobic interface.Thus,the presence of dipoles,not hydrophobicity plays a key role in aggregation observed at hydrophobic interfaces.

A hinge migration mechanism unlocks the evolution of green-to-red photoconversion in GFP-like proteins

DOE PAGES

Kim, Hanseong; Zou, Taisong; Modi, Chintan; ...

2014-12-31

In proteins, functional divergence involves mutations that modify structure and dynamics. In this paper, we provide experimental evidence for an evolutionary mechanism driven solely by long-range dynamic motions without significant backbone adjustments, catalytic group rearrangements, or changes in subunit assembly. Crystallographic structures were determined for several reconstructed ancestral proteins belonging to a GFP class frequently employed in superresolution microscopy. Their chain flexibility was analyzed using molecular dynamics and perturbation response scanning. The green-to-red photoconvertible phenotype appears to have arisen from a common green ancestor by migration of a knob-like anchoring region away from the active site diagonally across the βmore » barrel fold. The allosterically coupled mutational sites provide active site conformational mobility via epistasis. We propose that light-induced chromophore twisting is enhanced in a reverse-protonated subpopulation, activating internal acid-base chemistry and backbone cleavage to enlarge the chromophore. Finally, dynamics-driven hinge migration may represent a more general platform for the evolution of novel enzyme activities.« less

A de novo redesign of the WW domain

PubMed Central

Kraemer-Pecore, Christina M.; Lecomte, Juliette T.J.; Desjarlais, John R.

2003-01-01

We have used a sequence prediction algorithm and a novel sampling method to design protein sequences for the WW domain, a small β-sheet motif. The procedure, referred to as SPANS, designs sequences to be compatible with an ensemble of closely related polypeptide backbones, mimicking the inherent flexibility of proteins. Two designed sequences (termed SPANS-WW1 and SPANS-WW2), using only naturally occurring l-amino acids, were selected for study and the corresponding polypeptides were prepared in Escherichia coli. Circular dichroism data suggested that both purified polypeptides adopted secondary structure features related to those of the target without the aid of disulfide bridges or bound cofactors. The structure exhibited by SPANS-WW2 melted cooperatively by raising the temperature of the solution. Further analysis of this polypeptide by proton nuclear magnetic resonance spectroscopy demonstrated that at 5°C, it folds into a structure closely resembling a natural WW domain. This achievement constitutes one of a small number of successful de novo protein designs through fully automated computational methods and highlights the feasibility of including backbone flexibility in the design strategy. PMID:14500877

A de novo redesign of the WW domain.

PubMed

Kraemer-Pecore, Christina M; Lecomte, Juliette T J; Desjarlais, John R

2003-10-01

We have used a sequence prediction algorithm and a novel sampling method to design protein sequences for the WW domain, a small beta-sheet motif. The procedure, referred to as SPANS, designs sequences to be compatible with an ensemble of closely related polypeptide backbones, mimicking the inherent flexibility of proteins. Two designed sequences (termed SPANS-WW1 and SPANS-WW2), using only naturally occurring L-amino acids, were selected for study and the corresponding polypeptides were prepared in Escherichia coli. Circular dichroism data suggested that both purified polypeptides adopted secondary structure features related to those of the target without the aid of disulfide bridges or bound cofactors. The structure exhibited by SPANS-WW2 melted cooperatively by raising the temperature of the solution. Further analysis of this polypeptide by proton nuclear magnetic resonance spectroscopy demonstrated that at 5 degrees C, it folds into a structure closely resembling a natural WW domain. This achievement constitutes one of a small number of successful de novo protein designs through fully automated computational methods and highlights the feasibility of including backbone flexibility in the design strategy.

Tackling force-field bias in protein folding simulations: folding of Villin HP35 and Pin WW domains in explicit water.

PubMed

Mittal, Jeetain; Best, Robert B

2010-08-04

The ability to fold proteins on a computer has highlighted the fact that existing force fields tend to be biased toward a particular type of secondary structure. Consequently, force fields for folding simulations are often chosen according to the native structure, implying that they are not truly "transferable." Here we show that, while the AMBER ff03 potential is known to favor helical structures, a simple correction to the backbone potential (ff03( *)) results in an unbiased energy function. We take as examples the 35-residue alpha-helical Villin HP35 and 37 residue beta-sheet Pin WW domains, which had not previously been folded with the same force field. Starting from unfolded configurations, simulations of both proteins in Amber ff03( *) in explicit solvent fold to within 2.0 A RMSD of the experimental structures. This demonstrates that a simple backbone correction results in a more transferable force field, an important requirement if simulations are to be used to interpret folding mechanism. 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Characterization of cationic polymers by asymmetric flow field-flow fractionation and multi-angle light scattering-A comparison with traditional techniques.

PubMed

Wagner, Michael; Pietsch, Christian; Tauhardt, Lutz; Schallon, Anja; Schubert, Ulrich S

2014-01-17

In the field of nanomedicine, cationic polymers are the subject of intensive research and represent promising carriers for genetic material. The detailed characterization of these carriers is essential since the efficiency of gene delivery strongly depends on the properties of the used polymer. Common characterization methods such as size exclusion chromatography (SEC) or mass spectrometry (MS) suffer from problems, e.g. missing standards, or even failed for cationic polymers. As an alternative, asymmetrical flow field-flow fractionation (AF4) was investigated. Additionally, analytical ultracentrifugation (AUC) and (1)H NMR spectroscopy, as well-established techniques, were applied to evaluate the results obtained by AF4. In this study, different polymers of molar masses between 10 and 120kgmol(-1) with varying amine functionalities in the side chain or in the polymer backbone were investigated. To this end, some of the most successful gene delivery agents, namely linear poly(ethylene imine) (LPEI) (only secondary amines in the backbone), branched poly(ethylene imine) (B-PEI) (secondary and tertiary amino groups in the backbone, primary amine end groups), and poly(l-lysine) (amide backbone and primary amine side chains), were characterized. Moreover, poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA), poly(2-(amino)ethyl methacrylate) (PAEMA), and poly(2-(tert-butylamino)ethyl methacrylate) (PtBAEMA) as polymers with primary, secondary, and tertiary amines in the side chain, have been investigated. Reliable results were obtained for all investigated polymers by AF4. In addition, important factors for all methods were evaluated, e.g. the influence of different elution buffers and AF4 membranes. Besides this, the correct determination of the partial specific volume and the suppression of the polyelectrolyte effect are the most critical issues for AUC investigations. Copyright © 2013 Elsevier B.V. All rights reserved.

The topomer-sampling model of protein folding

PubMed Central

Debe, Derek A.; Carlson, Matt J.; Goddard, William A.

1999-01-01

Clearly, a protein cannot sample all of its conformations (e.g., ≈3100 ≈ 1048 for a 100 residue protein) on an in vivo folding timescale (<1 s). To investigate how the conformational dynamics of a protein can accommodate subsecond folding time scales, we introduce the concept of the native topomer, which is the set of all structures similar to the native structure (obtainable from the native structure through local backbone coordinate transformations that do not disrupt the covalent bonding of the peptide backbone). We have developed a computational procedure for estimating the number of distinct topomers required to span all conformations (compact and semicompact) for a polypeptide of a given length. For 100 residues, we find ≈3 × 107 distinct topomers. Based on the distance calculated between different topomers, we estimate that a 100-residue polypeptide diffusively samples one topomer every ≈3 ns. Hence, a 100-residue protein can find its native topomer by random sampling in just ≈100 ms. These results suggest that subsecond folding of modest-sized, single-domain proteins can be accomplished by a two-stage process of (i) topomer diffusion: random, diffusive sampling of the 3 × 107 distinct topomers to find the native topomer (≈0.1 s), followed by (ii) intratopomer ordering: nonrandom, local conformational rearrangements within the native topomer to settle into the precise native state. PMID:10077555

The Dynameomics Entropy Dictionary: A Large-Scale Assessment of Conformational Entropy across Protein Fold Space.

PubMed

Towse, Clare-Louise; Akke, Mikael; Daggett, Valerie

2017-04-27

Molecular dynamics (MD) simulations contain considerable information with regard to the motions and fluctuations of a protein, the magnitude of which can be used to estimate conformational entropy. Here we survey conformational entropy across protein fold space using the Dynameomics database, which represents the largest existing data set of protein MD simulations for representatives of essentially all known protein folds. We provide an overview of MD-derived entropies accounting for all possible degrees of dihedral freedom on an unprecedented scale. Although different side chains might be expected to impose varying restrictions on the conformational space that the backbone can sample, we found that the backbone entropy and side chain size are not strictly coupled. An outcome of these analyses is the Dynameomics Entropy Dictionary, the contents of which have been compared with entropies derived by other theoretical approaches and experiment. As might be expected, the conformational entropies scale linearly with the number of residues, demonstrating that conformational entropy is an extensive property of proteins. The calculated conformational entropies of folding agree well with previous estimates. Detailed analysis of specific cases identifies deviations in conformational entropy from the average values that highlight how conformational entropy varies with sequence, secondary structure, and tertiary fold. Notably, α-helices have lower entropy on average than do β-sheets, and both are lower than coil regions.

Dynamics of the GB3 loop regions from MD simulation: how much of it is real?

PubMed

Li, Tong; Jing, Qingqing; Yao, Lishan

2011-04-07

A total of 1.1 μs of molecular dynamics (MD) simulations were performed to study the structure and dynamics of protein GB3. The simulation motional amplitude of the loop regions is generally overestimated in comparison with the experimental backbone N-H order parameters S(2). Two-state behavior is observed for several residues in these regions, with the minor state population in the range of 3-13%. Further inspection suggests that the (φ, ψ) dihedral angles of the minor states deviate from the GB3 experimental values, implying the existence of nonnative states. After fitting the MD trajectories of these residues to the NMR RDCs, the minor state populations are significantly reduced by at least 80%, suggesting that MD simulations are strongly biased toward the minor states, thus overestimating the dynamics of the loop regions. The optimized trajectories produce intra, sequential H(N)-H(α) RDCs and intra (3)J(HNHα) that are not included in the trajectories fitting for these residues that are closer to the experimental data. Unlike GB3, 0.55 μs MD simulations of protein ubiquitin do not show distinctive minor states, and the derived NMR order parameters are better converged. Our findings indicate that the artifacts of the simulations depend on the specific system studied and that one should be cautious interpreting the enhanced dihedral dynamics from long MD simulations.

Intermolecular Structural Change for Thermoswitchable Polymeric Photosensitizer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Wooram; Park, Sin-Jung; Cho, Soojeong

2016-08-17

A switchable photosensitizer (PS), which can be activated at a spe-cific condition beside light, has tremendous advantages for photo-dynamic therapy (PDT). Herein, we developed a thermo-switchable polymeric photosensitizer (T-PPS) by conjugating PS (Pheophor-bide-a, PPb-a) to a temperature-responsive polymer backbone of biocompatible hydroxypropyl cellulose (HPC). Self-quenched PS molecules linked in close proximity by pi-pi stacking in T-PPS were easily transited to an active monomeric state by the tempera-ture induced phase transition of polymer backbones. The tempera-ture responsive inter-molecular interaction changes of PS molecules in T-PPS were demonstrated in synchrotron small-angle X-ray scattering (SAXS) and UV-Vis spectrophotometer analysis. The T-PPS allowed switchablemore » activation and synergistically enhanced cancer cell killing effect at the hyperthermia temperature (45 °C). Our developed T-PPS has the considerable potential not only as a new class of photomedicine in clinics but also as a biosensor based on temperature responsiveness.« less

Improved Atomistic Monte Carlo Simulations Demonstrate that Poly-L-Proline Adopts Heterogeneous Ensembles of Conformations of Semi-Rigid Segments Interrupted by Kinks

PubMed Central

Radhakrishnan, Aditya; Vitalis, Andreas; Mao, Albert H.; Steffen, Adam T.; Pappu, Rohit V.

2012-01-01

Poly-L-proline (PLP) polymers are useful mimics of biologically relevant proline-rich sequences. Biophysical and computational studies of PLP polymers in aqueous solutions are challenging because of the diversity of length scales and the slow time scales for conformational conversions. We describe an atomistic simulation approach that combines an improved ABSINTH implicit solvation model, with conformational sampling based on standard and novel Metropolis Monte Carlo moves. Refinements to forcefield parameters were guided by published experimental data for proline-rich systems. We assessed the validity of our simulation results through quantitative comparisons to experimental data that were not used in refining the forcefield parameters. Our analysis shows that PLP polymers form heterogeneous ensembles of conformations characterized by semi-rigid, rod-like segments interrupted by kinks, which result from a combination of internal cis peptide bonds, flexible backbone ψ-angles, and the coupling between ring puckering and backbone degrees of freedom. PMID:22329658

The Prediction of Botulinum Toxin Structure Based on in Silico and in Vitro Analysis

NASA Astrophysics Data System (ADS)

Suzuki, Tomonori; Miyazaki, Satoru

2011-01-01

Many of biological system mediated through protein-protein interactions. Knowledge of protein-protein complex structure is required for understanding the function. The determination of huge size and flexible protein-protein complex structure by experimental studies remains difficult, costly and five-consuming, therefore computational prediction of protein structures by homolog modeling and docking studies is valuable method. In addition, MD simulation is also one of the most powerful methods allowing to see the real dynamics of proteins. Here, we predict protein-protein complex structure of botulinum toxin to analyze its property. These bioinformatics methods are useful to report the relation between the flexibility of backbone structure and the activity.

Structural basis for new pattern of conserved amino acid residues related to chitin-binding in the antifungal peptide from the coconut rhinoceros beetle Oryctes rhinoceros.

PubMed

Hemmi, Hikaru; Ishibashi, Jun; Tomie, Tetsuya; Yamakawa, Minoru

2003-06-20

Scarabaecin isolated from hemolymph of the coconut rhinoceros beetle Oryctes rhinoceros is a 36-residue polypeptide that has antifungal activity. The solution structure of scarabaecin has been determined from twodimensional 1H NMR spectroscopic data and hybrid distance geometry-simulated annealing protocol calculation. Based on 492 interproton and 10 hydrogen-bonding distance restraints and 36 dihedral angle restraints, we obtained 20 structures. The average backbone root-mean-square deviation for residues 4-35 is 0.728 +/- 0.217 A from the mean structure. The solution structure consists of a two-stranded antiparallel beta-sheet connected by a type-I beta-turn after a short helical turn. All secondary structures and a conserved disulfide bond are located in the C-terminal half of the peptide, residues 18-36. Overall folding is stabilized by a combination of a disulfide bond, seven hydrogen bonds, and numerous hydrophobic interactions. The structural motif of the C-terminal half shares a significant tertiary structural similarity with chitin-binding domains of plant and invertebrate chitin-binding proteins, even though scarabaecin has no overall sequence similarity to other peptide/polypeptides including chitin-binding proteins. The length of its primary structure, the number of disulfide bonds, and the pattern of conserved functional residues binding to chitin in scarabaecin differ from those of chitin-binding proteins in other invertebrates and plants, suggesting that scarabaecin does not share a common ancestor with them. These results are thought to provide further strong experimental evidence to the hypothesis that chitin-binding proteins of invertebrates and plants are correlated by a convergent evolution process.

Prediction of Protein Structure by Template-Based Modeling Combined with the UNRES Force Field.

PubMed

Krupa, Paweł; Mozolewska, Magdalena A; Joo, Keehyoung; Lee, Jooyoung; Czaplewski, Cezary; Liwo, Adam

2015-06-22

A new approach to the prediction of protein structures that uses distance and backbone virtual-bond dihedral angle restraints derived from template-based models and simulations with the united residue (UNRES) force field is proposed. The approach combines the accuracy and reliability of template-based methods for the segments of the target sequence with high similarity to those having known structures with the ability of UNRES to pack the domains correctly. Multiplexed replica-exchange molecular dynamics with restraints derived from template-based models of a given target, in which each restraint is weighted according to the accuracy of the prediction of the corresponding section of the molecule, is used to search the conformational space, and the weighted histogram analysis method and cluster analysis are applied to determine the families of the most probable conformations, from which candidate predictions are selected. To test the capability of the method to recover template-based models from restraints, five single-domain proteins with structures that have been well-predicted by template-based methods were used; it was found that the resulting structures were of the same quality as the best of the original models. To assess whether the new approach can improve template-based predictions with incorrectly predicted domain packing, four such targets were selected from the CASP10 targets; for three of them the new approach resulted in significantly better predictions compared with the original template-based models. The new approach can be used to predict the structures of proteins for which good templates can be found for sections of the sequence or an overall good template can be found for the entire sequence but the prediction quality is remarkably weaker in putative domain-linker regions.

The unique fold and lability of the [2Fe-2S] clusters of NEET proteins mediate their key functions in health and disease.

PubMed

Karmi, Ola; Marjault, Henri-Baptiste; Pesce, Luca; Carloni, Paolo; Onuchic, Jose' N; Jennings, Patricia A; Mittler, Ron; Nechushtai, Rachel

2018-02-12

NEET proteins comprise a new class of [2Fe-2S] cluster proteins. In human, three genes encode for NEET proteins: cisd1 encodes mitoNEET (mNT), cisd2 encodes the Nutrient-deprivation autophagy factor-1 (NAF-1) and cisd3 encodes MiNT (Miner2). These recently discovered proteins play key roles in many processes related to normal metabolism and disease. Indeed, NEET proteins are involved in iron, Fe-S, and reactive oxygen homeostasis in cells and play an important role in regulating apoptosis and autophagy. mNT and NAF-1 are homodimeric and reside on the outer mitochondrial membrane. NAF-1 also resides in the membranes of the ER associated mitochondrial membranes (MAM) and the ER. MiNT is a monomer with distinct asymmetry in the molecular surfaces surrounding the clusters. Unlike its paralogs mNT and NAF-1, it resides within the mitochondria. NAF-1 and mNT share similar backbone folds to the plant homodimeric NEET protein (At-NEET), while MiNT's backbone fold resembles a bacterial MiNT protein. Despite the variation of amino acid composition among these proteins, all NEET proteins retained their unique CDGSH domain harboring their unique 3Cys:1His [2Fe-2S] cluster coordination through evolution. The coordinating exposed His was shown to convey the lability to the NEET proteins' [2Fe-2S] clusters. In this minireview, we discuss the NEET fold and its structural elements. Special attention is given to the unique lability of the NEETs' [2Fe-2S] cluster and the implication of the latter to the NEET proteins' cellular and systemic function in health and disease.

Construction and analysis of protein-protein interaction networks based on proteomics data of prostate cancer

PubMed Central

CHEN, CHEN; SHEN, HONG; ZHANG, LI-GUO; LIU, JIAN; CAO, XIAO-GE; YAO, AN-LIANG; KANG, SHAO-SAN; GAO, WEI-XING; HAN, HUI; CAO, FENG-HONG; LI, ZHI-GUO

2016-01-01

Currently, using human prostate cancer (PCa) tissue samples to conduct proteomics research has generated a large amount of data; however, only a very small amount has been thoroughly investigated. In this study, we manually carried out the mining of the full text of proteomics literature that involved comparisons between PCa and normal or benign tissue and identified 41 differentially expressed proteins verified or reported more than 2 times from different research studies. We regarded these proteins as seed proteins to construct a protein-protein interaction (PPI) network. The extended network included one giant network, which consisted of 1,264 nodes connected via 1,744 edges, and 3 small separate components. The backbone network was then constructed, which was derived from key nodes and the subnetwork consisting of the shortest path between seed proteins. Topological analyses of these networks were conducted to identify proteins essential for the genesis of PCa. Solute carrier family 2 (facilitated glucose transporter), member 4 (SLC2A4) had the highest closeness centrality located in the center of each network, and the highest betweenness centrality and largest degree in the backbone network. Tubulin, beta 2C (TUBB2C) had the largest degree in the giant network and subnetwork. In addition, using module analysis of the whole PPI network, we obtained a densely connected region. Functional annotation indicated that the Ras protein signal transduction biological process, mitogen-activated protein kinase (MAPK), neurotrophin and the gonadotropin-releasing hormone (GnRH) signaling pathway may play an important role in the genesis and development of PCa. Further investigation of the SLC2A4, TUBB2C proteins, and these biological processes and pathways may therefore provide a potential target for the diagnosis and treatment of PCa. PMID:27121963

Repacking the Core of T4 lysozyme by automated design.

PubMed

Mooers, Blaine H M; Datta, Deepshikha; Baase, Walter A; Zollars, Eric S; Mayo, Stephen L; Matthews, Brian W

2003-09-19

Automated protein redesign, as implemented in the program ORBIT, was used to redesign the core of phage T4 lysozyme. A total of 26 buried or partially buried sites in the C-terminal domain were allowed to vary both their sequence and side-chain conformation while the backbone and non-selected side-chains remained fixed. A variant with seven substitutions ("Core-7") was identified as having the most favorable energy. The redesign experiment was repeated with a penalty for the presence of methionine residues. In this case the redesigned protein ("Core-10") had ten amino acid changes. The two designed proteins, as well as the constituent single mutants, and several single-site revertants were over-expressed in Escherichia coli, purified, and subjected to crystallographic and thermal analyses. The thermodynamic and structural data show that some repacking was achieved although neither redesigned protein was more stable than the wild-type protein. The use of the methionine penalty was shown to be effective. Several of the side-chain rotamers in the predicted structure of Core-10 differ from those observed. Rather than changing to new rotamers predicted by the design process, side-chains tend to maintain conformations similar to those seen in the native molecule. In contrast, parts of the backbone change by up to 2.8A relative to both the designed structure and wild-type. Water molecules that are present within the lysozyme molecule were removed during the design process. In the redesigned protein the resultant cavities were, to some degree, re-occupied by side-chain atoms. In the observed structure, however, water molecules were still bound at or near their original sites. This suggests that it may be preferable to leave such water molecules in place during the design procedure. The results emphasize the specificity of the packing that occurs within the core of a typical protein. While point substitutions within the core are tolerated they almost always result in a loss of stability. Likewise, combinations of substitutions may also be tolerated but usually destabilize the protein. Experience with T4 lysozyme suggests that a general core repacking methodology with retention or enhancement of stability may be difficult to achieve without provision for shifts in the backbone.

Structural characterization of the P1+ intermediate state of the P-cluster of nitrogenase.

PubMed

Keable, Stephen M; Zadvornyy, Oleg A; Johnson, Lewis E; Ginovska, Bojana; Rasmussen, Andrew J; Danyal, Karamatullah; Eilers, Brian J; Prussia, Gregory A; LeVan, Axl X; Raugei, Simone; Seefeldt, Lance C; Peters, John W

2018-05-02

Nitrogenase is the enzyme that reduces atmospheric dinitrogen (N 2 ) to ammonia (NH 3 ) in biological systems. It catalyzes a series of single-electron transfers from the donor iron protein (Fe protein) to the molybdenum-iron protein (MoFe protein) that contains the iron-molybdenum cofactor (FeMo-co) sites where N 2 is reduced to NH 3 The [8Fe-7S] P-cluster in the MoFe protein functions in nitrogenase catalysis as an intermediate electron carrier between the external electron donor, the Fe protein, and the FeMo-co sites of the MoFe protein. Previous work has revealed that the P-cluster undergoes redox dependent structural changes and that the transition from the all-ferrous resting (P N ) state to the two electron oxidized P 2+ state is accompanied by protein serince hydroxyl and backbone amide ligation to Fe. In this work, the MoFe protein was poised at defined potentials with redox mediators in an electrochemical cell, and the three distinct structural states of the P-cluster (P 2+ , P 1+ , and P N ) were characterized by X-ray crystallography and confirmed by computational analysis. These analyses revealed that the three oxidation states differ in coordination implicating that the P 1+ state retains the serine hydroxyl coordination but lacks the backbone amide coordination observed in the P 2+ states. These results provide a complete picture of the redox-dependent ligand rearrangements of the three P-cluster redox states. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Quantum chemical calculations in the structural analysis of phloretin

NASA Astrophysics Data System (ADS)

Gómez-Zavaglia, Andrea

2009-07-01

In this work, a conformational search on the molecule of phloretin [2',4',6'-Trihydroxy-3-(4-hydroxyphenyl)-propiophenone] has been performed. The molecule of phloretin has eight dihedral angles, four of them taking part in the carbon backbone and the other four, related with the orientation of the hydroxyl groups. A systematic search involving a random variation of the dihedral angles has been used to generate input structures for the quantum chemical calculations. Calculations at the DFT(B3LYP)/6-311++G(d,p) level of theory permitted the identification of 58 local minima belonging to the C 1 symmetry point group. The molecular structures of the conformers have been analyzed using hierarchical cluster analysis. This method allowed us to group conformers according to their similarities, and thus, to correlate the conformers' stability with structural parameters. The dendrogram obtained from the hierarchical cluster analysis depicted two main clusters. Cluster I included all the conformers with relative energies lower than 25 kJ mol -1 and cluster II, the remaining conformers. The possibility of forming intramolecular hydrogen bonds resulted the main factor contributing for the stability. Accordingly, all conformers depicting intramolecular H-bonds belong to cluster I. These conformations are clearly favored when the carbon backbone is as planar as possible. The values of the νC dbnd O and νOH vibrational modes were compared among all the conformers of phloretin. The redshifts associated with intramolecular H-bonds were correlated with the H-bonds distances and energies.

Nature and properties of ionomer assemblies. II.

PubMed

Capek, Ignác

2005-12-30

The principle subject in the current paper is to summarize and characterize the ionomers based on polymers and copolymers such as polystyrene (PSt), polyisoprene (PIP), polybutadiene (PB), poly(styrene-b-isobutylene-b-styrene) (PSt-PIB-PSt), poly(butadiene-styrene) (PB-PSt), poly(ethylene terephthalate) (PET), poly(butylene adipate) (PBA), poly(butylene succinate) (PBSi), poly(dimethylcarbosiloxanes), polyurethane, etc. The self-assembly of ionomers, models concerning ionomer morphologies, physical and rheological properties of ionomer phase and percolation behavior of ionomers were discussed. The ionomer phase materials and dispersions have been characterized by differential scanning calorimetry (DSC), small-angle X-ray catering (SAXS), small-angle neutron scattering (SANS), Fourier transform infrared (FTIR) spectroscopy, transmission electron microscopy (TEM), etc. The wide range of compositions, molecular architectures, and morphologies present in ionomeric disperse systems are of great interest. The research is particularly devoted to the potential application of these materials and an understanding of the fundamental principles of the ionomers. They are extremely complex systems, sensitive to changes in structure and composition, and therefore not easily amenable to modeling and to the derivation of general patterns of behavior. The reviewed data indicate that a large number of parameters are important in influencing multiplet formation and clustering in random ionomers. Among these are the ion content, size of the polyion and counterion, dielectric constant of the host, T(g) of the polymer, rigidity or persistence length of the backbone, position of the ion pair relative to the backbone, steric constraints, amount and nature of added additive (plasticizer), thermal history, etc.

In silico folding of a three helix protein and characterization of its free-energy landscape in an all-atom force field.

PubMed

Herges, T; Wenzel, W

2005-01-14

We report the reproducible first-principles folding of the 40 amino-acid, three-helix headpiece of the HIV accessory protein in a recently developed all-atom free-energy force field. Six of 20 simulations using an adapted basin-hopping method converged to better than 3 A backbone rms deviation to the experimental structure. Using over 60 000 low-energy conformations of this protein, we constructed a decoy tree that completely characterizes its folding funnel.

In Silico Folding of a Three Helix Protein and Characterization of Its Free-Energy Landscape in an All-Atom Force Field

NASA Astrophysics Data System (ADS)

Herges, T.; Wenzel, W.

2005-01-01

We report the reproducible first-principles folding of the 40 amino-acid, three-helix headpiece of the HIV accessory protein in a recently developed all-atom free-energy force field. Six of 20 simulations using an adapted basin-hopping method converged to better than 3Å backbone rms deviation to the experimental structure. Using over 60 000 low-energy conformations of this protein, we constructed a decoy tree that completely characterizes its folding funnel.

α/β coiled coils

PubMed Central

Hartmann, Marcus D; Mendler, Claudia T; Bassler, Jens; Karamichali, Ioanna; Ridderbusch, Oswin; Lupas, Andrei N; Hernandez Alvarez, Birte

2016-01-01

Coiled coils are the best-understood protein fold, as their backbone structure can uniquely be described by parametric equations. This level of understanding has allowed their manipulation in unprecedented detail. They do not seem a likely source of surprises, yet we describe here the unexpected formation of a new type of fiber by the simple insertion of two or six residues into the underlying heptad repeat of a parallel, trimeric coiled coil. These insertions strain the supercoil to the breaking point, causing the local formation of short β-strands, which move the path of the chain by 120° around the trimer axis. The result is an α/β coiled coil, which retains only one backbone hydrogen bond per repeat unit from the parent coiled coil. Our results show that a substantially novel backbone structure is possible within the allowed regions of the Ramachandran space with only minor mutations to a known fold. DOI: http://dx.doi.org/10.7554/eLife.11861.001 PMID:26771248

Role of Pilus Proteins in Adherence and Invasion of Streptococcus agalactiae to the Lung and Cervical Epithelial Cells*

PubMed Central

Sharma, Puja; Lata, Hem; Arya, Deepak Kumar; Kashyap, Arun Kumar; Kumar, Hemant; Dua, Meenakshi; Ali, Arif; Johri, Atul Kumar

2013-01-01

Streptococcus agalactiae, or group B Streptococcus (GBS), is an important opportunistic pathogen that causes pneumonia, sepsis, and meningitis in neonates and severe diseases in immunocompromised adults. We have performed comparative genomics of prevalent GBS serotypes of Indian origin (i.e. Ia, III, V, and VII). Pilus-proteins were commonly found up-regulated, and their expression was studied by using antiserum for GBS80 (backbone protein of pilus island-I), GBS67 (ancillary protein of PI-2a), and SAN1518 (backbone protein of PI-2b) by whole cell and Western blot analysis. To check the role of pilus proteins in adherence and invasion, an inhibition assay was performed. Comparative immunoblotting experiments revealed that expression of pili proteins does not differ in geographically different selected serotypes, Ia and V, of India and the United States. In the case of A549 cells, we found that GBS VII invasion and adherence was inhibited by pilus protein-specific antiserum SAN1518 significantly (p < 0.001) by 88.5 and 91%, respectively. We found that mutant strains, deficient in the pilus proteins (Δgbs80 and Δsan1518) exhibit a significant decrease in adherence in the case of type Ia, III, and VII. In the case of type VII, we have found a 95% reduction in invasion when Δsan1518 was used with A549 cells. Because the pilus proteins were identified previously as vaccine candidates against GBS serotypes of developed countries, we also found their role in the attachment and invasion of GBS of Indian origin. Thus, the present work supports the idea of making a more effective pilus protein-based vaccine that can be used universally. PMID:23209289

Automated method to differentiate between native and mirror protein models obtained from contact maps.

PubMed

Kurczynska, Monika; Kotulska, Malgorzata

2018-01-01

Mirror protein structures are often considered as artifacts in modeling protein structures. However, they may soon become a new branch of biochemistry. Moreover, methods of protein structure reconstruction, based on their residue-residue contact maps, need methodology to differentiate between models of native and mirror orientation, especially regarding the reconstructed backbones. We analyzed 130 500 structural protein models obtained from contact maps of 1 305 SCOP domains belonging to all 7 structural classes. On average, the same numbers of native and mirror models were obtained among 100 models generated for each domain. Since their structural features are often not sufficient for differentiating between the two types of model orientations, we proposed to apply various energy terms (ETs) from PyRosetta to separate native and mirror models. To automate the procedure for differentiating these models, the k-means clustering algorithm was applied. Using total energy did not allow to obtain appropriate clusters-the accuracy of the clustering for class A (all helices) was no more than 0.52. Therefore, we tested a series of different k-means clusterings based on various combinations of ETs. Finally, applying two most differentiating ETs for each class allowed to obtain satisfying results. To unify the method for differentiating between native and mirror models, independent of their structural class, the two best ETs for each class were considered. Finally, the k-means clustering algorithm used three common ETs: probability of amino acid assuming certain values of dihedral angles Φ and Ψ, Ramachandran preferences and Coulomb interactions. The accuracies of clustering with these ETs were in the range between 0.68 and 0.76, with sensitivity and selectivity in the range between 0.68 and 0.87, depending on the structural class. The method can be applied to all fully-automated tools for protein structure reconstruction based on contact maps, especially those analyzing big sets of models.

Automated method to differentiate between native and mirror protein models obtained from contact maps

PubMed Central

Kurczynska, Monika

2018-01-01

Mirror protein structures are often considered as artifacts in modeling protein structures. However, they may soon become a new branch of biochemistry. Moreover, methods of protein structure reconstruction, based on their residue-residue contact maps, need methodology to differentiate between models of native and mirror orientation, especially regarding the reconstructed backbones. We analyzed 130 500 structural protein models obtained from contact maps of 1 305 SCOP domains belonging to all 7 structural classes. On average, the same numbers of native and mirror models were obtained among 100 models generated for each domain. Since their structural features are often not sufficient for differentiating between the two types of model orientations, we proposed to apply various energy terms (ETs) from PyRosetta to separate native and mirror models. To automate the procedure for differentiating these models, the k-means clustering algorithm was applied. Using total energy did not allow to obtain appropriate clusters–the accuracy of the clustering for class A (all helices) was no more than 0.52. Therefore, we tested a series of different k-means clusterings based on various combinations of ETs. Finally, applying two most differentiating ETs for each class allowed to obtain satisfying results. To unify the method for differentiating between native and mirror models, independent of their structural class, the two best ETs for each class were considered. Finally, the k-means clustering algorithm used three common ETs: probability of amino acid assuming certain values of dihedral angles Φ and Ψ, Ramachandran preferences and Coulomb interactions. The accuracies of clustering with these ETs were in the range between 0.68 and 0.76, with sensitivity and selectivity in the range between 0.68 and 0.87, depending on the structural class. The method can be applied to all fully-automated tools for protein structure reconstruction based on contact maps, especially those analyzing big sets of models. PMID:29787567

Solution structure of the IIAChitobiose-IIBChitobiose complex of the N,N'-diacetylchitobiose branch of the Escherichia coli phosphotransferase system.

PubMed

Jung, Young-Sang; Cai, Mengli; Clore, G Marius

2010-02-05

The solution structure of the IIA-IIB complex of the N,N'-diacetylchitobiose (Chb) transporter of the Escherichia coli phosphotransferase system has been solved by NMR. The active site His-89 of IIA(Chb) was mutated to Glu to mimic the phosphorylated state and the active site Cys-10 of IIB(Chb) was substituted by serine to prevent intermolecular disulfide bond formation. Binding is weak with a K(D) of approximately 1.3 mm. The two complementary interaction surfaces are largely hydrophobic, with the protruding active site loop (residues 9-16) of IIB(Chb) buried deep within the active site cleft formed at the interface of two adjacent subunits of the IIA(Chb) trimer. The central hydrophobic portion of the interface is surrounded by a ring of polar and charged residues that provide a relatively small number of electrostatic intermolecular interactions that serve to correctly align the two proteins. The conformation of the active site loop in unphosphorylated IIB(Chb) is inconsistent with the formation of a phosphoryl transition state intermediate because of steric hindrance, especially from the methyl group of Ala-12 of IIB(Chb). Phosphorylation of IIB(Chb) is accompanied by a conformational change within the active site loop such that its path from residues 11-13 follows a mirror-like image relative to that in the unphosphorylated state. This involves a transition of the phi/psi angles of Gly-13 from the right to left alpha-helical region, as well as smaller changes in the backbone torsion angles of Ala-12 and Met-14. The resulting active site conformation is fully compatible with the formation of the His-89-P-Cys-10 phosphoryl transition state without necessitating any change in relative translation or orientation of the two proteins within the complex.

Mirrors in the PDB: left-handed alpha-turns guide design with D-amino acids.

PubMed

Annavarapu, Srinivas; Nanda, Vikas

2009-09-22

Incorporating variable amino acid stereochemistry in molecular design has the potential to improve existing protein stability and create new topologies inaccessible to homochiral molecules. The Protein Data Bank has been a reliable, rich source of information on molecular interactions and their role in protein stability and structure. D-amino acids rarely occur naturally, making it difficult to infer general rules for how they would be tolerated in proteins through an analysis of existing protein structures. However, protein elements containing short left-handed turns and helices turn out to contain useful information. Molecular mechanisms used in proteins to stabilize left-handed elements by L-amino acids are structurally enantiomeric to potential synthetic strategies for stabilizing right-handed elements with D-amino acids. Propensities for amino acids to occur in contiguous alpha(L) helices correlate with published thermodynamic scales for incorporation of D-amino acids into alpha(R) helices. Two backbone rules for terminating a left-handed helix are found: an alpha(R) conformation is disfavored at the amino terminus, and a beta(R) conformation is disfavored at the carboxy terminus. Helix capping sidechain-backbone interactions are found which are unique to alpha(L) helices including an elevated propensity for L-Asn, and L-Thr at the amino terminus and L-Gln, L-Thr and L-Ser at the carboxy terminus. By examining left-handed alpha-turns containing L-amino acids, new interaction motifs for incorporating D-amino acids into right-handed alpha-helices are identified. These will provide a basis for de novo design of novel heterochiral protein folds.

Mirrors in the PDB: left-handed α-turns guide design with D-amino acids

PubMed Central

Annavarapu, Srinivas; Nanda, Vikas

2009-01-01

Background Incorporating variable amino acid stereochemistry in molecular design has the potential to improve existing protein stability and create new topologies inaccessible to homochiral molecules. The Protein Data Bank has been a reliable, rich source of information on molecular interactions and their role in protein stability and structure. D-amino acids rarely occur naturally, making it difficult to infer general rules for how they would be tolerated in proteins through an analysis of existing protein structures. However, protein elements containing short left-handed turns and helices turn out to contain useful information. Molecular mechanisms used in proteins to stabilize left-handed elements by L-amino acids are structurally enantiomeric to potential synthetic strategies for stabilizing right-handed elements with D-amino acids. Results Propensities for amino acids to occur in contiguous αL helices correlate with published thermodynamic scales for incorporation of D-amino acids into αR helices. Two backbone rules for terminating a left-handed helix are found: an αR conformation is disfavored at the amino terminus, and a βR conformation is disfavored at the carboxy terminus. Helix capping sidechain-backbone interactions are found which are unique to αL helices including an elevated propensity for L-Asn, and L-Thr at the amino terminus and L-Gln, L-Thr and L-Ser at the carboxy terminus. Conclusion By examining left-handed α-turns containing L-amino acids, new interaction motifs for incorporating D-amino acids into right-handed α-helices are identified. These will provide a basis for de novo design of novel heterochiral protein folds. PMID:19772623

Role of conserved nucleotides in building the 16S rRNA binding site of E. coli ribosomal protein S8.

PubMed Central

Allmang, C; Mougel, M; Westhof, E; Ehresmann, B; Ehresmann, C

1994-01-01

Ribosomal protein S8 specifically recognizes a helical and irregular region of 16S rRNA that is highly evolutionary constrained. Despite its restricted size, the precise conformation of this region remains a question of debate. Here, we used chemical probing to analyze the structural consequences of mutations in this RNA region. These data, combined with computer modelling and previously published data on protein binding were used to investigate the conformation of the RNA binding site. The experimental data confirm the model in which adenines A595, A640 and A642 bulge out in the deep groove. In addition to the already proposed non canonical U598-U641 interaction, the structure is stabilized by stacking interactions (between A595 and A640) and an array of hydrogen bonds involving bases and the sugar phosphate backbone. Mutations that alter the ability to form these interdependent interactions result in a local destabilization or reorganization. The specificity of recognition by protein S8 is provided by the irregular and distorted backbone and the two bulged adenines 640 and 642 in the deep groove. The third adenine (A595) is not a direct recognition site but must adopt a bulged position. The U598-U641 pair should not be directly in contact with the protein. Images PMID:7937081

The Ups and Downs of Repeated Cleavage and Internal Fragment Production in Top-Down Proteomics.

PubMed

Lyon, Yana A; Riggs, Dylan; Fornelli, Luca; Compton, Philip D; Julian, Ryan R

2018-01-01

Analysis of whole proteins by mass spectrometry, or top-down proteomics, has several advantages over methods relying on proteolysis. For example, proteoforms can be unambiguously identified and examined. However, from a gas-phase ion-chemistry perspective, proteins are enormous molecules that present novel challenges relative to peptide analysis. Herein, the statistics of cleaving the peptide backbone multiple times are examined to evaluate the inherent propensity for generating internal versus terminal ions. The raw statistics reveal an inherent bias favoring production of terminal ions, which holds true regardless of protein size. Importantly, even if the full suite of internal ions is generated by statistical dissociation, terminal ions are predicted to account for at least 50% of the total ion current, regardless of protein size, if there are three backbone dissociations or fewer. Top-down analysis should therefore be a viable approach for examining proteins of significant size. Comparison of the purely statistical analysis with actual top-down data derived from ultraviolet photodissociation (UVPD) and higher-energy collisional dissociation (HCD) reveals that terminal ions account for much of the total ion current in both experiments. Terminal ion production is more favored in UVPD relative to HCD, which is likely due to differences in the mechanisms controlling fragmentation. Importantly, internal ions are not found to dominate from either the theoretical or experimental point of view. Graphical abstract ᅟ.

The Ups and Downs of Repeated Cleavage and Internal Fragment Production in Top-Down Proteomics

NASA Astrophysics Data System (ADS)

Lyon, Yana A.; Riggs, Dylan; Fornelli, Luca; Compton, Philip D.; Julian, Ryan R.

2018-01-01

Analysis of whole proteins by mass spectrometry, or top-down proteomics, has several advantages over methods relying on proteolysis. For example, proteoforms can be unambiguously identified and examined. However, from a gas-phase ion-chemistry perspective, proteins are enormous molecules that present novel challenges relative to peptide analysis. Herein, the statistics of cleaving the peptide backbone multiple times are examined to evaluate the inherent propensity for generating internal versus terminal ions. The raw statistics reveal an inherent bias favoring production of terminal ions, which holds true regardless of protein size. Importantly, even if the full suite of internal ions is generated by statistical dissociation, terminal ions are predicted to account for at least 50% of the total ion current, regardless of protein size, if there are three backbone dissociations or fewer. Top-down analysis should therefore be a viable approach for examining proteins of significant size. Comparison of the purely statistical analysis with actual top-down data derived from ultraviolet photodissociation (UVPD) and higher-energy collisional dissociation (HCD) reveals that terminal ions account for much of the total ion current in both experiments. Terminal ion production is more favored in UVPD relative to HCD, which is likely due to differences in the mechanisms controlling fragmentation. Importantly, internal ions are not found to dominate from either the theoretical or experimental point of view. [Figure not available: see fulltext.

Statistical theory for protein combinatorial libraries. Packing interactions, backbone flexibility, and the sequence variability of a main-chain structure.

PubMed

Kono, H; Saven, J G

2001-02-23

Combinatorial experiments provide new ways to probe the determinants of protein folding and to identify novel folding amino acid sequences. These types of experiments, however, are complicated both by enormous conformational complexity and by large numbers of possible sequences. Therefore, a quantitative computational theory would be helpful in designing and interpreting these types of experiment. Here, we present and apply a statistically based, computational approach for identifying the properties of sequences compatible with a given main-chain structure. Protein side-chain conformations are included in an atom-based fashion. Calculations are performed for a variety of similar backbone structures to identify sequence properties that are robust with respect to minor changes in main-chain structure. Rather than specific sequences, the method yields the likelihood of each of the amino acids at preselected positions in a given protein structure. The theory may be used to quantify the characteristics of sequence space for a chosen structure without explicitly tabulating sequences. To account for hydrophobic effects, we introduce an environmental energy that it is consistent with other simple hydrophobicity scales and show that it is effective for side-chain modeling. We apply the method to calculate the identity probabilities of selected positions of the immunoglobulin light chain-binding domain of protein L, for which many variant folding sequences are available. The calculations compare favorably with the experimentally observed identity probabilities.

Reduced dimensionality (3,2)D NMR experiments and their automated analysis: implications to high-throughput structural studies on proteins.

PubMed

Reddy, Jithender G; Kumar, Dinesh; Hosur, Ramakrishna V

2015-02-01

Protein NMR spectroscopy has expanded dramatically over the last decade into a powerful tool for the study of their structure, dynamics, and interactions. The primary requirement for all such investigations is sequence-specific resonance assignment. The demand now is to obtain this information as rapidly as possible and in all types of protein systems, stable/unstable, soluble/insoluble, small/big, structured/unstructured, and so on. In this context, we introduce here two reduced dimensionality experiments – (3,2)D-hNCOcanH and (3,2)D-hNcoCAnH – which enhance the previously described 2D NMR-based assignment methods quite significantly. Both the experiments can be recorded in just about 2-3 h each and hence would be of immense value for high-throughput structural proteomics and drug discovery research. The applicability of the method has been demonstrated using alpha-helical bovine apo calbindin-D9k P43M mutant (75 aa) protein. Automated assignment of this data using AUTOBA has been presented, which enhances the utility of these experiments. The backbone resonance assignments so derived are utilized to estimate secondary structures and the backbone fold using Web-based algorithms. Taken together, we believe that the method and the protocol proposed here can be used for routine high-throughput structural studies of proteins. Copyright © 2014 John Wiley & Sons, Ltd.

Knoto-ID: a tool to study the entanglement of open protein chains using the concept of knotoids.

PubMed

Dorier, Julien; Goundaroulis, Dimos; Benedetti, Fabrizio; Stasiak, Andrzej

2018-05-02

The backbone of most proteins forms an open curve. To study their entanglement, a common strategy consists in searching for the presence of knots in their backbones using topological invariants. However, this approach requires to close the curve into a loop, which alters the geometry of curve. Knoto-ID allows evaluating the entanglement of open curves without the need to close them, using the recent concept of knotoids which is a generalization of the classical knot theory to open curves. Knoto-ID can analyse the global topology of the full chain as well as the local topology by exhaustively studying all subchains or only determining the knotted core. Knoto-ID permits to localize topologically non-trivial protein folds that are not detected by informatics tools detecting knotted protein folds. Knoto-ID is written in C ++ and includes R (www.R-project.org) scripts to generate plots of projections maps, fingerprint matrices and disk matrices. Knoto-ID is distributed under the GNU General Public License (GPL), version 2 or any later version and is available at https://github.com/sib-swiss/Knoto-ID. A binary distribution for Mac OS X, Linux and Windows with detailed user guide and examples can be obtained from https://www.vital-it.ch/software/Knoto-ID. julien.dorier@sib.swiss.

Improved modeling of side-chain--base interactions and plasticity in protein--DNA interface design.

PubMed

Thyme, Summer B; Baker, David; Bradley, Philip

2012-06-08

Combinatorial sequence optimization for protein design requires libraries of discrete side-chain conformations. The discreteness of these libraries is problematic, particularly for long, polar side chains, since favorable interactions can be missed. Previously, an approach to loop remodeling where protein backbone movement is directed by side-chain rotamers predicted to form interactions previously observed in native complexes (termed "motifs") was described. Here, we show how such motif libraries can be incorporated into combinatorial sequence optimization protocols and improve native complex recapitulation. Guided by the motif rotamer searches, we made improvements to the underlying energy function, increasing recapitulation of native interactions. To further test the methods, we carried out a comprehensive experimental scan of amino acid preferences in the I-AniI protein-DNA interface and found that many positions tolerated multiple amino acids. This sequence plasticity is not observed in the computational results because of the fixed-backbone approximation of the model. We improved modeling of this diversity by introducing DNA flexibility and reducing the convergence of the simulated annealing algorithm that drives the design process. In addition to serving as a benchmark, this extensive experimental data set provides insight into the types of interactions essential to maintain the function of this potential gene therapy reagent. Published by Elsevier Ltd.

Access of Hydrogen-Radicals to the Peptide-Backbone as a Measure for Estimating the Flexibility of Proteins Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

PubMed Central

Takayama, Mitsuo; Nagoshi, Keishiro; Iimuro, Ryunosuke; Inatomi, Kazuma

2014-01-01

A factor for estimating the flexibility of proteins is described that uses a cleavage method of “in-source decay (ISD)” coupled with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). The MALDI-ISD spectra of bovine serum albumin (BSA), myoglobin and thioredoxin show discontinuous intense ion peaks originating from one-side preferential cleavage at the N-Cα bond of Xxx-Asp, Xxx-Asn, Xxx-Cys and Gly-Xxx residues. Consistent with these observations, Asp, Asn and Gly residues are also identified by other flexibility measures such as B-factor, turn preference, protection and fluorescence decay factors, while Asp, Asn, Cys and Gly residues are identified by turn preference factor based on X-ray crystallography. The results suggest that protein molecules embedded in/on MALDI matrix crystals partly maintain α-helix and that the reason some of the residues are more susceptible to ISD (Asp, Asn, Cys and Gly) and others less so (Ile and Val) is because of accessibility of the peptide backbone to hydrogen-radicals from matrix molecules. The hydrogen-radical accessibility in MALDI-ISD could therefore be adopted as a factor for measuring protein flexibility. PMID:24828203

Classification of pseudo pairs between nucleotide bases and amino acids by analysis of nucleotide-protein complexes.

PubMed

Kondo, Jiro; Westhof, Eric

2011-10-01

Nucleotide bases are recognized by amino acid residues in a variety of DNA/RNA binding and nucleotide binding proteins. In this study, a total of 446 crystal structures of nucleotide-protein complexes are analyzed manually and pseudo pairs together with single and bifurcated hydrogen bonds observed between bases and amino acids are classified and annotated. Only 5 of the 20 usual amino acid residues, Asn, Gln, Asp, Glu and Arg, are able to orient in a coplanar fashion in order to form pseudo pairs with nucleotide bases through two hydrogen bonds. The peptide backbone can also form pseudo pairs with nucleotide bases and presents a strong bias for binding to the adenine base. The Watson-Crick side of the nucleotide bases is the major interaction edge participating in such pseudo pairs. Pseudo pairs between the Watson-Crick edge of guanine and Asp are frequently observed. The Hoogsteen edge of the purine bases is a good discriminatory element in recognition of nucleotide bases by protein side chains through the pseudo pairing: the Hoogsteen edge of adenine is recognized by various amino acids while the Hoogsteen edge of guanine is only recognized by Arg. The sugar edge is rarely recognized by either the side-chain or peptide backbone of amino acid residues.

Classification of pseudo pairs between nucleotide bases and amino acids by analysis of nucleotide–protein complexes

PubMed Central

Kondo, Jiro; Westhof, Eric

2011-01-01

Nucleotide bases are recognized by amino acid residues in a variety of DNA/RNA binding and nucleotide binding proteins. In this study, a total of 446 crystal structures of nucleotide–protein complexes are analyzed manually and pseudo pairs together with single and bifurcated hydrogen bonds observed between bases and amino acids are classified and annotated. Only 5 of the 20 usual amino acid residues, Asn, Gln, Asp, Glu and Arg, are able to orient in a coplanar fashion in order to form pseudo pairs with nucleotide bases through two hydrogen bonds. The peptide backbone can also form pseudo pairs with nucleotide bases and presents a strong bias for binding to the adenine base. The Watson–Crick side of the nucleotide bases is the major interaction edge participating in such pseudo pairs. Pseudo pairs between the Watson–Crick edge of guanine and Asp are frequently observed. The Hoogsteen edge of the purine bases is a good discriminatory element in recognition of nucleotide bases by protein side chains through the pseudo pairing: the Hoogsteen edge of adenine is recognized by various amino acids while the Hoogsteen edge of guanine is only recognized by Arg. The sugar edge is rarely recognized by either the side-chain or peptide backbone of amino acid residues. PMID:21737431

1H, 13C, and 15N backbone assignment and secondary structure of the receptor-binding domain of vascular endothelial growth factor.

PubMed Central

Fairbrother, W. J.; Champe, M. A.; Christinger, H. W.; Keyt, B. A.; Starovasnik, M. A.

1997-01-01

Nearly complete sequence-specific 1H, 13C, and 15N resonance assignments are reported for the backbone atoms of the receptor-binding domain of vascular endothelial growth factor (VEGF), a 23-kDa homodimeric protein that is a major regulator of both normal and pathological angiogenesis. The assignment strategy relied on the use of seven 3D triple-resonance experiments [HN(CO)CA, HNCA, HNCO, (HCA)CONH, HN(COCA)HA, HN(CA)HA, and CBCA-(CO)NH] and a 3D 15N-TOCSY-HSQC experiment recorded on a 0.5 mM (12 mg/mL) sample at 500 MHz, pH 7.0, 45 degrees C. Under these conditions, 15N relaxation data show that the protein has a rotational correlation time of 15.0 ns. Despite this unusually long correlation time, assignments were obtained for 94 of the 99 residues; 8 residues lack amide 1H and 15N assignments, presumably due to rapid exchange of the amide 1H with solvent under the experimental conditions used. The secondary structure of the protein was deduced from the chemical shift indices of the 1H alpha, 13C alpha, 13C beta, and 13CO nuclei, and from analysis of backbone NOEs observed in a 3D 15N-NOESY-HSQC spectrum. Two helices and a significant amount of beta-sheet structure were identified, in general agreement with the secondary structure found in a recently determined crystal structure of a similar VEGF construct [Muller YA et al., 1997, Proc Natl Acad Sci USA 94:7192-7197]. PMID:9336848

Structure and dynamics of a detergent-solubilized membrane protein: measurement of amide hydrogen exchange rates in M13 coat protein by /sub 1/H NMR spectroscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

O'Neil, J.D.J.; Sykes, B.D.

The coat protein of bacteriophage M13 is inserted into the inner membrane of Escherichia coli where it exists as an integral membrane protein during the reproductive cycle of the phage. The protein sequence consists of a highly hydrophobic 19-residue central segment flanked by an acidic 20-residue N-terminus and a basic 11-residue C-terminus. The authors have measured backbone amide hydrogen exchange of the protein solubilized in perdeuteriated sodium dodecyl sulfate using /sup 1/H nuclear magnetic resonance (NMR) spectroscopy. Direct proton exchange-out measurements in D/sub 2/O at 24 /sup 0/C were used to follow the exchange of the slowest amides in themore » protein. Multiple exponential fitting of the exchange data showed that these amides exchanged in two kinetic sets with exchange rates that differed by more than 100-fold. Steady-state saturation-transfer techniques were also used to measure exchange. These methods showed that 15-20 amides in the protein are very stable at 55/sup 0/C and that bout 30 amides have exchange rates retarded by at least 10/sup 5/-fold at 24/sup 0/C. Saturation-transfer studies also showed that the pH dependence of exchange in the hydrophilic termini was unusual. Relaxation and solid-state NMR experiments have previously shown that the majority of the protein backbone is rigid on the picosecond to microsecond time scale, except for the extreme ends of the molecule which are mobile. The hydrogen exchange results, which are sensitive to a much longer time scale, suggest a stable core with a progressive increase in amplitude or frequency of motions as the ends of the protein are approached.« less

Sulfation and Cation Effects on the Conformational Properties of the Glycan Backbone of Chondroitin Sulfate Disaccharides

PubMed Central

Faller, Christina E.; Guvench, Olgun

2015-01-01

Chondroitin sulfate (CS) is one of several glycosaminoglycans that are major components of proteoglycans. A linear polymer consisting of repeats of the disaccharide -4GlcAβ1-3GalNAcβ1-, CS undergoes differential sulfation resulting in five unique sulfation patterns. Because of the dimer repeat, the CS glycosidic “backbone” has two distinct sets of conformational degrees of freedom defined by pairs of dihedral angles: (ϕ1, ψ1) about the β1-3 glycosidic linkage and (ϕ2, ψ2) about the β1-4 glycosidic linkage. Differential sulfation and the possibility of cation binding, combined with the conformational flexibility and biological diversity of CS, complicate experimental efforts to understand CS three-dimensional structures at atomic resolution. Therefore, all-atom explicit-solvent molecular dynamics simulations with Adaptive Biasing Force sampling of the CS backbone were applied to obtain high resolution, high precision free energies of CS disaccharides as a function of all possible backbone geometries. All ten disaccharides (β1-3 vs. β1-4 linkage x five different sulfation patterns) were studied; additionally, ion effects were investigated by considering each disaccharide in the presence of either neutralizing sodium or calcium cations. GlcAβ1-3GalNAc disaccharides have a single, broad, thermodynamically important free-energy minimum whereas GalNAcβ1-4GlcA disaccharides have two such minima. Calcium cations but not sodium cations bind to the disaccharides, and binding is primarily to the GlcA –COO− moiety as opposed to sulfate groups. This binding alters the glycan backbone thermodynamics in instances where a calcium cation bound to –COO− can act to bridge and stabilize an interaction with an adjacent sulfate group, whereas, in the absence of this cation, the proximity of a sulfate group to –COO− results in two like charges being both desolvated and placed adjacent to each other and is found to be destabilizing. In addition to providing information on sulfation and cation effects, the present results can be applied to building models of CS polymers and as a point of comparison in studies of CS polymer backbone dynamics and thermodynamics. PMID:25906376

Proteomics analysis of Fusarium proliferatum under various initial pH during fumonisin production.

PubMed

Li, Taotao; Gong, Liang; Wang, Yong; Chen, Feng; Gupta, Vijai Kumar; Jian, Qijie; Duan, Xuewu; Jiang, Yueming

2017-07-05

Fusarium proliferatum as a fungal pathogen can produce fumonisin which causes a great threat to animal and human health. Proteomic approach was a useful tool for investigation into mycotoxin biosynthesis in fungal pathogens. In this study, we analyzed the fumonisin content and mycelium proteins of Fusarium proliferatum cultivated under the initial pH5 and 10. Fumonisin production after 10days was significantly induced in culture condition at pH10 than pH5. Ninety nine significantly differently accumulated protein spots under the two pH conditions were detected using two dimensional polyacrylamide gel electrophoresis and 89 of these proteins were successfully identified by MALDI-TOF/TOF and LC-ESI-MS/MS analysis. Among these 89 proteins, 45 were up-regulated at pH10 while 44 were up-accumulated at pH5. At pH10, these proteins were found to involve in the modification of fumonisin backbone including up-regulated polyketide synthase, cytochrome P450, S-adenosylmethionine synthase and O-methyltransferase, which might contribute to the induction of fumonisin production. At pH5, these up-regulated proteins such as l-amino-acid oxidase, isocitrate dehydrogenase and citrate lyase might inhibit the condensation of fumonisin backbone, resulting in reduced production of fumonisins. These results may help us to understand the molecular mechanism of the fumonisin synthesis in F. proliferatum. To extend our understanding of the mechanism of the fumonisin biosynthesis of F. proliferatum, we reported the fumonisin production in relation to the differential proteins of F. proliferatum mycelium under two pH culture conditions. Among these 89 identified spots, 45 were up-accumulated at pH10 while 44 were up-accumulated at pH5. Our results revealed that increased fumonisin production at pH10 might be related to the induction of fumonisin biosynthesis caused by up-regulation of polyketide synthase, cytochrome P450, S-adenosylmethionine synthase and O-methyltransferase. Meanwhile, the up-regulation of l-amino-acid oxidase, isocitrate dehydrogenase and citrate lyase at pH5 might be related to the inhibition of the condensation of fumonisin backbone, resulting in reduced production of fumonisin. These results may help us to understand better the molecular mechanism of the fumonisin synthesis in F. proliferatum and then broaden the current knowledge of the mechanism of the fumonisin biosynthesis. Copyright © 2017 Elsevier B.V. All rights reserved.

Measurement of energy landscape roughness of folded and unfolded proteins

PubMed Central

Milanesi, Lilia; Waltho, Jonathan P.; Hunter, Christopher A.; Shaw, Daniel J.; Beddard, Godfrey S.; Reid, Gavin D.; Dev, Sagarika; Volk, Martin

2012-01-01

The dynamics of protein conformational changes, from protein folding to smaller changes, such as those involved in ligand binding, are governed by the properties of the conformational energy landscape. Different techniques have been used to follow the motion of a protein over this landscape and thus quantify its properties. However, these techniques often are limited to short timescales and low-energy conformations. Here, we describe a general approach that overcomes these limitations. Starting from a nonnative conformation held by an aromatic disulfide bond, we use time-resolved spectroscopy to observe nonequilibrium backbone dynamics over nine orders of magnitude in time, from picoseconds to milliseconds, after photolysis of the disulfide bond. We find that the reencounter probability of residues that initially are in close contact decreases with time following an unusual power law that persists over the full time range and is independent of the primary sequence. Model simulations show that this power law arises from subdiffusional motion, indicating a wide distribution of trapping times in local minima of the energy landscape, and enable us to quantify the roughness of the energy landscape (4–5 kBT). Surprisingly, even under denaturing conditions, the energy landscape remains highly rugged with deep traps (>20 kBT) that result from multiple nonnative interactions and are sufficient for trapping on the millisecond timescale. Finally, we suggest that the subdiffusional motion of the protein backbone found here may promote rapid folding of proteins with low contact order by enhancing contact formation between nearby residues. PMID:23150572

Conformational Transitions upon Ligand Binding: Holo-Structure Prediction from Apo Conformations

PubMed Central

Seeliger, Daniel; de Groot, Bert L.

2010-01-01

Biological function of proteins is frequently associated with the formation of complexes with small-molecule ligands. Experimental structure determination of such complexes at atomic resolution, however, can be time-consuming and costly. Computational methods for structure prediction of protein/ligand complexes, particularly docking, are as yet restricted by their limited consideration of receptor flexibility, rendering them not applicable for predicting protein/ligand complexes if large conformational changes of the receptor upon ligand binding are involved. Accurate receptor models in the ligand-bound state (holo structures), however, are a prerequisite for successful structure-based drug design. Hence, if only an unbound (apo) structure is available distinct from the ligand-bound conformation, structure-based drug design is severely limited. We present a method to predict the structure of protein/ligand complexes based solely on the apo structure, the ligand and the radius of gyration of the holo structure. The method is applied to ten cases in which proteins undergo structural rearrangements of up to 7.1 Å backbone RMSD upon ligand binding. In all cases, receptor models within 1.6 Å backbone RMSD to the target were predicted and close-to-native ligand binding poses were obtained for 8 of 10 cases in the top-ranked complex models. A protocol is presented that is expected to enable structure modeling of protein/ligand complexes and structure-based drug design for cases where crystal structures of ligand-bound conformations are not available. PMID:20066034

Experimental determination of torsion angles in the polypeptide backbone of the gramicidin A channel by solid state nuclear magnetic resonance.

PubMed

Teng, Q; Nicholson, L K; Cross, T A

1991-04-05

An analytical method for the determination of torsion angles from solid state 15N nuclear magnetic resonance (n.m.r.) spectroscopic data is demonstrated. Advantage is taken of the 15N-1H and 15N-13C dipolar interactions as well as the 15N chemical shift interaction in oriented samples. The membrane-bound channel conformation of gramicidin A has eluded an atomic resolution structure determination by more traditional approaches. Here, the torsion angles for the Ala3 site are determined by obtaining the n.m.r. data for both the Gly2-Ala3 and Ala3-Leu4 peptide linkages. Complete utilization of the orientational constraints derived from these orientation-dependent nuclear spin interactions in restricting the conformational space is most effectively achieved by utilizing spherical trigonometry. Two possible sets of torsion angles for the Ala3 site are obtained (phi, psi = -129 degrees, 153 degrees and -129 degrees, 122 degrees), both of which are consistent with a right-handed beta-helix. Other functional and computational evidence strongly supports the set for which the carbonyl oxygen atom of the Ala3-Leu4 linkage is rotated into the channel lumen.

Secondary Structure Switch

ERIC Educational Resources Information Center

King, Angela G.

2006-01-01

Neurogenerative diseases like Alzheimer's disease and Parkinson's disease involve a transformation between two peptide and protein structures of alpha-helices and beta-sheets, where the peptide backbone can also participate in metal ion binding in addition to histidine residues. However, the complete absence of change in conformation of Coiled…

Backbone hydration determines the folding signature of amino acid residues.

PubMed

Bignucolo, Olivier; Leung, Hoi Tik Alvin; Grzesiek, Stephan; Bernèche, Simon

2015-04-08

The relation between the sequence of a protein and its three-dimensional structure remains largely unknown. A lasting dream is to elucidate the side-chain-dependent driving forces that govern the folding process. Different structural data suggest that aromatic amino acids play a particular role in the stabilization of protein structures. To better understand the underlying mechanism, we studied peptides of the sequence EGAAXAASS (X = Gly, Ile, Tyr, Trp) through comparison of molecular dynamics (MD) trajectories and NMR residual dipolar coupling (RDC) measurements. The RDC data for aromatic substitutions provide evidence for a kink in the peptide backbone. Analysis of the MD simulations shows that the formation of internal hydrogen bonds underlying a helical turn is key to reproduce the experimental RDC values. The simulations further reveal that the driving force leading to such helical-turn conformations arises from the lack of hydration of the peptide chain on either side of the bulky aromatic side chain, which can potentially act as a nucleation point initiating the folding process.

The dominant interaction between peptide and urea is electrostatic in nature: a molecular dynamics simulation study.

PubMed

Tobi, Dror; Elber, Ron; Thirumalai, Devarajan

2003-03-01

The conformational equilibrium of a blocked valine peptide in water and aqueous urea solution is studied using molecular dynamics simulations. Pair correlation functions indicate enhanced concentration of urea near the peptide. Stronger hydrogen bonding of urea-peptide compared to water-peptide is observed with preference for helical conformation. The potential of mean force, computed using umbrella sampling, shows only small differences between urea and water solvation that are difficult to quantify. The changes in solvent structure around the peptide are explained by favorable electrostatic interactions (hydrogen bonds) of urea with the peptide backbone. There is no evidence for significant changes in hydrophobic interactions in the two conformations of the peptide in urea solution. Our simulations suggest that urea denatures proteins by preferentially forming hydrogen bonds to the peptide backbone, reducing the barrier for exposing protein residues to the solvent, and reaching the unfolded state. Copyright 2003 Wiley Periodicals, Inc. Biopolymers: 359-369, 2003

Sequence-specific sup 1 H NMR resonance assignments of Bacillus subtilis HPr: Use of spectra obtained from mutants to resolve spectral overlap

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wittekind, M.; Klevit, R.E.; Reizer, J.

1990-08-07

On the basis of an analysis of two-dimensional {sup 1}H NMR spectra, the complete sequence-specific {sup 1}H NMR assignments are presented for the phosphocarrier protein HPr from the Gram-positive bacterium Bacillus subtilis. During the assignment procedure, extensive use was made of spectra obtained from point mutants of HPr in order to resolve spectral overlap and to provide verification of assignments. Regions of regular secondary structure were identified by characteristic patterns of sequential backbone proton NOEs and slowly exchanging amide protons. B subtilis HPr contains four {beta}-strands that form a single antiparallel {beta}-sheet and two well-defined {alpha}-helices. There are two stretchesmore » of extended backbone structure, one of which contains the active site His{sub 15}. The overall fold of the protein is very similar to that of Escherichia coli HPr determined by NMR studies.« less

Representing environment-induced helix-coil transitions in a coarse grained peptide model

NASA Astrophysics Data System (ADS)

Dalgicdir, Cahit; Globisch, Christoph; Sayar, Mehmet; Peter, Christine

2016-10-01

Coarse grained (CG) models are widely used in studying peptide self-assembly and nanostructure formation. One of the recurrent challenges in CG modeling is the problem of limited transferability, for example to different thermodynamic state points and system compositions. Understanding transferability is generally a prerequisite to knowing for which problems a model can be reliably used and predictive. For peptides, one crucial transferability question is whether a model reproduces the molecule's conformational response to a change in its molecular environment. This is of particular importance since CG peptide models often have to resort to auxiliary interactions that aid secondary structure formation. Such interactions take care of properties of the real system that are per se lost in the coarse graining process such as dihedral-angle correlations along the backbone or backbone hydrogen bonding. These auxiliary interactions may then easily overstabilize certain conformational propensities and therefore destroy the ability of the model to respond to stimuli and environment changes, i.e. they impede transferability. In the present paper we have investigated a short peptide with amphiphilic EALA repeats which undergoes conformational transitions between a disordered and a helical state upon a change in pH value or due to the presence of a soft apolar/polar interface. We designed a base CG peptide model that does not carry a specific (backbone) bias towards a secondary structure. This base model was combined with two typical approaches of ensuring secondary structure formation, namely a C α -C α -C α -C α pseudodihedral angle potential or a virtual site interaction that mimics hydrogen bonding. We have investigated the ability of the two resulting CG models to represent the environment-induced conformational changes in the helix-coil equilibrium of EALA. We show that with both approaches a CG peptide model can be obtained that is environment-transferable and that correctly represents the peptide's conformational response to different stimuli compared to atomistic reference simulations. The two types of auxiliary interactions lead to different kinetic behavior as well as to different structural properties for fully formed helices and folding intermediates, and we discuss the advantages and disadvantages of these approaches.

de Vries liquid crystals based on a chiral 5-phenylpyrimidine benzoate core with a tri- and tetra-carbosilane backbone

NASA Astrophysics Data System (ADS)

Sreenilayam, S. P.; Rodriguez-Lojo, D.; Agra-Kooijman, D. M.; Vij, J. K.; Panov, V. P.; Panov, A.; Fisch, M. R.; Kumar, Satyendra; Stevenson, P. J.

2018-02-01

New chiral de Vries smectic liquid-crystalline compounds are designed, synthesized, and investigated for perspective applications in defect-free bistable surface-stabilized ferroelectric liquid-crystal displays. In these compounds, a 5-phenyl-pyrimidine benzoate core is terminated on one side by a tri- or tetra-carbosilane group linked through an alkoxy group and an alkyl spacer and on the opposite side terminated by a chiral 2-octanol group. The stereogenic center contains either a methyl or perfluoromethyl functional group. These compounds exhibit Iso-Sm A*-Sm C*-Sm X -Cr phases under cooling from the isotropic state. Measurements of the temperature-dependent smectic layer spacing by x-ray diffraction experiments combined with the measured apparent optical tilt angle and the birefringence reveal that Sm A* phase in these compounds is of the de Vries type. In addition, the chiral compound with a tetra-carbosilane backbone, DR277, exhibits good de Vries properties with the Sm C* phase exhibited over a wide temperature range. By varying the carbosilane end group, the de Vries properties are enhanced, that is, the layer shrinkage of ˜1.9 % for the tri-carbosilane DR276 is reduced to ˜0.9 % for tetra-carbosilane DR277 at 10°C below Sm A* to Sm C* transition temperature, TAC. For DR277, the reduction factor R ≈0.22 for T =(TAC-10 )°C is reasonably low and the apparent optical tilt angle θapp=35.1°, hence this compound is a "good de Vries smectic" LC. Therefore, synthesis of the chiral mesogen with an even higher number of carbosilane groups may lead to a further reduction or even zero-layer shrinkage exhibited at TAC with Sm C* phase extending over a wide temperature range close to the room temperature for perspective suitability in device applications. Our results for 5-phenyl-pyrimidine benzoate core-based compounds support a recently drawn conclusion by Schubert et al. [J. Mater. Chem. C 4, 8483 (2016), 10.1039/C6TC03120J] from a different compound, namely that a carbosilane backbone in chiral mesogens strongly influences the de Vries properties.

Conformational interconversions in peptide beta-turns: analysis of turns in proteins and computational estimates of barriers.

PubMed

Gunasekaran, K; Gomathi, L; Ramakrishnan, C; Chandrasekhar, J; Balaram, P

1998-12-18

The two most important beta-turn features in peptides and proteins are the type I and type II turns, which differ mainly in the orientation of the central peptide unit. Facile conformational interconversion is possible, in principle, by a flip of the central peptide unit. Homologous crystal structures afford an opportunity to structurally characterize both possible conformational states, thus allowing identification of sites that are potentially stereochemically mobile. A representative data set of 250 high-resolution (

Principal component analysis on a torus: Theory and application to protein dynamics.

PubMed

Sittel, Florian; Filk, Thomas; Stock, Gerhard

2017-12-28

A dimensionality reduction method for high-dimensional circular data is developed, which is based on a principal component analysis (PCA) of data points on a torus. Adopting a geometrical view of PCA, various distance measures on a torus are introduced and the associated problem of projecting data onto the principal subspaces is discussed. The main idea is that the (periodicity-induced) projection error can be minimized by transforming the data such that the maximal gap of the sampling is shifted to the periodic boundary. In a second step, the covariance matrix and its eigendecomposition can be computed in a standard manner. Adopting molecular dynamics simulations of two well-established biomolecular systems (Aib 9 and villin headpiece), the potential of the method to analyze the dynamics of backbone dihedral angles is demonstrated. The new approach allows for a robust and well-defined construction of metastable states and provides low-dimensional reaction coordinates that accurately describe the free energy landscape. Moreover, it offers a direct interpretation of covariances and principal components in terms of the angular variables. Apart from its application to PCA, the method of maximal gap shifting is general and can be applied to any other dimensionality reduction method for circular data.

Solid-state NMR Study Reveals Collagen I Structural Modifications of Amino Acid Side Chains upon Fibrillogenesis*

PubMed Central

De Sa Peixoto, Paulo; Laurent, Guillaume; Azaïs, Thierry; Mosser, Gervaise

2013-01-01

In vivo, collagen I, the major structural protein in human body, is found assembled into fibrils. In the present work, we study a high concentrated collagen sample in its soluble, fibrillar, and denatured states using one and two dimensional {1H}-13C solid-state NMR spectroscopy. We interpret 13C chemical shift variations in terms of dihedral angle conformation changes. Our data show that fibrillogenesis increases the side chain and backbone structural complexity. Nevertheless, only three to five rotameric equilibria are found for each amino acid residue, indicating a relatively low structural heterogeneity of collagen upon fibrillogenesis. Using side chain statistical data, we calculate equilibrium constants for a great number of amino acid residues. Moreover, based on a 13C quantitative spectrum, we estimate the percentage of residues implicated in each equilibrium. Our data indicate that fibril formation greatly affects hydroxyproline and proline prolyl pucker ring conformation. Finally, we discuss the implication of these structural data and propose a model in which the attractive force of fibrillogenesis comes from a structural reorganization of 10 to 15% of the amino acids. These results allow us to further understand the self-assembling process and fibrillar structure of collagen. PMID:23341452

Principal component analysis on a torus: Theory and application to protein dynamics

NASA Astrophysics Data System (ADS)

Sittel, Florian; Filk, Thomas; Stock, Gerhard

2017-12-01

A dimensionality reduction method for high-dimensional circular data is developed, which is based on a principal component analysis (PCA) of data points on a torus. Adopting a geometrical view of PCA, various distance measures on a torus are introduced and the associated problem of projecting data onto the principal subspaces is discussed. The main idea is that the (periodicity-induced) projection error can be minimized by transforming the data such that the maximal gap of the sampling is shifted to the periodic boundary. In a second step, the covariance matrix and its eigendecomposition can be computed in a standard manner. Adopting molecular dynamics simulations of two well-established biomolecular systems (Aib9 and villin headpiece), the potential of the method to analyze the dynamics of backbone dihedral angles is demonstrated. The new approach allows for a robust and well-defined construction of metastable states and provides low-dimensional reaction coordinates that accurately describe the free energy landscape. Moreover, it offers a direct interpretation of covariances and principal components in terms of the angular variables. Apart from its application to PCA, the method of maximal gap shifting is general and can be applied to any other dimensionality reduction method for circular data.

Molecular dynamics simulations of trans- and cis- N-acetyl- N'-methylamides of XaaPro dipeptides

NASA Astrophysics Data System (ADS)

Hoon Choi, Seung; Yun Yu, Jeong; Kwang Shin, Jae; Shik Jhon, Mu

1994-07-01

The occurrence of cis imide bonds in proteins is much higher than that of cis amide bonds due to the unique properties of proline. In order to examine the relationship between the high occurrence of these cis imide bonds and the residues preceding the proline, we perform molecular dynamics simulations of trans- and cis- N-acetyl- N'-methylamides of XaaPro dipeptides (AcXaaProNHMe). We investigate the conformational energies and structures of trans- and cis-AcXaa where Xaa has 12 amino acids in the vacuum state and 5 amino acids in the solution state. It is found that the occurrence of the cis imide bonds is strongly affected by the residue preceding the proline, and the dihedral angles (φ,ψ) of the backbone in AcXaaProNHMe are influenced by the configuration of the imide bond. We also find that the equilibrium properties of XaaPro in solution simulations are more similar to the statistics of X-ray crystallographic data than are those in vacuum simulations and solvation causes a remarkable change in the conformation of the pyrrolidine ring from the endo to the exo form.

Transition paths of Met-enkephalin from Markov state modeling of a molecular dynamics trajectory.

PubMed

Banerjee, Rahul; Cukier, Robert I

2014-03-20

Conformational states and their interconversion pathways of the zwitterionic form of the pentapeptide Met-enkephalin (MetEnk) are identified. An explicit solvent molecular dynamics (MD) trajectory is used to construct a Markov state model (MSM) based on dihedral space clustering of the trajectory, and transition path theory (TPT) is applied to identify pathways between open and closed conformers. In the MD trajectory, only four of the eight backbone dihedrals exhibit bistable behavior. Defining a conformer as the string XXXX with X = "+" or "-" denoting, respectively, positive or negative values of a given dihedral angle and obtaining the populations of these conformers shows that only four conformers are highly populated, implying a strong correlation among these dihedrals. Clustering in dihedral space to construct the MSM finds the same four bistable dihedral angles. These state populations are very similar to those found directly from the MD trajectory. TPT is used to obtain pathways, parametrized by committor values, in dihedral state space that are followed in transitioning from closed to open states. Pathway costs are estimated by introducing a kinetics-based procedure that orders pathways from least (shortest) to greater cost paths. The least costly pathways in dihedral space are found to only involve the same XXXX set of dihedral angles, and the conformers accessed in the closed to open transition pathways are identified. For these major pathways, a correlation between reaction path progress (committors) and the end-to-end distance is identified. A dihedral space principal component analysis of the MD trajectory shows that the first three modes capture most of the overall fluctuation, and pick out the same four dihedrals having essentially all the weight in those modes. A MSM based on root-mean-square backbone clustering was also carried out, with good agreement found with dihedral clustering for the static information, but with results that differ significantly for the pathway analysis.

Relative stability of major types of beta-turns as a function of amino acid composition: a study based on Ab initio energetic and natural abundance data.

PubMed

Perczel, András; Jákli, Imre; McAllister, Michael A; Csizmadia, Imre G

2003-06-06

Folding properties of small globular proteins are determined by their amino acid sequence (primary structure). This holds both for local (secondary structure) and for global conformational features of linear polypeptides and proteins composed from natural amino acid derivatives. It thus provides the rational basis of structure prediction algorithms. The shortest secondary structure element, the beta-turn, most typically adopts either a type I or a type II form, depending on the amino acid composition. Herein we investigate the sequence-dependent folding stability of both major types of beta-turns using simple dipeptide models (-Xxx-Yyy-). Gas-phase ab initio properties of 16 carefully selected and suitably protected dipeptide models (for example Val-Ser, Ala-Gly, Ser-Ser) were studied. For each backbone fold most probable side-chain conformers were considered. Fully optimized 321G RHF molecular structures were employed in medium level [B3LYP/6-311++G(d,p)//RHF/3-21G] energy calculations to estimate relative populations of the different backbone conformers. Our results show that the preference for beta-turn forms as calculated by quantum mechanics and observed in Xray determined proteins correlates significantly.

Disulfide bridge regulates ligand-binding site selectivity in liver bile acid-binding proteins.

PubMed

Cogliati, Clelia; Tomaselli, Simona; Assfalg, Michael; Pedò, Massimo; Ferranti, Pasquale; Zetta, Lucia; Molinari, Henriette; Ragona, Laura

2009-10-01

Bile acid-binding proteins (BABPs) are cytosolic lipid chaperones that play central roles in driving bile flow, as well as in the adaptation to various pathological conditions, contributing to the maintenance of bile acid homeostasis and functional distribution within the cell. Understanding the mode of binding of bile acids with their cytoplasmic transporters is a key issue in providing a model for the mechanism of their transfer from the cytoplasm to the nucleus, for delivery to nuclear receptors. A number of factors have been shown to modulate bile salt selectivity, stoichiometry, and affinity of binding to BABPs, e.g. chemistry of the ligand, protein plasticity and, possibly, the formation of disulfide bridges. Here, the effects of the presence of a naturally occurring disulfide bridge on liver BABP ligand-binding properties and backbone dynamics have been investigated by NMR. Interestingly, the disulfide bridge does not modify the protein-binding stoichiometry, but has a key role in modulating recognition at both sites, inducing site selectivity for glycocholic and glycochenodeoxycholic acid. Protein conformational changes following the introduction of a disulfide bridge are small and located around the inner binding site, whereas significant changes in backbone motions are observed for several residues distributed over the entire protein, both in the apo form and in the holo form. Site selectivity appears, therefore, to be dependent on protein mobility rather than being governed by steric factors. The detected properties further establish a parallelism with the behaviour of human ileal BABP, substantiating the proposal that BABPs have parallel functions in hepatocytes and enterocytes.

Alpha-Helical Protein Domains Unify Strength and Robustness through Hierarchical Nanostructures

DTIC Science & Technology

2009-01-23

backbone atom (hydrogen donor) of peptide i + 4 in the polypeptide chain. Consequently, at each convolution , 3.5 H- bonds are found in a parallel...signaling and deformation behavior of cytoskeletal protein networks in cells (e.g. intermediate filaments vimentin and lamin as well as actin [7, 8... convolution . The Hierarchical Bell model enables one to predict the strength of different hierarchical bond arrangements as a function of the

Conformational study of the hydroxyproline-O-glycosidic linkage: sugar-peptide orientation and prolyl amide isomerization in (α/β)-galactosylated 4(R/S)-hydroxyproline.

PubMed

Naziga, Emmanuel B; Schweizer, Frank; Wetmore, Stacey D

2012-01-19

Glycosylation is a frequent post-translational modification of proteins that has been shown to influence protein structure and function. Glycosylation of hydroxyproline occurs widely in plants, but is absent in humans and animals. Previous experimental studies on model amides have indicated that α/β-galactosylation of 4R-hydroxyproline (Hyp) has no measurable effect on prolyl amide isomerization, while a 7% increase in the trans isomer population, as well as a 25-50% increase in the isomerization rate, was observed for the 4S stereoisomer (hyp). In this work, molecular dynamics simulations in explicit water and implicit solvent DFT optimizations are used to examine the structure of the hydroxyproline-O-galactosyl linkage and the effect of glycosylation on the structure and cis/trans isomerization of the peptide backbone. The calculations show two major minima with respect to the glycosidic linkage in all compounds. The C(γ)-exo puckering observed in 4R compounds projects the sugar away from the peptide backbone, while a twisted C(γ)-endo/C(β)-exo pucker in the 4S compounds brings the peptide and sugar rings together and leads to an intramolecular hydrogen-bonding interaction that is sometimes bridged by a water molecule. This hydrogen bond changes the conformation of the peptide backbone, inducing a favorable n → π* interaction between the oxygen lone pair from the prolyl N-terminal amide and the C═O, which explains the observed increase in trans isomer population in α/β-galactosylated 4S-hydroxyproline. Our results provide the first molecular level information about this important glycosidic linkage, as well as provide an explanation for the previously observed increase in trans isomer population in 4S-hyp compounds. Moreover, this study provides evidence that sugar-mediated long-range hydrogen bonding between hydroxyl groups and the carbonyl peptide backbone can modify the properties of N-terminal prolyl cis/trans isomerization in peptides.

Origin of higher affinity to RNA of the N-terminal RNA-binding domain than that of the C-terminal one of a mouse neural protein, musashi1, as revealed by comparison of their structures, modes of interaction, surface electrostatic potentials, and backbone dynamics.

PubMed

Miyanoiri, Youhei; Kobayashi, Hisanori; Imai, Takao; Watanabe, Michinao; Nagata, Takashi; Uesugi, Seiichi; Okano, Hideyuki; Katahira, Masato

2003-10-17

Musashi1 is an RNA-binding protein abundantly expressed in the developing mouse central nervous system. Its restricted expression in neural precursor cells suggests that it is involved in maintenance of the character of progenitor cells. Musashi1 contains two ribonucleoprotein-type RNA-binding domains (RBDs), RBD1 and RBD2, the affinity to RNA of RBD1 being much higher than that of RBD2. We previously reported the structure and mode of interaction with RNA of RBD2. Here, we have determined the structure and mode of interaction with RNA of RBD1. We have also analyzed the surface electrostatic potential and backbone dynamics of both RBDs. The two RBDs exhibit the same ribo-nucleoprotein-type fold and commonly make contact with RNA on the beta-sheet side. On the other hand, there is a remarkable difference in surface electrostatic potential, the beta-sheet of RBD1 being positively charged, which is favorable for binding negatively charged RNA, but that of RBD2 being almost neutral. There is also a difference in backbone dynamics, the central portion of the beta-sheet of RBD1 being flexible, but that of RBD2 not being flexible. The flexibility of RBD1 may be utilized in the recognition process to facilitate an induced fit. Thus, comparative studies have revealed the origin of the higher affinity of RBD1 than that of RBD2 and indicated that the affinity of an RBD to RNA is not governed by its fold alone but is also determined by its surface electrostatic potential and/or backbone dynamics. The biological role of RBD2 with lower affinity is also discussed.

The VSGB 2.0 Model: A Next Generation Energy Model for High Resolution Protein Structure Modeling

PubMed Central

Li, Jianing; Abel, Robert; Zhu, Kai; Cao, Yixiang; Zhao, Suwen; Friesner, Richard A.

2011-01-01

A novel energy model (VSGB 2.0) for high resolution protein structure modeling is described, which features an optimized implicit solvent model as well as physics-based corrections for hydrogen bonding, π-π interactions, self-contact interactions and hydrophobic interactions. Parameters of the VSGB 2.0 model were fit to a crystallographic database of 2239 single side chain and 100 11–13 residue loop predictions. Combined with an advanced method of sampling and a robust algorithm for protonation state assignment, the VSGB 2.0 model was validated by predicting 115 super long loops up to 20 residues. Despite the dramatically increasing difficulty in reconstructing longer loops, a high accuracy was achieved: all of the lowest energy conformations have global backbone RMSDs better than 2.0 Å from the native conformations. Average global backbone RMSDs of the predictions are 0.51, 0.63, 0.70, 0.62, 0.80, 1.41, and 1.59 Å for 14, 15, 16, 17, 18, 19, and 20 residue loop predictions, respectively. When these results are corrected for possible statistical bias as explained in the text, the average global backbone RMSDs are 0.61, 0.71, 0.86, 0.62, 1.06, 1.67, and 1.59 Å. Given the precision and robustness of the calculations, we believe that the VSGB 2.0 model is suitable to tackle “real” problems, such as biological function modeling and structure-based drug discovery. PMID:21905107

Structural and Biophysical Characterization of the Mycobacterium tuberculosis Protein Rv0577, a Protein Associated with Neutral Red Staining of Virulent Tuberculosis Strains and Homologue of the Streptomyces coelicolor Protein KbpA [Structural and Biophysical Characterization of Rv0577, a Protein Associated with Neutral Red Staining of Virulent Strains of Mycobacterium tuberculosis and homolog of the Streptomyces coelicolor protein KbpA

DOE PAGES

Buchko, Garry W.; Echols, Nathaniel; Flynn, E. Megan; ...

2017-07-10

Mycobacterium tuberculosis protein Rv0577 is a prominent antigen in tuberculosis patients, the component responsible for neutral red staining of virulent strains of M. tuberculosis, a putative component in a methylglyoxal detoxification pathway, and an agonist of toll-like receptor 2. It also has an amino acid sequence that is 36% identical to that of Streptomyces coelicolor AfsK-binding protein A (KbpA), a component in the complex secondary metabolite pathways in the Streptomyces genus. To gain insight into the biological function of Rv0577 and the family of KpbA kinase regulators, the crystal structure for Rv0577 was determined to a resolution of 1.75 Å,more » binding properties with neutral red and deoxyadenosine were surveyed, backbone dynamics were measured, and thermal stability was assayed by circular dichroism spectroscopy. The protein is composed of four approximate repeats with a βαβββ topology arranged radially in consecutive pairs to form two continuous eight-strand β-sheets capped on both ends with an α-helix. The two β-sheets intersect in the center at roughly a right angle and form two asymmetric deep “saddles” that may serve to bind ligands. Nuclear magnetic resonance chemical shift perturbation experiments show that neutral red and deoxyadenosine bind to Rv0577. Binding to deoxyadenosine is weaker with an estimated dissociation constants of 4.1 ± 0.3 mM for saddle 1. Heteronuclear steady-state { 1H}– 15N nuclear Overhauser effect, T 1, and T 2 values were generally uniform throughout the sequence with only a few modest pockets of differences. As a result, circular dichroism spectroscopy characterization of the thermal stability of Rv0577 indicated irreversible unfolding upon heating with an estimated melting temperature of 56 °C.« less

Structural and Biophysical Characterization of the Mycobacterium tuberculosis Protein Rv0577, a Protein Associated with Neutral Red Staining of Virulent Tuberculosis Strains and Homologue of the Streptomyces coelicolor Protein KbpA [Structural and Biophysical Characterization of Rv0577, a Protein Associated with Neutral Red Staining of Virulent Strains of Mycobacterium tuberculosis and homolog of the Streptomyces coelicolor protein KbpA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Buchko, Garry W.; Echols, Nathaniel; Flynn, E. Megan

Mycobacterium tuberculosis protein Rv0577 is a prominent antigen in tuberculosis patients, the component responsible for neutral red staining of virulent strains of M. tuberculosis, a putative component in a methylglyoxal detoxification pathway, and an agonist of toll-like receptor 2. It also has an amino acid sequence that is 36% identical to that of Streptomyces coelicolor AfsK-binding protein A (KbpA), a component in the complex secondary metabolite pathways in the Streptomyces genus. To gain insight into the biological function of Rv0577 and the family of KpbA kinase regulators, the crystal structure for Rv0577 was determined to a resolution of 1.75 Å,more » binding properties with neutral red and deoxyadenosine were surveyed, backbone dynamics were measured, and thermal stability was assayed by circular dichroism spectroscopy. The protein is composed of four approximate repeats with a βαβββ topology arranged radially in consecutive pairs to form two continuous eight-strand β-sheets capped on both ends with an α-helix. The two β-sheets intersect in the center at roughly a right angle and form two asymmetric deep “saddles” that may serve to bind ligands. Nuclear magnetic resonance chemical shift perturbation experiments show that neutral red and deoxyadenosine bind to Rv0577. Binding to deoxyadenosine is weaker with an estimated dissociation constants of 4.1 ± 0.3 mM for saddle 1. Heteronuclear steady-state { 1H}– 15N nuclear Overhauser effect, T 1, and T 2 values were generally uniform throughout the sequence with only a few modest pockets of differences. As a result, circular dichroism spectroscopy characterization of the thermal stability of Rv0577 indicated irreversible unfolding upon heating with an estimated melting temperature of 56 °C.« less

Apoprotein Structure and Metal Binding Characterization of a de Novo Designed Peptide, α3DIV, that Sequesters Toxic Heavy Metals.

PubMed

Plegaria, Jefferson S; Dzul, Stephen P; Zuiderweg, Erik R P; Stemmler, Timothy L; Pecoraro, Vincent L

2015-05-12

De novo protein design is a biologically relevant approach that provides a novel process in elucidating protein folding and modeling the metal centers of metalloproteins in a completely unrelated or simplified fold. An integral step in de novo protein design is the establishment of a well-folded scaffold with one conformation, which is a fundamental characteristic of many native proteins. Here, we report the NMR solution structure of apo α3DIV at pH 7.0, a de novo designed three-helix bundle peptide containing a triscysteine motif (Cys18, Cys28, and Cys67) that binds toxic heavy metals. The structure comprises 1067 NOE restraints derived from multinuclear multidimensional NOESY, as well as 138 dihedral angles (ψ, φ, and χ1). The backbone and heavy atoms of the 20 lowest energy structures have a root mean square deviation from the mean structure of 0.79 (0.16) Å and 1.31 (0.15) Å, respectively. When compared to the parent structure α3D, the substitution of Leu residues to Cys enhanced the α-helical content of α3DIV while maintaining the same overall topology and fold. In addition, solution studies on the metalated species illustrated metal-induced stability. An increase in the melting temperatures was observed for Hg(II), Pb(II), or Cd(II) bound α3DIV by 18-24 °C compared to its apo counterpart. Further, the extended X-ray absorption fine structure analysis on Hg(II)-α3DIV produced an average Hg(II)-S bond length at 2.36 Å, indicating a trigonal T-shaped coordination environment. Overall, the structure of apo α3DIV reveals an asymmetric distorted triscysteine metal binding site, which offers a model for native metalloregulatory proteins with thiol-rich ligands that function in regulating toxic heavy metals, such as ArsR, CadC, MerR, and PbrR.

Molprobity's ultimate rotamer-library distributions for model validation.

PubMed

Hintze, Bradley J; Lewis, Steven M; Richardson, Jane S; Richardson, David C

2016-09-01

Here we describe the updated MolProbity rotamer-library distributions derived from an order-of-magnitude larger and more stringently quality-filtered dataset of about 8000 (vs. 500) protein chains, and we explain the resulting changes and improvements to model validation as seen by users. To include only side-chains with satisfactory justification for their given conformation, we added residue-specific filters for electron-density value and model-to-density fit. The combined new protocol retains a million residues of data, while cleaning up false-positive noise in the multi- χ datapoint distributions. It enables unambiguous characterization of conformational clusters nearly 1000-fold less frequent than the most common ones. We describe examples of local interactions that favor these rare conformations, including the role of authentic covalent bond-angle deviations in enabling presumably strained side-chain conformations. Further, along with favored and outlier, an allowed category (0.3-2.0% occurrence in reference data) has been added, analogous to Ramachandran validation categories. The new rotamer distributions are used for current rotamer validation in MolProbity and PHENIX, and for rotamer choice in PHENIX model-building and refinement. The multi-dimensional χ distributions and Top8000 reference dataset are freely available on GitHub. These rotamers are termed "ultimate" because data sampling and quality are now fully adequate for this task, and also because we believe the future of conformational validation should integrate side-chain with backbone criteria. Proteins 2016; 84:1177-1189. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Native tandem and ion mobility mass spectrometry highlight structural and modular similarities in clustered-regularly-interspaced shot-palindromic-repeats (CRISPR)-associated protein complexes from Escherichia coli and Pseudomonas aeruginosa.

PubMed

van Duijn, Esther; Barbu, Ioana M; Barendregt, Arjan; Jore, Matthijs M; Wiedenheft, Blake; Lundgren, Magnus; Westra, Edze R; Brouns, Stan J J; Doudna, Jennifer A; van der Oost, John; Heck, Albert J R

2012-11-01

The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) immune system of bacteria and archaea provides acquired resistance against viruses and plasmids, by a strategy analogous to RNA-interference. Key components of the defense system are ribonucleoprotein complexes, the composition of which appears highly variable in different CRISPR/Cas subtypes. Previous studies combined mass spectrometry, electron microscopy, and small angle x-ray scattering to demonstrate that the E. coli Cascade complex (405 kDa) and the P. aeruginosa Csy-complex (350 kDa) are similar in that they share a central spiral-shaped hexameric structure, flanked by associating proteins and one CRISPR RNA. Recently, a cryo-electron microscopy structure of Cascade revealed that the CRISPR RNA molecule resides in a groove of the hexameric backbone. For both complexes we here describe the use of native mass spectrometry in combination with ion mobility mass spectrometry to assign a stable core surrounded by more loosely associated modules. Via computational modeling subcomplex structures were proposed that relate to the experimental IMMS data. Despite the absence of obvious sequence homology between several subunits, detailed analysis of sub-complexes strongly suggests analogy between subunits of the two complexes. Probing the specific association of E. coli Cascade/crRNA to its complementary DNA target reveals a conformational change. All together these findings provide relevant new information about the potential assembly process of the two CRISPR-associated complexes.

Crystal Structure of the Chromodomain Helicase DNA-binding Protein 1 (Chd1) DNA-binding Domain in Complex with DNA*

PubMed Central

Sharma, Amit; Jenkins, Katherine R.; Héroux, Annie; Bowman, Gregory D.

2011-01-01

Chromatin remodelers are ATP-dependent machines that dynamically alter the chromatin packaging of eukaryotic genomes by assembling, sliding, and displacing nucleosomes. The Chd1 chromatin remodeler possesses a C-terminal DNA-binding domain that is required for efficient nucleosome sliding and believed to be essential for sensing the length of DNA flanking the nucleosome core. The structure of the Chd1 DNA-binding domain was recently shown to consist of a SANT and SLIDE domain, analogous to the DNA-binding domain of the ISWI family, yet the details of how Chd1 recognized DNA were not known. Here we present the crystal structure of the Saccharomyces cerevisiae Chd1 DNA-binding domain in complex with a DNA duplex. The bound DNA duplex is straight, consistent with the preference exhibited by the Chd1 DNA-binding domain for extranucleosomal DNA. Comparison of this structure with the recently solved ISW1a DNA-binding domain bound to DNA reveals that DNA lays across each protein at a distinct angle, yet contacts similar surfaces on the SANT and SLIDE domains. In contrast to the minor groove binding seen for Isw1 and predicted for Chd1, the SLIDE domain of the Chd1 DNA-binding domain contacts the DNA major groove. The majority of direct contacts with the phosphate backbone occur only on one DNA strand, suggesting that Chd1 may not strongly discriminate between major and minor grooves. PMID:22033927

Backbone resonance assignments for G protein α(i3) subunit in the GDP-bound state.

PubMed

Mase, Yoko; Yokogawa, Mariko; Osawa, Masanori; Shimada, Ichio

2014-10-01

Guanine-nucleotide binding proteins (G proteins) serve as molecular switches in signaling pathways, by coupling the activation of G protein-coupled receptors (GPCRs) at the cell surface to intracellular responses. In the resting state, G protein forms a heterotrimer, consisting of the G protein α subunit with GDP (Gα·GDP) and the G protein βγ subunit (Gβγ). Ligand binding to GPCRs promotes the GDP-GTP exchange on Gα, leading to the dissociation of the GTP-bound form of Gα (Gα·GTP) and Gβγ. Then, Gα·GTP and Gβγ bind to their downstream effector enzymes or ion channels and regulate their activities, leading to a variety of cellular responses. Finally, Gα hydrolyzes the bound GTP to GDP and returns to the resting state by re-associating with Gβγ. The G proteins are classified with four major families based on the amino acid sequences of Gα: i/o, s, q/11, and 12/13. Here, we established the backbone resonance assignments of human Gαi3, a member of the i/o family with a molecular weight of 41 K, in complex with GDP. The chemical shifts were compared with those of Gα(i3) in complex with a GTP-analogue, GTPγS, which we recently reported, indicating that the residues with significant chemical shift differences are mostly consistent with the regions with the structural differences between the GDP- and GTPγS-bound states, as indicated in the crystal structures. The assignments of Gα(i3)·GDP would be useful for the analyses of the dynamics of Gα(i3) and its interactions with various target molecules.

Insights into the folding pathway of the Engrailed Homeodomain protein using replica exchange molecular dynamics simulations.

PubMed

Koulgi, Shruti; Sonavane, Uddhavesh; Joshi, Rajendra

2010-11-01

Protein folding studies were carried out by performing microsecond time scale simulations on the ultrafast/fast folding protein Engrailed Homeodomain (EnHD) from Drosophila melanogaster. It is a three-helix bundle protein consisting of 54 residues (PDB ID: 1ENH). The positions of the helices are 8-20 (Helix I), 26-36 (Helix II) and 40-53 (Helix III). The second and third helices together form a Helix-Turn-Helix (HTH) motif which belongs to the family of DNA binding proteins. The molecular dynamics (MD) simulations were performed using replica exchange molecular dynamics (REMD). REMD is a method that involves simulating a protein at different temperatures and performing exchanges at regular time intervals. These exchanges were accepted or rejected based on the Metropolis criterion. REMD was performed using the AMBER FF03 force field with the generalised Born solvation model for the temperature range 286-373 K involving 30 replicas. The extended conformation of the protein was used as the starting structure. A simulation of 600 ns per replica was performed resulting in an overall simulation time of 18 μs. The protein was seen to fold close to the native state with backbone root mean square deviation (RMSD) of 3.16 Å. In this low RMSD structure, the Helix I was partially formed with a backbone RMSD of 3.37 Å while HTH motif had an RMSD of 1.81 Å. Analysis suggests that EnHD folds to its native structure via an intermediate in which the HTH motif is formed. The secondary structure development occurs first followed by tertiary packing. The results were in good agreement with the experimental findings. Copyright © 2010 Elsevier Inc. All rights reserved.

Solution structures and backbone dynamics of Escherichia coli rhodanese PspE in its sulfur-free and persulfide-intermediate forms: implications for the catalytic mechanism of rhodanese.

PubMed

Li, Hongwei; Yang, Fan; Kang, Xue; Xia, Bin; Jin, Changwen

2008-04-15

Rhodanese catalyzes the sulfur-transfer reaction that transfers sulfur from thiosulfate to cyanide by a double-displacement mechanism, in which an active cysteine residue plays a central role. Previous studies indicated that the phage-shock protein E (PspE) from Escherichia coli is a rhodanese composed of a single active domain and is the only accessible rhodanese among the three single-domain rhodaneses in E. coli. To understand the catalytic mechanism of rhodanese at the molecular level, we determined the solution structures of the sulfur-free and persulfide-intermediate forms of PspE by nuclear magnetic resonance (NMR) spectroscopy and identified the active site by NMR titration experiments. To obtain further insights into the catalytic mechanism, we studied backbone dynamics by NMR relaxation experiments. Our results demonstrated that the overall structures in both sulfur-free and persulfide-intermediate forms are highly similar, suggesting that no significant conformational changes occurred during the catalytic reaction. However, the backbone dynamics revealed that the motional properties of PspE in its sulfur-free form are different from the persulfide-intermediate state. The conformational exchanges are largely enhanced in the persulfide-intermediate form of PspE, especially around the active site. The present structural and biochemical studies in combination with backbone dynamics provide further insights in understanding the catalytic mechanism of rhodanese.

Backbone Brackets and Arginine Tweezers delineate Class I and Class II aminoacyl tRNA synthetases

PubMed Central

Haupt, V. Joachim; Schroeder, Michael; Labudde, Dirk

2018-01-01

The origin of the machinery that realizes protein biosynthesis in all organisms is still unclear. One key component of this machinery are aminoacyl tRNA synthetases (aaRS), which ligate tRNAs to amino acids while consuming ATP. Sequence analyses revealed that these enzymes can be divided into two complementary classes. Both classes differ significantly on a sequence and structural level, feature different reaction mechanisms, and occur in diverse oligomerization states. The one unifying aspect of both classes is their function of binding ATP. We identified Backbone Brackets and Arginine Tweezers as most compact ATP binding motifs characteristic for each Class. Geometric analysis shows a structural rearrangement of the Backbone Brackets upon ATP binding, indicating a general mechanism of all Class I structures. Regarding the origin of aaRS, the Rodin-Ohno hypothesis states that the peculiar nature of the two aaRS classes is the result of their primordial forms, called Protozymes, being encoded on opposite strands of the same gene. Backbone Brackets and Arginine Tweezers were traced back to the proposed Protozymes and their more efficient successors, the Urzymes. Both structural motifs can be observed as pairs of residues in contemporary structures and it seems that the time of their addition, indicated by their placement in the ancient aaRS, coincides with the evolutionary trace of Proto- and Urzymes. PMID:29659563

Synthesis, characterization and electroluminescence of two highly-twisted non-doped blue light-emitting materials

NASA Astrophysics Data System (ADS)

Gong, Xiaojie; Pan, Yipeng; Xie, Xiang; Tong, Tong; Chen, Runfeng; Gao, Deqing

2018-04-01

Two pyrene derivatives, substituted with 2-methylnaphthalene units on 1,3-position and 1,6-position of pyrene backbones, were designed and synthesized. DFT calculation confirmed that the two molecules were highly twisted and the dihedral angles between pyrene backbone and naphthalene unit were over 80°, being attributed to the steric hindrance of ortho-methyl group and the substitution position of pyrene itself. As a result, the intermolecular aggregation was greatly inhibited in the solid state, being beneficial for suppressing the fluorescence quenching. By analyzing the optical and thermal properties, it was found that the π-π conjugation extension could be adjusted and a balance for high fluorescent efficiency and avoiding quenching at the same time could be reached, which may guide the molecular design in the future. The electroluminescence properties of the non-doped devices were enhanced with the double hole-transporting layers by optimizing the energy level matching. The stable blue EL emission, with the Commission Internationaled'Eclairage (CIEx,y) color coordinates of (0.15, 0.13) and (0.15, 0.11) at 7, 8, 9 and 10 V respectively, was obtained.

Role of monomer sequence and backbone chemistry in polypeptoid copolymers for marine antifouling coatings

NASA Astrophysics Data System (ADS)

Patterson, Anastasia; Wenning, Brandon; Rizis, Georgios; Calabrese, David; Finlay, John; Franco, Sofia; Clare, Anthony; Kramer, Edward; Ober, Christopher; Segalman, Rachel

The design rules elucidated in this work suggest that antifouling coatings bearing pendant peptoid side chains perform better overall in marine fouling tests than those with peptide side chains, with extremely low attachment of N. incerta and high removal of U. linza. This difference in performance is likely due to the lack of a hydrogen bond donor in the peptoid backbone. Furthermore, we show that the bulk polymer material of these hierarchical coatings (based on PEO or PDMS) plays a key role in determining both surface presentation and fouling release performance. We demonstrate these trends utilizing a modular coating based on a triblock copolymer consisting of polystyrene and a vinyl-containing midblock, to which sequence-defined pendant oligomers (peptides or peptoids with sequences of oligo-PEO and fluoroalkyl groups) are attached via thiol-ene ``click'' chemistry. Surface presentation was analyzed with X-ray photoelectron spectroscopy and captive bubble water contact angle, and antifouling performance was evaluated with attachment and removal bioassays of the marine macroalga U. linza and diatom N. incerta. NSF GRFP and ONR PECASE.

Avian Nanostructured Tissues as Models for New Defensive Coatings and Photonic Crystal Fibers

DTIC Science & Technology

2012-03-31

promiscuous binding capacity of chitin , the chemical backbone of the arthropod cuticle (Kumar 2000). This polysaccharide binds many proteins and other...properties. The greater refractive index contrast between light and dark layers afforded by chitin may allow Arthropoda to attain brighter and more 71

The Application of Ligand-Mapping Molecular Dynamics Simulations to the Rational Design of Peptidic Modulators of Protein-Protein Interactions.

PubMed

Tan, Yaw Sing; Spring, David R; Abell, Chris; Verma, Chandra S

2015-07-14

A computational ligand-mapping approach to detect protein surface pockets that interact with hydrophobic moieties is presented. In this method, we incorporated benzene molecules into explicit solvent molecular dynamics simulations of various protein targets. The benzene molecules successfully identified the binding locations of hydrophobic hot-spot residues and all-hydrocarbon cross-links from known peptidic ligands. They also unveiled cryptic binding sites that are occluded by side chains and the protein backbone. Our results demonstrate that ligand-mapping molecular dynamics simulations hold immense promise to guide the rational design of peptidic modulators of protein-protein interactions, including that of stapled peptides, which show promise as an exciting new class of cell-penetrating therapeutic molecules.

Chemical Denaturants Smoothen Ruggedness on the Free Energy Landscape of Protein Folding.

PubMed

Malhotra, Pooja; Jethva, Prashant N; Udgaonkar, Jayant B

2017-08-08

To characterize experimentally the ruggedness of the free energy landscape of protein folding is challenging, because the distributed small free energy barriers are usually dominated by one, or a few, large activation free energy barriers. This study delineates changes in the roughness of the free energy landscape by making use of the observation that a decrease in ruggedness is accompanied invariably by an increase in folding cooperativity. Hydrogen exchange (HX) coupled to mass spectrometry was used to detect transient sampling of local energy minima and the global unfolded state on the free energy landscape of the small protein single-chain monellin. Under native conditions, local noncooperative openings result in interconversions between Boltzmann-distributed intermediate states, populated on an extremely rugged "uphill" energy landscape. The cooperativity of these interconversions was increased by selectively destabilizing the native state via mutations, and further by the addition of a chemical denaturant. The perturbation of stability alone resulted in seven backbone amide sites exchanging cooperatively. The size of the cooperatively exchanging and/or unfolding unit did not depend on the extent of protein destabilization. Only upon the addition of a denaturant to a destabilized mutant variant did seven additional backbone amide sites exchange cooperatively. Segmentwise analysis of the HX kinetics of the mutant variants further confirmed that the observed increase in cooperativity was due to the smoothing of the ruggedness of the free energy landscape of folding of the protein by the chemical denaturant.

Knowledge-based prediction of protein backbone conformation using a structural alphabet.

PubMed

Vetrivel, Iyanar; Mahajan, Swapnil; Tyagi, Manoj; Hoffmann, Lionel; Sanejouand, Yves-Henri; Srinivasan, Narayanaswamy; de Brevern, Alexandre G; Cadet, Frédéric; Offmann, Bernard

2017-01-01

Libraries of structural prototypes that abstract protein local structures are known as structural alphabets and have proven to be very useful in various aspects of protein structure analyses and predictions. One such library, Protein Blocks, is composed of 16 standard 5-residues long structural prototypes. This form of analyzing proteins involves drafting its structure as a string of Protein Blocks. Predicting the local structure of a protein in terms of protein blocks is the general objective of this work. A new approach, PB-kPRED is proposed towards this aim. It involves (i) organizing the structural knowledge in the form of a database of pentapeptide fragments extracted from all protein structures in the PDB and (ii) applying a knowledge-based algorithm that does not rely on any secondary structure predictions and/or sequence alignment profiles, to scan this database and predict most probable backbone conformations for the protein local structures. Though PB-kPRED uses the structural information from homologues in preference, if available. The predictions were evaluated rigorously on 15,544 query proteins representing a non-redundant subset of the PDB filtered at 30% sequence identity cut-off. We have shown that the kPRED method was able to achieve mean accuracies ranging from 40.8% to 66.3% depending on the availability of homologues. The impact of the different strategies for scanning the database on the prediction was evaluated and is discussed. Our results highlight the usefulness of the method in the context of proteins without any known structural homologues. A scoring function that gives a good estimate of the accuracy of prediction was further developed. This score estimates very well the accuracy of the algorithm (R2 of 0.82). An online version of the tool is provided freely for non-commercial usage at http://www.bo-protscience.fr/kpred/.

Fluorinated bottlebrush polymers based on poly(trifluoroethyl methacrylate): Synthesis and characterizations

DOE PAGES

Xu, Yuewen; Wang, Weiyu; Wang, Yangyang; ...

2015-11-25

Bottlebrush polymers are densely grafted polymers with long side-chains attached to a linear polymeric backbone. Their unusual structures endow them with a number of unique and potentially useful properties in solution, in thin films, and in bulk. Despite the many studies of bottlebrushes that have been reported, the structure–property relationships for this class of materials are still poorly understood. In this contribution, we report the synthesis and characterization of fluorinated bottlebrush polymers based on poly(2,2,2-trifluoroethyl methacrylate). The synthesis was achieved by atom transfer radical polymerization (ATRP) using an α-bromoisobutyryl bromide functionalized norbornene initiator, followed by ring-opening metathesis polymerization (ROMP) usingmore » a third generation Grubbs’ catalyst (G3). Rheological characterization revealed that the bottlebrush polymer backbones remained unentangled as indicated by the lack of a rubbery plateau in the modulus. By tuning the size of the backbone of the bottlebrush polymers, near-spherical and elongated particles representing single brush molecular morphologies were observed in a good solvent as evidenced by TEM imaging, suggesting a semi-flexible nature of their backbones in dilute solutions. Thin films of bottlebrush polymers exhibited noticeably higher static water contact angles as compared to that of the macromonomer reaching the hydrophobic regime, where little differences were observed between each bottlebrush polymer. Further investigation by AFM revealed that the surface of the macromonomer film was relatively smooth; in contrast, the surface of bottlebrush polymers displayed certain degrees of nano-scale roughness (R q = 0.8–2.4 nm). The enhanced hydrophobicity of these bottlebrushes likely results from the preferential enrichment of the fluorine containing end groups at the periphery of the molecules and the film surface due to the side chain crowding effect. Furthermore, our results provide key information towards the design of architecturally tailored fluorinated polymers with desirable properties.« less

Tertiary alphabet for the observable protein structural universe.

PubMed

Mackenzie, Craig O; Zhou, Jianfu; Grigoryan, Gevorg

2016-11-22

Here, we systematically decompose the known protein structural universe into its basic elements, which we dub tertiary structural motifs (TERMs). A TERM is a compact backbone fragment that captures the secondary, tertiary, and quaternary environments around a given residue, comprising one or more disjoint segments (three on average). We seek the set of universal TERMs that capture all structure in the Protein Data Bank (PDB), finding remarkable degeneracy. Only ∼600 TERMs are sufficient to describe 50% of the PDB at sub-Angstrom resolution. However, more rare geometries also exist, and the overall structural coverage grows logarithmically with the number of TERMs. We go on to show that universal TERMs provide an effective mapping between sequence and structure. We demonstrate that TERM-based statistics alone are sufficient to recapitulate close-to-native sequences given either NMR or X-ray backbones. Furthermore, sequence variability predicted from TERM data agrees closely with evolutionary variation. Finally, locations of TERMs in protein chains can be predicted from sequence alone based on sequence signatures emergent from TERM instances in the PDB. For multisegment motifs, this method identifies spatially adjacent fragments that are not contiguous in sequence-a major bottleneck in structure prediction. Although all TERMs recur in diverse proteins, some appear specialized for certain functions, such as interface formation, metal coordination, or even water binding. Structural biology has benefited greatly from previously observed degeneracies in structure. The decomposition of the known structural universe into a finite set of compact TERMs offers exciting opportunities toward better understanding, design, and prediction of protein structure.

Tertiary alphabet for the observable protein structural universe

PubMed Central

Mackenzie, Craig O.; Zhou, Jianfu; Grigoryan, Gevorg

2016-01-01

Here, we systematically decompose the known protein structural universe into its basic elements, which we dub tertiary structural motifs (TERMs). A TERM is a compact backbone fragment that captures the secondary, tertiary, and quaternary environments around a given residue, comprising one or more disjoint segments (three on average). We seek the set of universal TERMs that capture all structure in the Protein Data Bank (PDB), finding remarkable degeneracy. Only ∼600 TERMs are sufficient to describe 50% of the PDB at sub-Angstrom resolution. However, more rare geometries also exist, and the overall structural coverage grows logarithmically with the number of TERMs. We go on to show that universal TERMs provide an effective mapping between sequence and structure. We demonstrate that TERM-based statistics alone are sufficient to recapitulate close-to-native sequences given either NMR or X-ray backbones. Furthermore, sequence variability predicted from TERM data agrees closely with evolutionary variation. Finally, locations of TERMs in protein chains can be predicted from sequence alone based on sequence signatures emergent from TERM instances in the PDB. For multisegment motifs, this method identifies spatially adjacent fragments that are not contiguous in sequence—a major bottleneck in structure prediction. Although all TERMs recur in diverse proteins, some appear specialized for certain functions, such as interface formation, metal coordination, or even water binding. Structural biology has benefited greatly from previously observed degeneracies in structure. The decomposition of the known structural universe into a finite set of compact TERMs offers exciting opportunities toward better understanding, design, and prediction of protein structure. PMID:27810958

The Structure of a Thermophilic Kinase Shapes Fitness upon Random Circular Permutation.

PubMed

Jones, Alicia M; Mehta, Manan M; Thomas, Emily E; Atkinson, Joshua T; Segall-Shapiro, Thomas H; Liu, Shirley; Silberg, Jonathan J

2016-05-20

Proteins can be engineered for synthetic biology through circular permutation, a sequence rearrangement in which native protein termini become linked and new termini are created elsewhere through backbone fission. However, it remains challenging to anticipate a protein's functional tolerance to circular permutation. Here, we describe new transposons for creating libraries of randomly circularly permuted proteins that minimize peptide additions at their termini, and we use transposase mutagenesis to study the tolerance of a thermophilic adenylate kinase (AK) to circular permutation. We find that libraries expressing permuted AKs with either short or long peptides amended to their N-terminus yield distinct sets of active variants and present evidence that this trend arises because permuted protein expression varies across libraries. Mapping all sites that tolerate backbone cleavage onto AK structure reveals that the largest contiguous regions of sequence that lack cleavage sites are proximal to the phosphotransfer site. A comparison of our results with a range of structure-derived parameters further showed that retention of function correlates to the strongest extent with the distance to the phosphotransfer site, amino acid variability in an AK family sequence alignment, and residue-level deviations in superimposed AK structures. Our work illustrates how permuted protein libraries can be created with minimal peptide additions using transposase mutagenesis, and it reveals a challenge of maintaining consistent expression across permuted variants in a library that minimizes peptide additions. Furthermore, these findings provide a basis for interpreting responses of thermophilic phosphotransferases to circular permutation by calibrating how different structure-derived parameters relate to retention of function in a cellular selection.

Protein expression and isotopic enrichment based on induction of the Entner-Doudoroff pathway in Escherichia coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Refaeli, Bosmat; Goldbourt, Amir, E-mail: amirgo@post.tau.ac.il

2012-10-12

Highlights: Black-Right-Pointing-Pointer The Entner-Doudoroff pathway is induced during protein expression in E. coli. Black-Right-Pointing-Pointer 1-{sup 13}C-gluconate and {sup 15}NH{sub 4}Cl provide a carbonyl-amide protein backbone labeling scheme. Black-Right-Pointing-Pointer The enrichment pattern is determined by nuclear magnetic resonance. -- Abstract: The Entner-Doudoroff pathway is known to exist in many organisms including bacteria, archea and eukarya. Although the common route for carbon catabolism in Escherichia coli is the Embden-Meyerhof-Parnas pathway, it was shown that gluconate catabolism in E. coli occurs via the Entner-Doudoroff pathway. We demonstrate here that by supplying BL21(DE3) competent E.coli cells with gluconate in a minimal growth medium, proteinmore » expression can be induced. Nuclear magnetic resonance data of over-expressed ubiquitin show that by using [1-{sup 13}C]-gluconate as the only carbon source, and {sup 15}N-enriched ammonium chloride, sparse isotopic enrichment in the form of a spin-pair carbonyl-amide backbone enrichment is obtained. The specific amino acid labeling pattern is analyzed and is shown to be compatible with Entner-Doudoroff metabolism. Isotopic enrichment serves as a key factor in the biophysical characterization of proteins by various methods including nuclear magnetic resonance, mass spectrometry, infrared spectroscopy and more. Therefore, the method presented here can be applied to study proteins by obtaining sparse enrichment schemes that are not based on the regular glycolytic pathway, or to study the Entner-Doudoroff metabolism during protein expression.« less

Characterization of the sensor domain of QseE histidine kinase from Escherichia coli.

PubMed

Yeo, Kwon Joo; Park, Jin-Wan; Kim, Eun-Hee; Jeon, Young Ho; Hwang, Kwang Yeon; Cheong, Hae-Kap

2016-10-01

In enterohemorrhagic Escherichia coli (EHEC), the QseEF two-component system causes attaching and effacing (AE) lesion on epithelial cells. QseE histidine kinase senses the host hormone epinephrine, sulfate, and phosphate; it also regulates QseF response regulator, which activates LEE gene that encodes AE lesion. In order to understand the recognition of ligand molecules and signal transfer mechanism in pathogenic bacteria, structural studies of the sensor domain of QseE of Escherichia coli should be conducted. In this study, we describe the overexpression, purification, and structural and biophysical properties of the sensor domain of QseE. The fusion protein had a 6×His tag at its N-terminus; this protein was overexpressed as inclusion bodies in E. coli BL21 (DE3). The protein was denatured in 7M guanidine hydrochloride and refolded by dialysis. The purification of the refolded protein was carried out using Ni-NTA affinity column and size-exclusion chromatography. Thereafter, the characteristics of the refolded protein were determined from NMR, CD, and MALS spectroscopies. In a pH range of 7.4-5.0, the folded protein existed in a monomeric form with a predominantly helical structure. (1)H-(15)N HSQC NMR spectra shows that approximately 93% backbone amide peaks are detected at pH 5.0, suggesting that the number of backbone signals is sufficient for NMR studies. These data might provide an opportunity for structural and functional studies of the sensor domain of QseE. Copyright © 2016 Elsevier Inc. All rights reserved.

Kinetics of Contact Formation and End-to-End Distance Distributions of Swollen Disordered Peptides

PubMed Central

Soranno, Andrea; Longhi, Renato; Bellini, Tommaso; Buscaglia, Marco

2009-01-01

Unstructured polypeptide chains are subject to various degrees of swelling or compaction depending on the combination of solvent condition and amino acid sequence. Highly denatured proteins generally behave like random-coils with excluded volume repulsion, whereas in aqueous buffer more compact conformations have been observed for the low-populated unfolded state of globular proteins as well as for naturally disordered sequences. To quantitatively account for the different mechanisms inducing the swelling of polypeptides, we have examined three 14-residues peptides in aqueous buffer and in denaturant solutions, including the well characterized AGQ repeat as a reference and two variants, in which we have successively introduced charged side chains and removed the glycines. Quenching of the triplet state of tryptophan by close contact with cysteine has been used in conjunction with Förster resonance energy transfer to study the equilibrium and kinetic properties of the peptide chains. The experiments enable accessing end-to-end root mean-square distance, probability of end-to-end contact formation and intrachain diffusion coefficient. The data can be coherently interpreted on the basis of a simple chain model with backbone angles obtained from a library of coil segments of proteins and hard sphere repulsion at each Cα position. In buffered water, we find that introducing charges in a glycine-rich sequence induces a mild chain swelling and a significant speed-up of the intrachain dynamics, whereas the removal of the glycines results in almost a two-fold increase of the chain volume and a drastic slowing down. In denaturants we observe a pronounced swelling of all the chains, with significant differences between the effect of urea and guanidinium chloride. PMID:19217868

Structural consequences of metallothionein dimerization: solution structure of the isolated Cd4-alpha-domain and comparison with the holoprotein dimer.

PubMed

Ejnik, John W; Muñoz, Amalia; DeRose, Eugene; Shaw, C Frank; Petering, David H

2003-07-22

The NMR determination of the structure of Cd(7)-metallothionein was done previously using a relatively large protein concentration that favors dimer formation. The reactivity of the protein is also affected under this condition. To examine the influence of protein concentration on metallothionein conformation, the isolated Cd(4)-alpha-domain was prepared from rabbit metallothionein-2 (MT 2), and its three-dimensional structure was determined by heteronuclear, (1)H-(111)Cd, and homonuclear, (1)H-(1)H NMR, correlation experiments. The three-dimensional structure was refined using distance and angle constraints derived from these two-dimensional NMR data sets and a distance geometry/simulated annealing protocol. The backbone superposition of the alpha-domain from rabbit holoprotein Cd(7)-MT 2 and the isolated rabbit Cd(4)-alpha was measured at a RMSD of 2.0 A. Nevertheless, the conformations of the two Cd-thiolate clusters were distinctly different at two of the cadmium centers. In addition, solvent access to the sulfhydryl ligands of the isolated Cd(4)-alpha cluster was 130% larger due to this small change in cluster geometry. To probe whether these differences were an artifact of the structure calculation, the Cd(4)-alpha-domain structure in rabbit Cd(7)-MT 2 was redetermined, using the previously defined set of NOEs and the present calculation protocol. All calculations employed the same ionic radius for Cd(2+) and same cadmium-thiolate bond distance. The newly calculated structure matched the original with an RMSD of 1.24 A. It is hypothesized that differences in the two alpha-domain structures result from a perturbation of the holoprotein structure because of head-to-tail dimerization under the conditions of the NMR experiments.

Neighbor-Dependent Ramachandran Probability Distributions of Amino Acids Developed from a Hierarchical Dirichlet Process Model

PubMed Central

Mitra, Rajib; Jordan, Michael I.; Dunbrack, Roland L.

2010-01-01

Distributions of the backbone dihedral angles of proteins have been studied for over 40 years. While many statistical analyses have been presented, only a handful of probability densities are publicly available for use in structure validation and structure prediction methods. The available distributions differ in a number of important ways, which determine their usefulness for various purposes. These include: 1) input data size and criteria for structure inclusion (resolution, R-factor, etc.); 2) filtering of suspect conformations and outliers using B-factors or other features; 3) secondary structure of input data (e.g., whether helix and sheet are included; whether beta turns are included); 4) the method used for determining probability densities ranging from simple histograms to modern nonparametric density estimation; and 5) whether they include nearest neighbor effects on the distribution of conformations in different regions of the Ramachandran map. In this work, Ramachandran probability distributions are presented for residues in protein loops from a high-resolution data set with filtering based on calculated electron densities. Distributions for all 20 amino acids (with cis and trans proline treated separately) have been determined, as well as 420 left-neighbor and 420 right-neighbor dependent distributions. The neighbor-independent and neighbor-dependent probability densities have been accurately estimated using Bayesian nonparametric statistical analysis based on the Dirichlet process. In particular, we used hierarchical Dirichlet process priors, which allow sharing of information between densities for a particular residue type and different neighbor residue types. The resulting distributions are tested in a loop modeling benchmark with the program Rosetta, and are shown to improve protein loop conformation prediction significantly. The distributions are available at http://dunbrack.fccc.edu/hdp. PMID:20442867

Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions.

PubMed

Krissinel, E; Henrick, K

2004-12-01

The present paper describes the SSM algorithm of protein structure comparison in three dimensions, which includes an original procedure of matching graphs built on the protein's secondary-structure elements, followed by an iterative three-dimensional alignment of protein backbone Calpha atoms. The SSM results are compared with those obtained from other protein comparison servers, and the advantages and disadvantages of different scores that are used for structure recognition are discussed. A new score, balancing the r.m.s.d. and alignment length Nalign, is proposed. It is found that different servers agree reasonably well on the new score, while showing considerable differences in r.m.s.d. and Nalign.

Backbone dynamics of a model membrane protein: assignment of the carbonyl carbon /sup 13/C NMR resonances in detergent-solubilized M13 coat protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Henry, G.D.; Weiner, J.H.; Sykes, B.D.

The major coat protein of the filamentous bacteriophage M13 is a 50-residue amphiphilic polypeptide which is inserted, as an integral membrane-spanning protein, in the inner membrane of the Escherichia coli host during infection. /sup 13/C was incorporated biosynthetically into a total of 23 of the peptide carbonyls using labeled amino acids (alanine, glycine, lysine, phenylalanine, and proline). The structure and dynamics of carbonyl-labeled M13 coat protein were monitored by /sup 13/C nuclear magnetic resonance (NMR) spectroscopy. Assignment of many resonances was achieved by using protease digestion, pH titration, or labeling of the peptide bond with both /sup 13/C and /supmore » 15/N. The carbonyl region of the natural-abundance /sup 13/C NMR spectrum of M13 coat protein in sodium dodecyl sulfate solution shows approximately eight backbone carbonyl resonances with line widths much narrower than the rest. Three of these more mobile residues correspond to assigned peaks (glycine-3, lysine-48, and alanine-49) in the individual amino acid spectra, and another almost certainly arises from glutamic acid-2. A ninth residue, alanine-1, also gives rise to a very narrow carbonyl resonance if the pH is well above or below the pK/sub a/ of the terminal amino group. These data suggest that only about four residues at either end of the protein experience large-amplitude spatial fluctuations; the rest of the molecule is essentially rigid on the time scale of the overall rotational tumbling of the protein-detergent complex. The relative exposure of different regions of detergent-bound protein was monitored by limited digestion with proteinase K. Comparable spectra and digestion patterns were obtained when the protein was solubilized in sodium deoxycholate, suggesting that the coat protein binds both amphiphiles in a similar fashion.« less

Predicting the helix packing of globular proteins by self-correcting distance geometry.

PubMed

Mumenthaler, C; Braun, W

1995-05-01

A new self-correcting distance geometry method for predicting the three-dimensional structure of small globular proteins was assessed with a test set of 8 helical proteins. With the knowledge of the amino acid sequence and the helical segments, our completely automated method calculated the correct backbone topology of six proteins. The accuracy of the predicted structures ranged from 2.3 A to 3.1 A for the helical segments compared to the experimentally determined structures. For two proteins, the predicted constraints were not restrictive enough to yield a conclusive prediction. The method can be applied to all small globular proteins, provided the secondary structure is known from NMR analysis or can be predicted with high reliability.

Conformational analysis of endomorphin-2 analogs with phenylalanine mimics by NMR and molecular modeling.

PubMed

Shao, Xuan; Gao, Yanfeng; Zhu, Chuanjun; Liu, Xuehui; Yao, Jinlong; Cui, Yuxin; Wang, Rui

2007-05-15

We investigated a series of conformations of endomorphin-2 (EM-2) analogs substituted by phenylglycine (Phg) and homophenylalanine (Hfe) in the position 3 or 4 by two-dimensional (1)H NMR spectroscopy and molecular modeling. Evaluating the aromatic interactions and the dihedral angles in these phenylalanine mimics, we have observed that the conformations in trans isomer have varied from extended to folded as bioactivity decreases. It is suggested that the flexibility of aromatic side chain affects the backbone of EM-2 to adopt folded structures, which may block the ligands in binding to micro-opioid receptor.

Genetic engineering combined with deep UV resonance Raman spectroscopy for structural characterization of amyloid-like fibrils.

PubMed

Sikirzhytski, Vitali; Topilina, Natalya I; Higashiya, Seiichiro; Welch, John T; Lednev, Igor K

2008-05-07

Elucidating the structure of the cross-beta core in large amyloid fibrils is a challenging problem in modern structural biology. For the first time, a set of de novo polypeptides was genetically engineered to form amyloid-like fibrils with similar morphology and yet different strand length. Differential ultraviolet Raman spectroscopy allowed for separation of the spectroscopic signatures of the highly ordered beta-sheet strands and turns of the fibril core. The relationship between Raman frequencies and Ramachandran dihedral angles of the polypeptide backbone indicates the nature of the beta-sheet and turn structural elements.

Conformational responses to changes in the state of ionization of titrable groups in proteins

NASA Astrophysics Data System (ADS)

Richman, Daniel Eric

Electrostatic energy links the structural properties of proteins with some of their important biological functions, including catalysis, energy transduction, and binding and recognition. Accurate calculation of electrostatic energy is essential for predicting and for analyzing function from structure. All proteins have many ionizable residues at the protein-water interface. These groups tend to have ionization equilibria (pK a values) shifted slightly relative to their values in water. In contrast, groups buried in the hydrophobic interior usually have highly anomalous p Ka values. These shifts are what structure-based calculations have to reproduce to allow examination of contributions from electrostatics to stability, solubility and interactions of proteins. Electrostatic energies are challenging to calculate accurately because proteins are heterogeneous dielectric materials. Any individual ionizable group can experience very different local environments with different dielectric properties. The studies in this thesis examine the hypothesis that proteins reorganize concomitant with changes in their state of ionization. It appears that the pKa value measured experimentally reflects the average of pKa values experienced in the different electrostatic environments corresponding to different conformational microstates. Current computational models fail to sample conformational reorganization of the backbone correctly. Staphyloccocal nuclease (SNase) was used as a model protein in nuclear magnetic resonance (NMR) spectroscopy studies to characterize the conformational rearrangements of the protein coupled to changes in the ionization state of titrable groups. One set of experiments tests the hypothesis that proton binding to surface Asp and Glu side chains drives local unfolding by stabilizing less-native, more water-solvated conformations in which the side chains have normalized pKa values. Increased backbone flexibility in the ps-ns timescale, hydrogen bond (H-bond) breaking on at least the mus timescale, and segmental unfolding were detected near titrating groups as pH decreased into the acidic range. The study identified local structural features and stabilities that modulate the magnitude of electrostatic effects. The data demonstrate that computational approaches to pK a calculations for surface groups must account for local fluctuations spanning a wide range of timescales. A comparative NMR spectroscopy study with the L25K and L125K variants of SNase, each with a Lys residue buried in the hydrophobic interior of the protein, determined locations, timescales, and amplitudes of backbone conformational reorganization coupled with ionization of the buried Lys residues. The L25K protein exhibited an ensemble of local fluctuations of the beta barrel in the hundreds of mus timescale and an ensemble of subglobally unfolded beta-barrel states in the hundreds of ms timescale with strong pH dependence. The L125K protein exhibited fluctuations of the helix around site 125 in the mus timescale, with negligible pH dependence. These data illustrate the diverse timescales and local structural properties of conformational reorganization coupled to ionization of buried groups, and the challenge to structure-based electrostatics calculations, which must capture these long-timescale processes.

Determining Functional Aptamer-Protein Interaction by Biolayer Interferometry.

PubMed

Lou, Xinhui; Egli, Martin; Yang, Xianbin

2016-12-01

Short single-stranded nucleic acids called aptamers are widely being explored as recognition molecules of high affinity and specificity for binding a wide range of target molecules, particularly protein targets. In biolayer interferometry (BLI), a simple Dip-and-Read approach in which the aptamer-coated biosensors are dipped into microplate wells is used to study the interactions between an aptamer and its target protein. Here we describe the protocol for the analysis of the interaction between a well-characterized anti-thrombin RNA aptamer with thrombin (Basic Protocol). We also report on the protocol for the affinity screening of a panel of anti-thrombin RNA aptamers with a single phosphorodithioate (PS2) modification, whereby the position of the modification along the RNA backbone is varied systematically (Support Protocol). The PS2 modification uses two sulfur atoms to replace two non-bridging oxygen atoms at an internucleotide phosphodiester backbone linkage. The PS2-modified RNAs are nuclease resistant and several in vitro and in vivo assays have demonstrated their biological activity. For example, combining the PS2 with the 2'-OMe modification affords increased loading of modified small interfering RNA (siRNA) duplexes into the RNA-induced silencing complex (RISC) as well as enhanced gene-silencing antitumor activity. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

Temperature-dependent instability of the cTnI subunit in NIST SRM2921 characterized by tryptic peptide mapping.

PubMed

van der Burgt, Yuri E M; Cobbaert, Christa M; Dalebout, Hans; Smit, Nico; Deelder, André M

2012-08-01

In this study temperature-dependent instability of the cTnI subunit of the three-protein complex NIST SRM2921 was demonstrated using a mass spectrometric tryptic peptide mapping approach. The results were compared to the cTnI subunit obtained as a protein standard from Calbiochem with identical amino acid sequence. Both the three-protein complex from NIST as well as the cTnI subunit were incubated at elevated temperatures and then evaluated with respect to the primary sequence. The corresponding peptide maps were analyzed using LC-MS/MS. From a Mascot database search in combination with "semiTrypsin" tolerance it was found that two peptide backbone cleavages had occurred in subunit cTnI in NIST SRM2921 material upon incubation at 37°C, namely between amino acids at 148/149 and 194/195. The Calbiochem standard did not show increased levels of "unexpected" peptides in tryptic peptide maps. One of the two peptide backbone cleavages could also be monitored using a "single-step" MALDI-MS approach, i.e. without the need for peptide separation. The amount of degradation appeared rather constant in replicate temperature-instability experiments. However, for accurate quantification internal labelled standards are needed. Copyright © 2012 Elsevier B.V. All rights reserved.

NMR conformational properties of an Anthrax Lethal Factor domain studied by multiple amino acid-selective labeling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vourtsis, Dionysios J.; Chasapis, Christos T.; Pairas, George

2014-07-18

Highlights: • A polypeptide, N-ALF{sub 233}, was overexpressed in E. coli and successfully isolated. • We produced {sup 2}H/{sup 15}N/{sup 13}C labeled protein samples. • Amino acid selective approaches were applied. • We acquired several heteronuclear NMR spectra, to complete the backbone assignment. • Prediction of the secondary structure was performed. - Abstract: NMR-based structural biology urgently needs cost- and time-effective methods to assist both in the process of acquiring high-resolution NMR spectra and their subsequent analysis. Especially for bigger proteins (>20 kDa) selective labeling is a frequently used means of sequence-specific assignment. In this work we present the successfulmore » overexpression of a polypeptide of 233 residues, corresponding to the structured part of the N-terminal domain of Anthrax Lethal Factor, using Escherichia coli expression system. The polypeptide was subsequently isolated in pure, soluble form and analyzed structurally by solution NMR spectroscopy. Due to the non-satisfying quality and resolution of the spectra of this 27 kDa protein, an almost complete backbone assignment became feasible only by the combination of uniform and novel amino acid-selective labeling schemes. Moreover, amino acid-type selective triple-resonance NMR experiments proved to be very helpful.« less

RITA Mimics: Synthesis and Mechanistic Evaluation of Asymmetric Linked Trithiazoles.

PubMed

Pietkiewicz, Adrian L; Zhang, Yuqi; Rahimi, Marwa N; Stramandinoli, Michael; Teusner, Matthew; McAlpine, Shelli R

2017-04-13

The established cytotoxic agent RITA contains a thiophene-furan-thiophene backbone and two terminal alcohol groups. Herein we investigate the effect of using thiazoles as the backbone in RITA-like molecules and modifying the terminal groups of these trithiazoles, thereby generating 41 unique structures. Incorporating side chains with varied steric bulk allowed us to investigate how size and a stereocenter impacted biological activity. Subjecting compounds to growth inhibition assays on HCT-116 cells showed that the most potent compounds 7d , 7e , and 7h had GI 50 values of 4.4, 4.4, and 3.4 μM, respectively, versus RITA (GI 50 of 800 nM). Analysis of these compounds in apoptosis assays proved that 7d , 7e , and 7h were as effective as RITA at inducing apoptosis. Evaluating the impact of 7h on proteins targeted by RITA (p53, c-Myc, and Mcl-1) indicated that it acts via a different mechanism of action to that of RITA. RITA suppressed Mcl-1 protein via p53, whereas compound 7h suppressed Mcl-1 expression via an alternative mechanism independent of p53.

Coarse Graining to Investigate Membrane Induced Peptide Folding of Anticancer Peptides

NASA Astrophysics Data System (ADS)

Ganesan, Sai; Xu, Hongcheng; Matysiak, Silvina

Information about membrane induced peptide folding mechanisms using all-atom molecular dynamics simulations is a challenge due to time and length scale issues.We recently developed a low resolution Water Explicit Polarizable PROtein coarse-grained Model by adding oppositely charged dummy particles inside protein backbone beads.These two dummy particles represent a fluctuating dipole,thus introducing structural polarization into the coarse-grained model.With this model,we were able to achieve significant α- β secondary structure content de novo,without any added bias.We extended the model to zwitterionic and anionic lipids,by adding oppositely charged dummy particles inside polar beads, to capture the ability of the head group region to form hydrogen bonds.We use zwitterionic POPC and anionic POPS as our model lipids, and a cationic anticancer peptide,SVS1,as our model peptide.We have characterized the driving forces for SVS1 folding on lipid bilayers with varying anionic and zwitterionic lipid compositions.Based on our results, dipolar interactions between peptide backbone and lipid head groups contribute to stabilize folded conformations.Cooperativity in folding is induced by both intra peptide and membrane-peptide interaction.

Donkey's milk detailed lipid composition.

PubMed

Gastaldi, Daniela; Bertino, Enrico; Monti, Giovanna; Baro, Cristina; Fabris, Claudio; Lezo, Antonela; Medana, Claudio; Baiocchi, Claudio; Mussap, Michele; Galvano, Fabio; Conti, Amedeo

2010-01-01

Donkey's milk (DM) has recently aroused scientific interest, above all among paediatric allergologists. A deeper knowledge of both proteins and fats in donkey's milk is necessary to evaluate the immunological, physiological and nutritional properties. By using the most refined techniques for fatty acids analysis, the paper offers a detailed comparative analysis of the lipid fractions of DM as well as of human and cow milk, also indicating the distribution of fatty-acid moieties among sn-1/3 and sn-2 positions of the glycerol backbone. In DM the position of fatty acids on glycerol backbone, above all of long chain saturated fatty acids, is very similar to that of human milk: this fact, in conjunction with the relatively high contents of medium-chain triglycerides, makes the lipids in DM, through quantitatively reduced, highly bioavailable. The high PUFA n-3 content of donkey's milk, and especially its low n-6/n-3 ratio, acquires particular interest in subjects affected by cow's milk protein allergy. Whole DM might also constitute the basis for formulas suitable for subjects in the first year of life.

Protein Topology Determines Cysteine Oxidation Fate: The Case of Sulfenyl Amide Formation among Protein Families

PubMed Central

Defelipe, Lucas A.; Lanzarotti, Esteban; Gauto, Diego; Marti, Marcelo A.; Turjanski, Adrián G.

2015-01-01

Cysteine residues have a rich chemistry and play a critical role in the catalytic activity of a plethora of enzymes. However, cysteines are susceptible to oxidation by Reactive Oxygen and Nitrogen Species, leading to a loss of their catalytic function. Therefore, cysteine oxidation is emerging as a relevant physiological regulatory mechanism. Formation of a cyclic sulfenyl amide residue at the active site of redox-regulated proteins has been proposed as a protection mechanism against irreversible oxidation as the sulfenyl amide intermediate has been identified in several proteins. However, how and why only some specific cysteine residues in particular proteins react to form this intermediate is still unknown. In the present work using in-silico based tools, we have identified a constrained conformation that accelerates sulfenyl amide formation. By means of combined MD and QM/MM calculation we show that this conformation positions the NH backbone towards the sulfenic acid and promotes the reaction to yield the sulfenyl amide intermediate, in one step with the concomitant release of a water molecule. Moreover, in a large subset of the proteins we found a conserved beta sheet-loop-helix motif, which is present across different protein folds, that is key for sulfenyl amide production as it promotes the previous formation of sulfenic acid. For catalytic activity, in several cases, proteins need the Cysteine to be in the cysteinate form, i.e. a low pKa Cys. We found that the conserved motif stabilizes the cysteinate by hydrogen bonding to several NH backbone moieties. As cysteinate is also more reactive toward ROS we propose that the sheet-loop-helix motif and the constraint conformation have been selected by evolution for proteins that need a reactive Cys protected from irreversible oxidation. Our results also highlight how fold conservation can be correlated to redox chemistry regulation of protein function. PMID:25741692

Structural basis for the enhanced stability of protein model compounds and peptide backbone unit in ammonium ionic liquids.

PubMed

Vasantha, T; Attri, Pankaj; Venkatesu, Pannuru; Devi, R S Rama

2012-10-04

Protein folding/unfolding is a fascinating study in the presence of cosolvents, which protect/disrupt the native structure of protein, respectively. The structure and stability of proteins and their functional groups may be modulated by the addition of cosolvents. Ionic liquids (ILs) are finding a vast array of applications as novel cosolvents for a wide variety of biochemical processes that include protein folding. Here, the systematic and quantitative apparent transfer free energies (ΔG'(tr)) of protein model compounds from water to ILs through solubility measurements as a function of IL concentration at 25 °C have been exploited to quantify and interpret biomolecular interactions between model compounds of glycine peptides (GPs) with ammonium based ILs. The investigated aqueous systems consist of zwitterionic glycine peptides: glycine (Gly), diglycine (Gly(2)), triglycine (Gly(3)), tetraglycine (Gly(4)), and cyclic glycylglycine (c(GG)) in the presence of six ILs such as diethylammonium acetate (DEAA), diethylammonium hydrogen sulfate (DEAS), triethylammonium acetate (TEAA), triethylammonium hydrogen sulfate (TEAS), triethylammonium dihydrogen phosphate (TEAP), and trimethylammonium acetate (TMAA). We have observed positive values of ΔG'(tr) for GPs from water to ILs, indicating that interactions between ILs and GPs are unfavorable, which leads to stabilization of the structure of model protein compounds. Moreover, our experimental data ΔG'(tr) is used to obtain transfer free energies (Δg'(tr)) of the peptide backbone unit (or glycyl unit) (-CH(2)C═ONH-), which is the most numerous group in globular proteins, from water to IL solutions. To obtain the mechanism events of the ILs' role in enhancing the stability of the model compounds, we have further obtained m-values for GPs from solubility limits. These results explicitly elucidate that all alkyl ammonium ILs act as stabilizers for model compounds through the exclusion of ILs from model compounds of proteins and also reflect the effect of alkyl chain on the stability of protein model compounds.

The Broken Ring: Reduced Aromaticity in Lys-Trp Cations and High pH Tautomer Correlates with Lower Quantum Yield and Shorter Lifetimes

PubMed Central

2015-01-01

Several nonradiative processes compete with tryptophan fluorescence emission. The difficulty in spectral interpretation lies in associating specific molecular environmental features with these processes and thereby utilizing the fluorescence spectral data to identify the local environment of tryptophan. Here, spectroscopic and molecular modeling study of Lys-Trp dipeptide charged species shows that backbone-ring interactions are undistinguished. Instead, quantum mechanical ground state isosurfaces reveal variations in indole π electron distribution and density that parallel charge (as a function of pK1, pK2, and pKR) on the backbone and residues. A pattern of aromaticity-associated quantum yield and fluorescence lifetime changes emerges. Where quantum yield is high, isosurfaces have a charge distribution similar to the highest occupied molecular orbital (HOMO) of indole, which is the dominant fluorescent ground state of the 1La transition dipole moment. Where quantum yield is low, isosurface charge distribution over the ring is uneven, diminished, and even found off ring. At pH 13, the indole amine is deprotonated, and Lys-Trp quantum yield is extremely low due to tautomer structure that concentrates charge on the indole amine; the isosurface charge distribution bears scant resemblance to the indole HOMO. Such greatly diminished fluorescence has been observed for proteins where the indole nitrogen is hydrogen bonded, lending credence to the association of aromaticity changes with diminished quantum yield in proteins as well. Thus tryptophan ground state isosurfaces are an indicator of indole aromaticity, signaling the partition of excitation energy between radiative and nonradiative processes. PMID:24882092

Rosetta FlexPepDock ab-initio: simultaneous folding, docking and refinement of peptides onto their receptors.

PubMed

Raveh, Barak; London, Nir; Zimmerman, Lior; Schueler-Furman, Ora

2011-04-29

Flexible peptides that fold upon binding to another protein molecule mediate a large number of regulatory interactions in the living cell and may provide highly specific recognition modules. We present Rosetta FlexPepDock ab-initio, a protocol for simultaneous docking and de-novo folding of peptides, starting from an approximate specification of the peptide binding site. Using the Rosetta fragments library and a coarse-grained structural representation of the peptide and the receptor, FlexPepDock ab-initio samples efficiently and simultaneously the space of possible peptide backbone conformations and rigid-body orientations over the receptor surface of a given binding site. The subsequent all-atom refinement of the coarse-grained models includes full side-chain modeling of both the receptor and the peptide, resulting in high-resolution models in which key side-chain interactions are recapitulated. The protocol was applied to a benchmark in which peptides were modeled over receptors in either their bound backbone conformations or in their free, unbound form. Near-native peptide conformations were identified in 18/26 of the bound cases and 7/14 of the unbound cases. The protocol performs well on peptides from various classes of secondary structures, including coiled peptides with unusual turns and kinks. The results presented here significantly extend the scope of state-of-the-art methods for high-resolution peptide modeling, which can now be applied to a wide variety of peptide-protein interactions where no prior information about the peptide backbone conformation is available, enabling detailed structure-based studies and manipulation of those interactions. © 2011 Raveh et al.

Rosetta FlexPepDock ab-initio: Simultaneous Folding, Docking and Refinement of Peptides onto Their Receptors

PubMed Central

Raveh, Barak; London, Nir; Zimmerman, Lior; Schueler-Furman, Ora

2011-01-01

Flexible peptides that fold upon binding to another protein molecule mediate a large number of regulatory interactions in the living cell and may provide highly specific recognition modules. We present Rosetta FlexPepDock ab-initio, a protocol for simultaneous docking and de-novo folding of peptides, starting from an approximate specification of the peptide binding site. Using the Rosetta fragments library and a coarse-grained structural representation of the peptide and the receptor, FlexPepDock ab-initio samples efficiently and simultaneously the space of possible peptide backbone conformations and rigid-body orientations over the receptor surface of a given binding site. The subsequent all-atom refinement of the coarse-grained models includes full side-chain modeling of both the receptor and the peptide, resulting in high-resolution models in which key side-chain interactions are recapitulated. The protocol was applied to a benchmark in which peptides were modeled over receptors in either their bound backbone conformations or in their free, unbound form. Near-native peptide conformations were identified in 18/26 of the bound cases and 7/14 of the unbound cases. The protocol performs well on peptides from various classes of secondary structures, including coiled peptides with unusual turns and kinks. The results presented here significantly extend the scope of state-of-the-art methods for high-resolution peptide modeling, which can now be applied to a wide variety of peptide-protein interactions where no prior information about the peptide backbone conformation is available, enabling detailed structure-based studies and manipulation of those interactions. PMID:21572516

Independent Metrics for Protein Backbone and Side-Chain Flexibility: Time Scales and Effects of Ligand Binding.

PubMed

Fuchs, Julian E; Waldner, Birgit J; Huber, Roland G; von Grafenstein, Susanne; Kramer, Christian; Liedl, Klaus R

2015-03-10

Conformational dynamics are central for understanding biomolecular structure and function, since biological macromolecules are inherently flexible at room temperature and in solution. Computational methods are nowadays capable of providing valuable information on the conformational ensembles of biomolecules. However, analysis tools and intuitive metrics that capture dynamic information from in silico generated structural ensembles are limited. In standard work-flows, flexibility in a conformational ensemble is represented through residue-wise root-mean-square fluctuations or B-factors following a global alignment. Consequently, these approaches relying on global alignments discard valuable information on local dynamics. Results inherently depend on global flexibility, residue size, and connectivity. In this study we present a novel approach for capturing positional fluctuations based on multiple local alignments instead of one single global alignment. The method captures local dynamics within a structural ensemble independent of residue type by splitting individual local and global degrees of freedom of protein backbone and side-chains. Dependence on residue type and size in the side-chains is removed via normalization with the B-factors of the isolated residue. As a test case, we demonstrate its application to a molecular dynamics simulation of bovine pancreatic trypsin inhibitor (BPTI) on the millisecond time scale. This allows for illustrating different time scales of backbone and side-chain flexibility. Additionally, we demonstrate the effects of ligand binding on side-chain flexibility of three serine proteases. We expect our new methodology for quantifying local flexibility to be helpful in unraveling local changes in biomolecular dynamics.

Predicting disease-related proteins based on clique backbone in protein-protein interaction network.

PubMed

Yang, Lei; Zhao, Xudong; Tang, Xianglong

2014-01-01

Network biology integrates different kinds of data, including physical or functional networks and disease gene sets, to interpret human disease. A clique (maximal complete subgraph) in a protein-protein interaction network is a topological module and possesses inherently biological significance. A disease-related clique possibly associates with complex diseases. Fully identifying disease components in a clique is conductive to uncovering disease mechanisms. This paper proposes an approach of predicting disease proteins based on cliques in a protein-protein interaction network. To tolerate false positive and negative interactions in protein networks, extending cliques and scoring predicted disease proteins with gene ontology terms are introduced to the clique-based method. Precisions of predicted disease proteins are verified by disease phenotypes and steadily keep to more than 95%. The predicted disease proteins associated with cliques can partly complement mapping between genotype and phenotype, and provide clues for understanding the pathogenesis of serious diseases.

Expanded turn conformations: characterization and sequence-structure correspondence in alpha-turns with implications in helix folding.

PubMed

Dasgupta, Bhaskar; Pal, Lipika; Basu, Gautam; Chakrabarti, Pinak

2004-05-01

Like the beta-turns, which are characterized by a limiting distance between residues two positions apart (i, i+3), a distance criterion (involving residues at positions i and i+4) is used here to identify alpha-turns from a database of known protein structures. At least 15 classes of alpha-turns have been enumerated based on the location in the phi,psi space of the three central residues (i+1 to i+3)-one of the major being the class AAA, where the residues occupy the conventional helical backbone torsion angles. However, moving towards the C-terminal end of the turn, there is a shift in the phi,psi angles towards more negative phi, such that the electrostatic repulsion between two consecutive carbonyl oxygen atoms is reduced. Except for the last position (i+4), there is not much similarity in residue composition at different positions of hydrogen and non-hydrogen bonded AAA turns. The presence or absence of Pro at i+1 position of alpha- and beta-turns has a bearing on whether the turn is hydrogen-bonded or without a hydrogen bond. In the tertiary structure, alpha-turns are more likely to be found in beta-hairpin loops. The residue composition at the beginning of the hydrogen bonded AAA alpha-turn has similarity with type I beta-turn and N-terminal positions of helices, but the last position matches with the C-terminal capping position of helices, suggesting that the existence of a "helix cap signal" at i+4 position prevents alpha-turns from growing into helices. Our results also provide new insights into alpha-helix nucleation and folding. Copyright 2004 Wiley-Liss, Inc.

Backbone and stereospecific (13)C methyl Ile (δ1), Leu and Val side-chain chemical shift assignments of Crc.

PubMed

Sharma, Rakhi; Sahu, Bhubanananda; Ray, Malay K; Deshmukh, Mandar V

2015-04-01

Carbon catabolite repression (CCR) allows bacteria to selectively assimilate a preferred compound among a mixture of several potential carbon sources, thus boosting growth and economizing the cost of adaptability to variable nutrients in the environment. The RNA-binding catabolite repression control (Crc) protein acts as a global post-transcriptional regulator of CCR in Pseudomonas species. Crc triggers repression by inhibiting the expression of genes involved in transport and catabolism of non-preferred substrates, thus indirectly favoring assimilation of preferred one. We report here a nearly complete backbone and stereospecific (13)C methyl side-chain chemical shift assignments of Ile (δ1), Leu and Val of Crc (~ 31 kDa) from Pseudomonas syringae Lz4W.

NMR backbone assignments of the tyrosine kinase domain of human fibroblast growth factor receptor 3 in apo state and in complex with inhibitor PD173074.

PubMed

Sanfelice, Domenico; Koss, Hans; Bunney, Tom D; Thompson, Gary S; Farrell, Brendan; Katan, Matilda; Breeze, Alexander L

2018-03-26

Fibroblast growth factors receptors (FGFR) are transmembrane protein tyrosine kinases involved in many cellular process, including growth, differentiation and angiogenesis. Dysregulation of FGFR enzymatic activity is associated with developmental disorders and cancers; therefore FGFRs have become attractive targets for drug discovery, with a number of agents in late-stage clinical trials. Here, we present the backbone resonance assignments of FGFR3 tyrosine kinase domain in the ligand-free form and in complex with the canonical FGFR kinase inhibitor PD173074. Analysis of chemical shift changes upon inhibitor binding highlights a characteristic pattern of allosteric network perturbations that is of relevance for future drug discovery activities aimed at development of conformationally-selective FGFR inhibitors.

The structure of a thermophilic kinase shapes fitness upon random circular permutation

PubMed Central

Jones, Alicia M.; Mehta, Manan M.; Thomas, Emily E.; Atkinson, Joshua T.; Segall-Shapiro, Thomas H.; Liu, Shirley; Silberg, Jonathan J.

2016-01-01

Proteins can be engineered for synthetic biology through circular permutation, a sequence rearrangement where native protein termini become linked and new termini are created elsewhere through backbone fission. However, it remains challenging to anticipate a protein’s functional tolerance to circular permutation. Here, we describe new transposons for creating libraries of randomly circularly permuted proteins that minimize peptide additions at their termini, and we use transposase mutagenesis to study the tolerance of a thermophilic adenylate kinase (AK) to circular permutation. We find that libraries expressing permuted AK with either short or long peptides amended to their N-terminus yield distinct sets of active variants and present evidence that this trend arises because permuted protein expression varies across libraries. Mapping all sites that tolerate backbone cleavage onto AK structure reveals that the largest contiguous regions of sequence that lack cleavage sites are proximal to the phosphotransfer site. A comparison of our results with a range of structure-derived parameters further showed that retention of function correlates to the strongest extent with the distance to the phosphotransfer site, amino acid variability in an AK family sequence alignment, and residue-level deviations in superimposed AK structures. Our work illustrates how permuted protein libraries can be created with minimal peptide additions using transposase mutagenesis, and they reveal a challenge of maintaining consistent expression across permuted variants in a library that minimizes peptide additions. Furthermore, these findings provide a basis for interpreting responses of thermophilic phosphotransferases to circular permutation by calibrating how different structure-derived parameters relate to retention of function in a cellular selection. PMID:26976658

PERMutation Using Transposase Engineering (PERMUTE): A Simple Approach for Constructing Circularly Permuted Protein Libraries.

PubMed

Jones, Alicia M; Atkinson, Joshua T; Silberg, Jonathan J

2017-01-01

Rearrangements that alter the order of a protein's sequence are used in the lab to study protein folding, improve activity, and build molecular switches. One of the simplest ways to rearrange a protein sequence is through random circular permutation, where native protein termini are linked together and new termini are created elsewhere through random backbone fission. Transposase mutagenesis has emerged as a simple way to generate libraries encoding different circularly permuted variants of proteins. With this approach, a synthetic transposon (called a permuteposon) is randomly inserted throughout a circularized gene to generate vectors that express different permuted variants of a protein. In this chapter, we outline the protocol for constructing combinatorial libraries of circularly permuted proteins using transposase mutagenesis, and we describe the different permuteposons that have been developed to facilitate library construction.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dyer, David H.; Vyazunova, Irina; Lorch, Jeffery M.

The sterol carrier protein-2 like 3 gene (AeSCP-2L3), a new member of the SCP-2 protein family, is identified from the yellow fever mosquito, Aedes aegypti. The predicted molecular weight of AeSCP-2L3 is 13.4 kDa with a calculated pI of 4.98. AeSCP-2L3 transcription occurs in the larval feeding stages and the mRNA levels decrease in pupae and adults. The highest levels of AeSCP-2L3 gene expression are found in the body wall, and possibly originated in the fat body. This is the first report of a mosquito SCP-2-like protein with prominent expression in tissue other than the midgut. The X-ray protein crystalmore » structure of AeSCP-2L3 reveals a bound C16 fatty acid whose acyl tail penetrates deeply into a hydrophobic cavity. Interestingly, the ligand-binding cavity is slightly larger than previously described for AeSCP-2 (Dyer et al. J Biol Chem 278:39085-39091, 2003) and AeSCP-2L2 (Dyer et al. J Lipid Res M700460-JLR200, 2007). There are also an additional 10 amino acids in SCP-2L3 that are not present in other characterized mosquito SCP-2s forming an extended loop between {beta}3 and {beta}4. Otherwise, the protein backbone is exceedingly similar to other SCP-2 and SCP-2-like proteins. In contrast to this observed high structural homology of members in the mosquito SCP2 family, the amino acid sequence identity between the members is less than 30%. The results from structural analysis imply that there have been evolutionary constraints that favor the SCP-2 C{alpha} backbone fold while the specificity of ligand binding can be altered.« less

15N backbone dynamics of the S-peptide from ribonuclease A in its free and S-protein bound forms: toward a site-specific analysis of entropy changes upon folding.

PubMed Central

Alexandrescu, A. T.; Rathgeb-Szabo, K.; Rumpel, K.; Jahnke, W.; Schulthess, T.; Kammerer, R. A.

1998-01-01

Backbone 15N relaxation parameters (R1, R2, 1H-15N NOE) have been measured for a 22-residue recombinant variant of the S-peptide in its free and S-protein bound forms. NMR relaxation data were analyzed using the "model-free" approach (Lipari & Szabo, 1982). Order parameters obtained from "model-free" simulations were used to calculate 1H-15N bond vector entropies using a recently described method (Yang & Kay, 1996), in which the form of the probability density function for bond vector fluctuations is derived from a diffusion-in-a-cone motional model. The average change in 1H-15N bond vector entropies for residues T3-S15, which become ordered upon binding of the S-peptide to the S-protein, is -12.6+/-1.4 J/mol.residue.K. 15N relaxation data suggest a gradient of decreasing entropy values moving from the termini toward the center of the free peptide. The difference between the entropies of the terminal and central residues is about -12 J/mol residue K, a value comparable to that of the average entropy change per residue upon complex formation. Similar entropy gradients are evident in NMR relaxation studies of other denatured proteins. Taken together, these observations suggest denatured proteins may contain entropic contributions from non-local interactions. Consequently, calculations that model the entropy of a residue in a denatured protein as that of a residue in a di- or tri-peptide, might over-estimate the magnitude of entropy changes upon folding. PMID:9521116

An all-atom structure-based potential for proteins: bridging minimal models with all-atom empirical forcefields.

PubMed

Whitford, Paul C; Noel, Jeffrey K; Gosavi, Shachi; Schug, Alexander; Sanbonmatsu, Kevin Y; Onuchic, José N

2009-05-01

Protein dynamics take place on many time and length scales. Coarse-grained structure-based (Go) models utilize the funneled energy landscape theory of protein folding to provide an understanding of both long time and long length scale dynamics. All-atom empirical forcefields with explicit solvent can elucidate our understanding of short time dynamics with high energetic and structural resolution. Thus, structure-based models with atomic details included can be used to bridge our understanding between these two approaches. We report on the robustness of folding mechanisms in one such all-atom model. Results for the B domain of Protein A, the SH3 domain of C-Src Kinase, and Chymotrypsin Inhibitor 2 are reported. The interplay between side chain packing and backbone folding is explored. We also compare this model to a C(alpha) structure-based model and an all-atom empirical forcefield. Key findings include: (1) backbone collapse is accompanied by partial side chain packing in a cooperative transition and residual side chain packing occurs gradually with decreasing temperature, (2) folding mechanisms are robust to variations of the energetic parameters, (3) protein folding free-energy barriers can be manipulated through parametric modifications, (4) the global folding mechanisms in a C(alpha) model and the all-atom model agree, although differences can be attributed to energetic heterogeneity in the all-atom model, and (5) proline residues have significant effects on folding mechanisms, independent of isomerization effects. Because this structure-based model has atomic resolution, this work lays the foundation for future studies to probe the contributions of specific energetic factors on protein folding and function.

An All-atom Structure-Based Potential for Proteins: Bridging Minimal Models with All-atom Empirical Forcefields

PubMed Central

Whitford, Paul C.; Noel, Jeffrey K.; Gosavi, Shachi; Schug, Alexander; Sanbonmatsu, Kevin Y.; Onuchic, José N.

2012-01-01

Protein dynamics take place on many time and length scales. Coarse-grained structure-based (Gō) models utilize the funneled energy landscape theory of protein folding to provide an understanding of both long time and long length scale dynamics. All-atom empirical forcefields with explicit solvent can elucidate our understanding of short time dynamics with high energetic and structural resolution. Thus, structure-based models with atomic details included can be used to bridge our understanding between these two approaches. We report on the robustness of folding mechanisms in one such all-atom model. Results for the B domain of Protein A, the SH3 domain of C-Src Kinase and Chymotrypsin Inhibitor 2 are reported. The interplay between side chain packing and backbone folding is explored. We also compare this model to a Cα structure-based model and an all-atom empirical forcefield. Key findings include 1) backbone collapse is accompanied by partial side chain packing in a cooperative transition and residual side chain packing occurs gradually with decreasing temperature 2) folding mechanisms are robust to variations of the energetic parameters 3) protein folding free energy barriers can be manipulated through parametric modifications 4) the global folding mechanisms in a Cα model and the all-atom model agree, although differences can be attributed to energetic heterogeneity in the all-atom model 5) proline residues have significant effects on folding mechanisms, independent of isomerization effects. Since this structure-based model has atomic resolution, this work lays the foundation for future studies to probe the contributions of specific energetic factors on protein folding and function. PMID:18837035

Technological advances in site-directed spin labeling of proteins.

PubMed

Hubbell, Wayne L; López, Carlos J; Altenbach, Christian; Yang, Zhongyu

2013-10-01

Molecular flexibility over a wide time range is of central importance to the function of many proteins, both soluble and membrane. Revealing the modes of flexibility, their amplitudes, and time scales under physiological conditions is the challenge for spectroscopic methods, one of which is site-directed spin labeling EPR (SDSL-EPR). Here we provide an overview of some recent technological advances in SDSL-EPR related to investigation of structure, structural heterogeneity, and dynamics of proteins. These include new classes of spin labels, advances in measurement of long range distances and distance distributions, methods for identifying backbone and conformational fluctuations, and new strategies for determining the kinetics of protein motion. Copyright © 2013 Elsevier Ltd. All rights reserved.

NMR-based automated protein structure determination.

PubMed

Würz, Julia M; Kazemi, Sina; Schmidt, Elena; Bagaria, Anurag; Güntert, Peter

2017-08-15

NMR spectra analysis for protein structure determination can now in many cases be performed by automated computational methods. This overview of the computational methods for NMR protein structure analysis presents recent automated methods for signal identification in multidimensional NMR spectra, sequence-specific resonance assignment, collection of conformational restraints, and structure calculation, as implemented in the CYANA software package. These algorithms are sufficiently reliable and integrated into one software package to enable the fully automated structure determination of proteins starting from NMR spectra without manual interventions or corrections at intermediate steps, with an accuracy of 1-2 Å backbone RMSD in comparison with manually solved reference structures. Copyright © 2017 Elsevier Inc. All rights reserved.

Further optimization of a hybrid united-atom and coarse-grained force field for folding simulations: Improved backbone hydration and interactions between charged side chains

PubMed Central

Han, Wei; Schulten, Klaus

2012-01-01

PACE, a hybrid force field which couples united-atom protein models with coarse-grained (CG) solvent, has been further optimized, aiming to improve itse ciency for folding simulations. Backbone hydration parameters have been re-optimized based on hydration free energies of polyalanyl peptides through atomistic simulations. Also, atomistic partial charges from all-atom force fields were combined with PACE in order to provide a more realistic description of interactions between charged groups. Using replica exchange molecular dynamics (REMD), ab initio folding using the new PACE has been achieved for seven small proteins (16 – 23 residues) with different structural motifs. Experimental data about folded states, such as their stability at room temperature, melting point and NMR NOE constraints, were also well reproduced. Moreover, a systematic comparison of folding kinetics at room temperature has been made with experiments, through standard MD simulations, showing that the new PACE may speed up the actual folding kinetics 5-10 times. Together with the computational speedup benefited from coarse-graining, the force field provides opportunities to study folding mechanisms. In particular, we used the new PACE to fold a 73-residue protein, 3D, in multiple 10 – 30 μs simulations, to its native states (Cα RMSD ~ 0.34 nm). Our results suggest the potential applicability of the new PACE for the study of folding and dynamics of proteins. PMID:23204949

Electronic polarization stabilizes tertiary structure prediction of HP-36.

PubMed

Duan, Li L; Zhu, Tong; Zhang, Qing G; Tang, Bo; Zhang, John Z H

2014-04-01

Molecular dynamic (MD) simulations with both implicit and explicit solvent models have been carried out to study the folding dynamics of HP-36 protein. Starting from the extended conformation, the secondary structure of all three helices in HP-36 was formed in about 50 ns and remained stable in the remaining simulation. However, the formation of the tertiary structure was difficult. Although some intermediates were close to the native structure, the overall conformation was not stable. Further analysis revealed that the large structure fluctuation of loop and hydrophobic core regions was devoted mostly to the instability of the structure during MD simulation. The backbone root-mean-square deviation (RMSD) of the loop and hydrophobic core regions showed strong correlation with the backbone RMSD of the whole protein. The free energy landscape indicated that the distribution of main chain torsions in loop and turn regions was far away from the native state. Starting from an intermediate structure extracted from the initial AMBER simulation, HP-36 was found to generally fold to the native state under the dynamically adjusted polarized protein-specific charge (DPPC) simulation, while the peptide did not fold into the native structure when AMBER force filed was used. The two best folded structures were extracted and taken into further simulations in water employing AMBER03 charge and DPPC for 25 ns. Result showed that introducing polarization effect into interacting potential could stabilize the near-native protein structure.

GRID: a high-resolution protein structure refinement algorithm.

PubMed

Chitsaz, Mohsen; Mayo, Stephen L

2013-03-05

The energy-based refinement of protein structures generated by fold prediction algorithms to atomic-level accuracy remains a major challenge in structural biology. Energy-based refinement is mainly dependent on two components: (1) sufficiently accurate force fields, and (2) efficient conformational space search algorithms. Focusing on the latter, we developed a high-resolution refinement algorithm called GRID. It takes a three-dimensional protein structure as input and, using an all-atom force field, attempts to improve the energy of the structure by systematically perturbing backbone dihedrals and side-chain rotamer conformations. We compare GRID to Backrub, a stochastic algorithm that has been shown to predict a significant fraction of the conformational changes that occur with point mutations. We applied GRID and Backrub to 10 high-resolution (≤ 2.8 Å) crystal structures from the Protein Data Bank and measured the energy improvements obtained and the computation times required to achieve them. GRID resulted in energy improvements that were significantly better than those attained by Backrub while expending about the same amount of computational resources. GRID resulted in relaxed structures that had slightly higher backbone RMSDs compared to Backrub relative to the starting crystal structures. The average RMSD was 0.25 ± 0.02 Å for GRID versus 0.14 ± 0.04 Å for Backrub. These relatively minor deviations indicate that both algorithms generate structures that retain their original topologies, as expected given the nature of the algorithms. Copyright © 2012 Wiley Periodicals, Inc.

13C multiplet nuclear magnetic resonance relaxation-derived ring puckering and backbone dynamics in proline-containing glycine-based peptides.

PubMed Central

Mikhailov, D; Daragan, V A; Mayo, K H

1995-01-01

13CH2-multiplet nuclear magnetic resonance relaxation studies on proline (P)-containing glycine (G)-based peptides, GP, PG, GPG, PGG, and GPGG, provided numerous dipolar auto- and cross-correlation times for various motional model analyses of backbone and proline-ring bond rotations. Molecular dynamics simulations and bond rotation energy profiles were calculated to assess which motions could contribute most to observed relaxation phenomena. Results indicate that proline restricts backbone psi 1, psi 2, and phi 2 motions by 50% relative to those found for a polyglycine control peptide. psi 1 rotations are more restricted in the trans-proline isomer state than in the cis form. A two-state jump model best approximates proline ring puckering which in water could occur either by the C gamma endo-exo or by the C2 interconversion mechanism. The temperature dependence (5 degrees to 75 degrees C) of C beta, and C gamma, and C delta angular changes is rather flat, suggesting a near zero enthalpic contribution to the ring puckering process. In lower dielectric solvents, dimethylsulfoxide and methanol, which may mimic the hydrophobic environment within a protein, the endo-exo mechanism is preferred. PMID:7787039

Gum arabic glycoprotein is a twisted hairy rope. A new model based on O-galactosylhydroxyproline as the polysaccharide attachment site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu Qi; Fong, C.; Lamport, D.T.A.

1991-07-01

Separation of the wound exudate from Acacia senegal (L.) Willd., gum arabic, on a preparative Superose-6 column gave two major fractions: a high molecular weight gum arabic glyco-protein (GAGP) containing about 90% carbohydrate and a lower molecular weight heterogeneous gum arabic polysaccharide fraction. Hydrogen fluoride-deglycosylation of GAGP gave a large hydroxyproline-rich polypeptide backbone (dGAGP). Alkaline hydrolysis of GAGP showed that most of the carbohydrate was attached to the polypeptide backbone as small hydroxyproline (Hyp)-polysaccharide substituents. The data imply a rodlike molecule with numerous small polysaccharide substituents (attached to 24% of the Hyp residues), regularly arranged along a highly periodic polypeptidemore » backbone based, hypothetically, on a 10 to 12 residue repetitive peptide motif. Thus, a simple statistical model of the gum arabic glycoprotein predicts a repeating polysaccharide substituents will maximize intramolecular hydrogen bonding if aligned along the long axis of the molecule, forming in effect a twisted hairy rope. Electron micrographs of rotary shadowed GAGP molecules support that prediction and may also explain show such apparently large molecules can exit the cell by endwise reptation through the small pores of the primary cell wall.« less

Principal component analysis of molecular dynamics: On the use of Cartesian vs. internal coordinates

NASA Astrophysics Data System (ADS)

Sittel, Florian; Jain, Abhinav; Stock, Gerhard

2014-07-01

Principal component analysis of molecular dynamics simulations is a popular method to account for the essential dynamics of the system on a low-dimensional free energy landscape. Using Cartesian coordinates, first the translation and overall rotation need to be removed from the trajectory. Since the rotation depends via the moment of inertia on the molecule's structure, this separation is only straightforward for relatively rigid systems. Adopting millisecond molecular dynamics simulations of the folding of villin headpiece and the functional dynamics of BPTI provided by D. E. Shaw Research, it is demonstrated via a comparison of local and global rotational fitting that the structural dynamics of flexible molecules necessarily results in a mixing of overall and internal motion. Even for the small-amplitude functional motion of BPTI, the conformational distribution obtained from a Cartesian principal component analysis therefore reflects to some extend the dominant overall motion rather than the much smaller internal motion of the protein. Internal coordinates such as backbone dihedral angles, on the other hand, are found to yield correct and well-resolved energy landscapes for both examples. The virtues and shortcomings of the choice of various fitting schemes and coordinate sets as well as the generality of these results are discussed in some detail.

Principal component analysis of molecular dynamics: on the use of Cartesian vs. internal coordinates.

PubMed

Sittel, Florian; Jain, Abhinav; Stock, Gerhard

2014-07-07

Principal component analysis of molecular dynamics simulations is a popular method to account for the essential dynamics of the system on a low-dimensional free energy landscape. Using Cartesian coordinates, first the translation and overall rotation need to be removed from the trajectory. Since the rotation depends via the moment of inertia on the molecule's structure, this separation is only straightforward for relatively rigid systems. Adopting millisecond molecular dynamics simulations of the folding of villin headpiece and the functional dynamics of BPTI provided by D. E. Shaw Research, it is demonstrated via a comparison of local and global rotational fitting that the structural dynamics of flexible molecules necessarily results in a mixing of overall and internal motion. Even for the small-amplitude functional motion of BPTI, the conformational distribution obtained from a Cartesian principal component analysis therefore reflects to some extend the dominant overall motion rather than the much smaller internal motion of the protein. Internal coordinates such as backbone dihedral angles, on the other hand, are found to yield correct and well-resolved energy landscapes for both examples. The virtues and shortcomings of the choice of various fitting schemes and coordinate sets as well as the generality of these results are discussed in some detail.

Exploring TAR–RNA aptamer loop–loop interaction by X-ray crystallography, UV spectroscopy and surface plasmon resonance

PubMed Central

Lebars, Isabelle; Legrand, Pierre; Aimé, Ahissan; Pinaud, Noël; Fribourg, Sébastien; Di Primo, Carmelo

2008-01-01

In HIV-1, trans-activation of transcription of the viral genome is regulated by an imperfect hairpin, the trans-activating responsive (TAR) RNA element, located at the 5′ untranslated end of all viral transcripts. TAR acts as a binding site for viral and cellular proteins. In an attempt to identify RNA ligands that would interfere with the virus life-cycle by interacting with TAR, an in vitro selection was previously carried out. RNA hairpins that formed kissing-loop dimers with TAR were selected [Ducongé F. and Toulmé JJ (1999) RNA, 5:1605–1614]. We describe here the crystal structure of TAR bound to a high-affinity RNA aptamer. The two hairpins form a kissing complex and interact through six Watson–Crick base pairs. The complex adopts an overall conformation with an inter-helix angle of 28.1°, thus contrasting with previously reported solution and modelling studies. Structural analysis reveals that inter-backbone hydrogen bonds between ribose 2′ hydroxyl and phosphate oxygens at the stem-loop junctions can be formed. Thermal denaturation and surface plasmon resonance experiments with chemically modified 2′-O-methyl incorporated into both hairpins at key positions, clearly demonstrate the involvement of this intermolecular network of hydrogen bonds in complex stability. PMID:18996893

Resting myosin cross-bridge configuration in frog muscle thick filaments.

PubMed

Cantino, M; Squire, J

1986-02-01

Clear images of myosin filaments have been seen in shadowed freeze-fracture replicas of single fibers of relaxed frog semitendinosus muscles rapidly frozen using a dual propane jet freezing device. These images have been analyzed by optical diffraction and computer averaging and have been modelled to reveal details of the myosin head configuration on the right-handed, three-stranded helix of cross-bridges. Both the characteristic 430-A and 140-150-A repeats of the myosin cross-bridge array could be seen. The measured filament backbone diameter was 140-160 A, and the outer diameter of the cross-bridge array was 300 A. Evidence is presented that suggests that the observed images are consistent with a model in which both of the heads of one myosin molecule tilt in the same direction at an angle of approximately 50-70 degrees to the normal to the filament long axis and are slewed so that they lie alongside each other and their radially projected density lies along the three right-handed helical tracks. Any perturbation of the myosin heads away from their ideal lattice sites needed to account for x-ray reflections not predicted for a perfect helix must be essentially along the three helical tracks of cross-bridges. Little trace of the presence of non-myosin proteins could be seen.

Arabidopsis GUX Proteins Are Glucuronyltransferases Responsible for the Addition of Glucuronic Acid Side Chains onto Xylan

EPA Science Inventory

Xylan, the second most abundant cell wall polysaccharide, is composed of a linear backbone of β-(1,4)-linked xylosyl residues that are often substituted with sugar side chains, such as glucuronic acid (GlcA) and methylglucuronic acid (MeGlcA). It has recently been shown that muta...

Near-Native Protein Loop Sampling Using Nonparametric Density Estimation Accommodating Sparcity

PubMed Central

Day, Ryan; Lennox, Kristin P.; Sukhanov, Paul; Dahl, David B.; Vannucci, Marina; Tsai, Jerry

2011-01-01

Unlike the core structural elements of a protein like regular secondary structure, template based modeling (TBM) has difficulty with loop regions due to their variability in sequence and structure as well as the sparse sampling from a limited number of homologous templates. We present a novel, knowledge-based method for loop sampling that leverages homologous torsion angle information to estimate a continuous joint backbone dihedral angle density at each loop position. The φ,ψ distributions are estimated via a Dirichlet process mixture of hidden Markov models (DPM-HMM). Models are quickly generated based on samples from these distributions and were enriched using an end-to-end distance filter. The performance of the DPM-HMM method was evaluated against a diverse test set in a leave-one-out approach. Candidates as low as 0.45 Å RMSD and with a worst case of 3.66 Å were produced. For the canonical loops like the immunoglobulin complementarity-determining regions (mean RMSD <2.0 Å), the DPM-HMM method performs as well or better than the best templates, demonstrating that our automated method recaptures these canonical loops without inclusion of any IgG specific terms or manual intervention. In cases with poor or few good templates (mean RMSD >7.0 Å), this sampling method produces a population of loop structures to around 3.66 Å for loops up to 17 residues. In a direct test of sampling to the Loopy algorithm, our method demonstrates the ability to sample nearer native structures for both the canonical CDRH1 and non-canonical CDRH3 loops. Lastly, in the realistic test conditions of the CASP9 experiment, successful application of DPM-HMM for 90 loops from 45 TBM targets shows the general applicability of our sampling method in loop modeling problem. These results demonstrate that our DPM-HMM produces an advantage by consistently sampling near native loop structure. The software used in this analysis is available for download at http://www.stat.tamu.edu/~dahl/software/cortorgles/. PMID:22028638

Native Tandem and Ion Mobility Mass Spectrometry Highlight Structural and Modular Similarities in Clustered-Regularly-Interspaced Shot-Palindromic-Repeats (CRISPR)-associated Protein Complexes From Escherichia coli and Pseudomonas aeruginosa*

PubMed Central

van Duijn, Esther; Barbu, Ioana M.; Barendregt, Arjan; Jore, Matthijs M.; Wiedenheft, Blake; Lundgren, Magnus; Westra, Edze R.; Brouns, Stan J. J.; Doudna, Jennifer A.; van der Oost, John; Heck, Albert J. R.

2012-01-01

The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) immune system of bacteria and archaea provides acquired resistance against viruses and plasmids, by a strategy analogous to RNA-interference. Key components of the defense system are ribonucleoprotein complexes, the composition of which appears highly variable in different CRISPR/Cas subtypes. Previous studies combined mass spectrometry, electron microscopy, and small angle x-ray scattering to demonstrate that the E. coli Cascade complex (405 kDa) and the P. aeruginosa Csy-complex (350 kDa) are similar in that they share a central spiral-shaped hexameric structure, flanked by associating proteins and one CRISPR RNA. Recently, a cryo-electron microscopy structure of Cascade revealed that the CRISPR RNA molecule resides in a groove of the hexameric backbone. For both complexes we here describe the use of native mass spectrometry in combination with ion mobility mass spectrometry to assign a stable core surrounded by more loosely associated modules. Via computational modeling subcomplex structures were proposed that relate to the experimental IMMS data. Despite the absence of obvious sequence homology between several subunits, detailed analysis of sub-complexes strongly suggests analogy between subunits of the two complexes. Probing the specific association of E. coli Cascade/crRNA to its complementary DNA target reveals a conformational change. All together these findings provide relevant new information about the potential assembly process of the two CRISPR-associated complexes. PMID:22918228

NMR Studies of the C-Terminus of alpha4 Reveal Possible Mechanism of Its Interaction with MID1 and Protein Phosphatase 2A

PubMed Central

Du, Haijuan; Massiah, Michael A.

2011-01-01

Alpha4 is a regulatory subunit of the protein phosphatase family of enzymes and plays an essential role in regulating the catalytic subunit of PP2A (PP2Ac) within the rapamycin-sensitive signaling pathway. Alpha4 also interacts with MID1, a microtubule-associated ubiquitin E3 ligase that appears to regulate the function of PP2A. The C-terminal region of alpha4 plays a key role in the binding interaction of PP2Ac and MID1. Here we report on the solution structure of a 45-amino acid region derived from the C-terminus of alpha4 (alpha45) that binds tightly to MID1. In aqueous solution, alpha45 has properties of an intrinsically unstructured peptide although chemical shift index and dihedral angle estimation based on chemical shifts of backbone atoms indicate the presence of a transient α-helix. Alpha45 adopts a helix-turn-helix HEAT-like structure in 1% SDS micelles, which may mimic a negatively charged surface for which alpha45 could bind. Alpha45 binds tightly to the Bbox1 domain of MID1 in aqueous solution and adopts a structure consistent with the helix-turn-helix structure observed in 1% SDS. The structure of alpha45 reveals two distinct surfaces, one that can interact with a negatively charged surface, which is present on PP2A, and one that interacts with the Bbox1 domain of MID1. PMID:22194938

Structure and Dynamics Analysis on Plexin-B1 Rho GTPase Binding Domain as a Monomer and Dimer

PubMed Central

2015-01-01

Plexin-B1 is a single-pass transmembrane receptor. Its Rho GTPase binding domain (RBD) can associate with small Rho GTPases and can also self-bind to form a dimer. In total, more than 400 ns of NAMD molecular dynamics simulations were performed on RBD monomer and dimer. Different analysis methods, such as root mean squared fluctuation (RMSF), order parameters (S2), dihedral angle correlation, transfer entropy, principal component analysis, and dynamical network analysis, were carried out to characterize the motions seen in the trajectories. RMSF results show that after binding, the L4 loop becomes more rigid, but the L2 loop and a number of residues in other regions become slightly more flexible. Calculating order parameters (S2) for CH, NH, and CO bonds on both backbone and side chain shows that the L4 loop becomes essentially rigid after binding, but part of the L1 loop becomes slightly more flexible. Backbone dihedral angle cross-correlation results show that loop regions such as the L1 loop including residues Q25 and G26, the L2 loop including residue R61, and the L4 loop including residues L89–R91, are highly correlated compared to other regions in the monomer form. Analysis of the correlated motions at these residues, such as Q25 and R61, indicate two signal pathways. Transfer entropy calculations on the RBD monomer and dimer forms suggest that the binding process should be driven by the L4 loop and C-terminal. However, after binding, the L4 loop functions as the motion responder. The signal pathways in RBD were predicted based on a dynamical network analysis method using the pathways predicted from the dihedral angle cross-correlation calculations as input. It is found that the shortest pathways predicted from both inputs can overlap, but signal pathway 2 (from F90 to R61) is more dominant and overlaps all of the routes of pathway 1 (from F90 to P111). This project confirms the allosteric mechanism in signal transmission inside the RBD network, which was in part proposed in the previous experimental study. PMID:24901636

Highly optical transparency and thermally stable polyimides containing pyridine and phenyl pendant.

PubMed

Yao, Jianan; Wang, Chunbo; Tian, Chengshuo; Zhao, Xiaogang; Zhou, Hongwei; Wang, Daming; Chen, Chunhai

2017-01-01

In order to obtain highly optical transparency polyimides, two novel aromatic diamine monomers containing pyridine and kinky structures, 1,1-bis[4-(5-amino-2-pyridinoxy)phenyl]diphenylmethane (BAPDBP) and 1,1-bis[4-(5-amino-2-pyridinoxy)phenyl]-1-phenylethane (BAPDAP), were designed and synthesized. Polyimides based on BAPDBP, BAPDAP, 2,2-bis[4-(5-amino-2-pyridinoxy)phenyl]propane (BAPDP) with various commercial dianhydrides were prepared for comparison and structure-property relationships study. The structures of the polyimides were characterized by Fourier transform infrared (FT-IR) spectrometer, wide-angle X-ray diffractograms (XRD) and elemental analysis. Film properties including solubility, optical transparency, water uptake, thermal and mechanical properties were also evaluated. The introduction of pyridine and kinky structure into the backbones that polyimides presented good optical properties with 91-97% transparent at 500 nm and a low cut-off wavelength at 353-398 nm. Moreover, phenyl pendant groups of the polyimides showed high glass transition temperatures ( T g ) in the range of 257-281 °C. These results suggest that the incorporating pyridine, kinky and bulky substituents to polymer backbone can improve the optical transparency effectively without sacrificing the thermal properties.

Complex lasso: new entangled motifs in proteins

NASA Astrophysics Data System (ADS)

Niemyska, Wanda; Dabrowski-Tumanski, Pawel; Kadlof, Michal; Haglund, Ellinor; Sułkowski, Piotr; Sulkowska, Joanna I.

2016-11-01

We identify new entangled motifs in proteins that we call complex lassos. Lassos arise in proteins with disulfide bridges (or in proteins with amide linkages), when termini of a protein backbone pierce through an auxiliary surface of minimal area, spanned on a covalent loop. We find that as much as 18% of all proteins with disulfide bridges in a non-redundant subset of PDB form complex lassos, and classify them into six distinct geometric classes, one of which resembles supercoiling known from DNA. Based on biological classification of proteins we find that lassos are much more common in viruses, plants and fungi than in other kingdoms of life. We also discuss how changes in the oxidation/reduction potential may affect the function of proteins with lassos. Lassos and associated surfaces of minimal area provide new, interesting and possessing many potential applications geometric characteristics not only of proteins, but also of other biomolecules.

CADB: Conformation Angles DataBase of proteins

PubMed Central

Sheik, S. S.; Ananthalakshmi, P.; Bhargavi, G. Ramya; Sekar, K.

2003-01-01

Conformation Angles DataBase (CADB) provides an online resource to access data on conformation angles (both main-chain and side-chain) of protein structures in two data sets corresponding to 25% and 90% sequence identity between any two proteins, available in the Protein Data Bank. In addition, the database contains the necessary crystallographic parameters. The package has several flexible options and display facilities to visualize the main-chain and side-chain conformation angles for a particular amino acid residue. The package can also be used to study the interrelationship between the main-chain and side-chain conformation angles. A web based JAVA graphics interface has been deployed to display the user interested information on the client machine. The database is being updated at regular intervals and can be accessed over the World Wide Web interface at the following URL: http://144.16.71.148/cadb/. PMID:12520049

BetaSCPWeb: side-chain prediction for protein structures using Voronoi diagrams and geometry prioritization

PubMed Central

Ryu, Joonghyun; Lee, Mokwon; Cha, Jehyun; Laskowski, Roman A.; Ryu, Seong Eon; Kim, Deok-Soo

2016-01-01

Many applications, such as protein design, homology modeling, flexible docking, etc. require the prediction of a protein's optimal side-chain conformations from just its amino acid sequence and backbone structure. Side-chain prediction (SCP) is an NP-hard energy minimization problem. Here, we present BetaSCPWeb which efficiently computes a conformation close to optimal using a geometry-prioritization method based on the Voronoi diagram of spherical atoms. Its outputs are visual, textual and PDB file format. The web server is free and open to all users at http://voronoi.hanyang.ac.kr/betascpweb with no login requirement. PMID:27151195

Structural insights into the evolution of a sexy protein: novel topology and restricted backbone flexibility in a hypervariable pheromone from the red-legged salamander, Plethodon shermani.

PubMed

Wilburn, Damien B; Bowen, Kathleen E; Doty, Kari A; Arumugam, Sengodagounder; Lane, Andrew N; Feldhoff, Pamela W; Feldhoff, Richard C

2014-01-01

In response to pervasive sexual selection, protein sex pheromones often display rapid mutation and accelerated evolution of corresponding gene sequences. For proteins, the general dogma is that structure is maintained even as sequence or function may rapidly change. This phenomenon is well exemplified by the three-finger protein (TFP) superfamily: a diverse class of vertebrate proteins co-opted for many biological functions - such as components of snake venoms, regulators of the complement system, and coordinators of amphibian limb regeneration. All of the >200 structurally characterized TFPs adopt the namesake "three-finger" topology. In male red-legged salamanders, the TFP pheromone Plethodontid Modulating Factor (PMF) is a hypervariable protein such that, through extensive gene duplication and pervasive sexual selection, individual male salamanders express more than 30 unique isoforms. However, it remained unclear how this accelerated evolution affected the protein structure of PMF. Using LC/MS-MS and multidimensional NMR, we report the 3D structure of the most abundant PMF isoform, PMF-G. The high resolution structural ensemble revealed a highly modified TFP structure, including a unique disulfide bonding pattern and loss of secondary structure, that define a novel protein topology with greater backbone flexibility in the third peptide finger. Sequence comparison, models of molecular evolution, and homology modeling together support that this flexible third finger is the most rapidly evolving segment of PMF. Combined with PMF sequence hypervariability, this structural flexibility may enhance the plasticity of PMF as a chemical signal by permitting potentially thousands of structural conformers. We propose that the flexible third finger plays a critical role in PMF:receptor interactions. As female receptors co-evolve, this flexibility may allow PMF to still bind its receptor(s) without the immediate need for complementary mutations. Consequently, this unique adaptation may establish new paradigms for how receptor:ligand pairs co-evolve, in particular with respect to sexual conflict.

Dynamics simulation on the flexibility and backbone motions of HP1 chromodomain bound to free and lysine 9-methylated histone H3 tails

NASA Astrophysics Data System (ADS)

Jiang, Yanke; Zou, Jianwei; Zeng, Min; Zhang, Na; Yu, Qingsen

Histone methylation has emerged as a central epigenetic modification with both activating and repressive roles in eukaryotic chromatin. Drosophila HP1 (heterochromatin-associated protein 1) is one of the chromodomain proteins that contain the essential aromatic residues as the recognition pocket for lysine methylated histone H3 tail. The aromatic cage indicates that the complex of chromodomain protein binding lysine methylated histone H3 tail can be seen as a typical host-guest system between protein and protein. About 10-ns molecular dynamics simulations have been carried out in this study to examine how the presence of mono-, trimethylated lysine 9 histone H3 tail (Me1K9, Me3K9 H3) influences the motions of HP1 protein receptor. The study shows that the conformation of HP1 protein free of H3 tail easily changes, whereas that of HP1 protein bound to methylated H3 tail does not. But the conformation of inserted Me1K9 H3 changes obviously as the Me1K recognition makes hydrogen-bonded interactions associated with the aromatic cage even more unstable than those in free HP1 protein. The conformational change of Me1K9 H3 is correlated with the motions of HP1 protein. As the recognition factor going from Me1K to Me3K produces a more favorable interaction for aromatic ring, hydrogen-bonded interactions associated with aromatic cage in Me3K9 H3-HP1 complex were observed to be much more stable than those in Me1K9 H3-HP1 complex and free HP1. Because of correlation, the flexibility of Me3K9 H3 decreases. The simulations indicate that both the MeK and the surrounding histone tail sequence are necessary features of recognition which significantly affect the flexibility and backbone motions of HP1 chromodomain. These findings confirm a regulatory mechanism of protein-protein interactions through a trimethylated post-translational modification.

Structure-Based Druggability Assessment of the Mammalian Structural Proteome with Inclusion of Light Protein Flexibility

PubMed Central

Loving, Kathryn A.; Lin, Andy; Cheng, Alan C.

2014-01-01

Advances reported over the last few years and the increasing availability of protein crystal structure data have greatly improved structure-based druggability approaches. However, in practice, nearly all druggability estimation methods are applied to protein crystal structures as rigid proteins, with protein flexibility often not directly addressed. The inclusion of protein flexibility is important in correctly identifying the druggability of pockets that would be missed by methods based solely on the rigid crystal structure. These include cryptic pockets and flexible pockets often found at protein-protein interaction interfaces. Here, we apply an approach that uses protein modeling in concert with druggability estimation to account for light protein backbone movement and protein side-chain flexibility in protein binding sites. We assess the advantages and limitations of this approach on widely-used protein druggability sets. Applying the approach to all mammalian protein crystal structures in the PDB results in identification of 69 proteins with potential druggable cryptic pockets. PMID:25079060

Wide bandgap OPV polymers based on pyridinonedithiophene unit with efficiency >5%

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schneider, Alexander M.; Lu, Luyao; Manley, Eric F.

2015-06-04

We report the properties of a new series of wide band gap photovoltaic polymers based on the N-alkyl 2-pyridone dithiophene (PDT) unit. These polymers are effective bulk heterojunction solar cell materials when blended with phenyl-C 71-butyric acid methyl ester (PC 71BM). They achieve power conversion efficiencies (up to 5.33%) high for polymers having such large bandgaps, ca. 2.0 eV (optical) and 2.5 eV (electrochemical). As a result, grazing incidence wide-angle X-ray scattering (GIWAXS) reveals strong correlations between π–π stacking distance and regularity, polymer backbone planarity, optical absorption maximum energy, and photovoltaic efficiency.

Reconstructing the equilibrium Boltzmann distribution from well-tempered metadynamics.

PubMed

Bonomi, M; Barducci, A; Parrinello, M

2009-08-01

Metadynamics is a widely used and successful method for reconstructing the free-energy surface of complex systems as a function of a small number of suitably chosen collective variables. This is achieved by biasing the dynamics of the system. The bias acting on the collective variables distorts the probability distribution of the other variables. Here we present a simple reweighting algorithm for recovering the unbiased probability distribution of any variable from a well-tempered metadynamics simulation. We show the efficiency of the reweighting procedure by reconstructing the distribution of the four backbone dihedral angles of alanine dipeptide from two and even one dimensional metadynamics simulation. 2009 Wiley Periodicals, Inc.

Molecular characterization of a Penicillium chrysogenum exo-rhamnogalacturonan lyase that is structurally distinct from other polysaccharide lyase family proteins.

PubMed

Iwai, Marin; Kawakami, Takuya; Ikemoto, Takeshi; Fujiwara, Daisuke; Takenaka, Shigeo; Nakazawa, Masami; Ueda, Mitsuhiro; Sakamoto, Tatsuji

2015-10-01

We previously described an endo-acting rhamnogalacturonan (RG) lyase, termed PcRGL4A, of Penicillium chrysogenum 31B. Here, we describe a second RG lyase, called PcRGLX. We determined the cDNA sequence of the Pcrglx gene, which encodes PcRGLX. Based on analyses using a BLAST search and a conserved domain search, PcRGLX was found to be structurally distinct from known RG lyases and might belong to a new polysaccharide lyase family together with uncharacterized fungal proteins of Nectria haematococca, Aspergillus oryzae, and Fusarium oxysporum. The Pcrglx cDNA gene product (rPcRGLX) expressed in Escherichia coli demonstrated specific activity against RG but not against homogalacturonan. Divalent cations were not essential for the enzymatic activity of rPcRGLX. rPcRGLX mainly released unsaturated galacturonosyl rhamnose (ΔGR) from RG backbones used as the substrate from the initial stage of the reaction, indicating that the enzyme can be classified as an exo-acting RG lyase (EC 4.2.2.24). This is the first report of an RG lyase with this mode of action in Eukaryota. rPcRGLX acted synergistically with PcRGL4A to degrade soybean RG and released ΔGR. This ΔGR was partially decorated with galactose (Gal) residues, indicating that rPcRGLX preferred oligomeric RGs to polymeric RGs, that the enzyme did not require Gal decoration of RG backbones for degradation, and that the enzyme bypassed the Gal side chains of RG backbones. These characteristics of rPcRGLX might be useful in the determination of complex structures of pectins.

Structural insights into the interactions of phorbol ester and bryostatin complexed with protein kinase C: a comparative molecular dynamics simulation study.

PubMed

Thangsunan, Patcharapong; Tateing, Suriya; Hannongbua, Supa; Suree, Nuttee

2016-07-01

Protein kinase C (PKC) isozymes are important regulatory enzymes that have been implicated in many diseases, including cancer, Alzheimer's disease, and in the eradication of HIV/AIDS. Given their potential clinical ramifications, PKC modulators, e.g. phorbol esters and bryostatin, are also of great interest in the drug development. However, structural details on the binding between PKC and its modulators, especially bryostatin - the highly potent and non-tumor promoting activator for PKCs, are still lacking. Here, we report the first comparative molecular dynamics study aimed at gaining structural insight into the mechanisms by which the PKC delta cys2 activator domain is used in its binding to phorbol ester and bryostatin-1. As anticipated in the phorbol ester binding, hydrogen bonds are formed through the backbone atoms of Thr242, Leu251, and Gly253 of PKC. However, the opposition of H-bond formation between Thr242 and Gly253 may cause the phorbol ester complex to become less stable when compared with the bryostatin binding. For the PKC delta-bryostatin complex, hydrogen bonds are formed between the Gly253 backbone carbonyl and the C30 carbomethoxy substituent of the ligand. Additionally, the indole Nε1 of the highly homologous Trp252 also forms an H-bond to the C20 ester group on bryostatin. Backbone fluctuations also suggest that this latter H-bond formation may abrogate the transient interaction between Trp252 and His269, thus dampening the fluctuations observed on the nearby Zn(2+)-coordinating residues. This new dynamic fluctuation dampening model can potentially benefit future design of new PKC modulators.

Characterization of signal bias at the GLP-1 receptor induced by backbone modification of GLP-1.

PubMed

Hager, Marlies V; Clydesdale, Lachlan; Gellman, Samuel H; Sexton, Patrick M; Wootten, Denise

2017-07-15

The glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor that is a major therapeutic target for the treatment of type 2 diabetes. Activation of this receptor promotes insulin secretion and blood glucose regulation. The GLP-1R can initiate signaling through several intracellular pathways upon activation by GLP-1. GLP-1R ligands that preferentially stimulate subsets among the natural signaling pathways ("biased agonists") could be useful as tools for elucidating the consequences of specific pathways and might engender therapeutic agents with tailored effects. Using HEK-293 cells recombinantly expressing human GLP-1R, we have previously reported that backbone modification of GLP-1, via replacement of selected α-amino acid residues with β-amino acid residues, generates GLP-1 analogues with distinctive preferences for promoting G protein activation versus β-arrestin recruitment. Here, we have explored the influence of cell background across these two parameters and expanded our analysis to include affinity and other key signaling pathways (intracellular calcium mobilization and ERK phosphorylation) using recombinant human GLP-1R expressed in a CHO cell background, which has been used extensively to demonstrate biased agonism of GLP-1R ligands. The new data indicate that α/β-peptide analogues of GLP-1 exhibit a range of distinct bias profiles relative to GLP-1 and that broad assessment of signaling endpoints is required to reveal the spectrum of behavior of modified peptides. These results support the view that backbone modification via α→β amino acid replacement can enable rapid discovery of peptide hormone analogues that display substantial signal bias at a cognate GPCR. Copyright © 2017 Elsevier Inc. All rights reserved.

From globules to crystals: a spectral study of poly(2-isopropyl-2-oxazoline) crystallization in hot water.

PubMed

Sun, Shengtong; Wu, Peiyi

2015-12-28

One easy strategy to comprehend the complex folding/crystallization behaviors of proteins is to study the self-assembly process of their synthetic polymeric analogues with similar properties owing to their simple structures and easy access to molecular design. Poly(2-isopropyl-2-oxazoline) (PIPOZ) is often regarded as an ideal pseudopeptide with similar two-step crystallization behavior to proteins, whose aqueous solution experiences successive lower critical solution temperature (LCST)-type liquid-liquid phase separation upon heating and irreversible crystallization when annealed above LCST for several hours. In this paper, by microscopic observations, IR and Raman spectroscopy in combination with 2D correlation analysis, we show that the second step of PIPOZ crystallization in hot water can be further divided into two apparent stages, i.e., nucleation and crystal growth, and perfect crystalline PIPOZ chains are found to only develop in the second stage. While all the groups exhibit changes in initial nucleation, only methylene groups on the backbone participate in the crystal growth stage. During nucleation, a group motion transfer is found from the side chain to the backbone, and nucleation is assumed to be mainly driven by the cleavage of bridging C=O···D-O-D···O=C hydrogen bonds followed by chain arrangement due to amide dipolar orientation. Nevertheless, during crystal growth, a further chain ordering process occurs resulting in the final formation of crystalline PIPOZ chains with partial trans conformation of backbones and alternative side chains on the two sides. The underlying crystallization mechanism of PIPOZ in hot water we present here may provide very useful information for understanding the crystallization of biomacromolecules in biological systems.

ISAMBARD: an open-source computational environment for biomolecular analysis, modelling and design.

PubMed

Wood, Christopher W; Heal, Jack W; Thomson, Andrew R; Bartlett, Gail J; Ibarra, Amaurys Á; Brady, R Leo; Sessions, Richard B; Woolfson, Derek N

2017-10-01

The rational design of biomolecules is becoming a reality. However, further computational tools are needed to facilitate and accelerate this, and to make it accessible to more users. Here we introduce ISAMBARD, a tool for structural analysis, model building and rational design of biomolecules. ISAMBARD is open-source, modular, computationally scalable and intuitive to use. These features allow non-experts to explore biomolecular design in silico. ISAMBARD addresses a standing issue in protein design, namely, how to introduce backbone variability in a controlled manner. This is achieved through the generalization of tools for parametric modelling, describing the overall shape of proteins geometrically, and without input from experimentally determined structures. This will allow backbone conformations for entire folds and assemblies not observed in nature to be generated de novo, that is, to access the 'dark matter of protein-fold space'. We anticipate that ISAMBARD will find broad applications in biomolecular design, biotechnology and synthetic biology. A current stable build can be downloaded from the python package index (https://pypi.python.org/pypi/isambard/) with development builds available on GitHub (https://github.com/woolfson-group/) along with documentation, tutorial material and all the scripts used to generate the data described in this paper. d.n.woolfson@bristol.ac.uk or chris.wood@bristol.ac.uk. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.

Amide or Amine: Determining the Origin of the 3300 cm−1 NH Mode in Protein SFG Spectra Using 15N Isotope Labels

PubMed Central

Weidner, Tobias; Breen, Nicholas F.; Drobny, Gary P.; Castner, David G.

2009-01-01

Sum frequency generation (SFG) vibrational spectroscopy has been employed in biomaterials research and protein adsorption studies with growing success in recent years. A number of studies focusing on understanding SFG spectra of proteins and peptides at different interfaces have laid the foundation for future, more complex studies. In many cases a strong NH mode near 3300 cm−1 is observed in the SFG spectra, but the relationship of this mode to the peptide structure is uncertain since it has been assigned to either a backbone amide mode or a side chain related amine resonance. A thorough understanding of the SFG spectra of these first model systems is an important first step for future experiments. To clarify the origin of the NH SFG mode we studied 15N isotopically labeled 14-amino acid amphiphilic model peptides composed of lysine (K) and leucine (L) in an α-helical secondary structure (LKα14) that were adsorbed onto charged surfaces in situ at the solid-liquid interface. 15N substitution at the terminal amine group of the lysine side chains resulted in a red-shift of the NH mode of 9 cm−1 on SiO2 and 13 cm−1 on CaF2. This clearly shows the 3300 cm−1 NH feature is associated with side chain NH stretches and not with backbone amide modes. PMID:19873996

Amide or amine: determining the origin of the 3300 cm(-1) NH mode in protein SFG spectra using 15N isotope labels.

PubMed

Weidner, Tobias; Breen, Nicholas F; Drobny, Gary P; Castner, David G

2009-11-26

Sum frequency generation (SFG) vibrational spectroscopy has been employed in biomaterials research and protein adsorption studies with growing success in recent years. A number of studies focusing on understanding SFG spectra of proteins and peptides at different interfaces have laid the foundation for future, more complex studies. In many cases, a strong NH mode near 3300 cm(-1) is observed in the SFG spectra, but the relationship of this mode to the peptide structure is uncertain, since it has been assigned to either a backbone amide mode or a side chain related amine resonance. A thorough understanding of the SFG spectra of these first model systems is an important first step for future experiments. To clarify the origin of the NH SFG mode, we studied (15)N isotopically labeled 14-amino acid amphiphilic model peptides composed of lysine (K) and leucine (L) in an alpha-helical secondary structure (LKalpha14) that were adsorbed onto charged surfaces in situ at the solid-liquid interface. (15)N substitution at the terminal amine group of the lysine side chains resulted in a red-shift of the NH mode of 9 cm(-1) on SiO(2) and 13 cm(-1) on CaF(2). This clearly shows the 3300 cm(-1) NH feature is associated with side chain NH stretches and not with backbone amide modes.

Structural variability of E. coli thioredoxin captured in the crystal structures of single-point mutants

PubMed Central

Noguera, Martín E.; Vazquez, Diego S.; Ferrer-Sueta, Gerardo; Agudelo, William A.; Howard, Eduardo; Rasia, Rodolfo M.; Manta, Bruno; Cousido-Siah, Alexandra; Mitschler, André; Podjarny, Alberto; Santos, Javier

2017-01-01

Thioredoxin is a ubiquitous small protein that catalyzes redox reactions of protein thiols. Additionally, thioredoxin from E. coli (EcTRX) is a widely-used model for structure-function studies. In a previous paper, we characterized several single-point mutants of the C-terminal helix (CTH) that alter global stability of EcTRX. However, spectroscopic signatures and enzymatic activity for some of these mutants were found essentially unaffected. A comprehensive structural characterization at the atomic level of these near-invariant mutants can provide detailed information about structural variability of EcTRX. We address this point through the determination of the crystal structures of four point-mutants, whose mutations occurs within or near the CTH, namely L94A, E101G, N106A and L107A. These structures are mostly unaffected compared with the wild-type variant. Notably, the E101G mutant presents a large region with two alternative traces for the backbone of the same chain. It represents a significant shift in backbone positions. Enzymatic activity measurements and conformational dynamics studies monitored by NMR and molecular dynamic simulations show that E101G mutation results in a small effect in the structural features of the protein. We hypothesize that these alternative conformations represent samples of the native-state ensemble of EcTRX, specifically the magnitude and location of conformational heterogeneity. PMID:28181556

Proline Substitution of Dimer Interface β-strand Residues as a Strategy for the Design of Functional Monomeric Proteins

PubMed Central

Joseph, Prem Raj B.; Poluri, Krishna Mohan; Gangavarapu, Pavani; Rajagopalan, Lavanya; Raghuwanshi, Sandeep; Richardson, Ricardo M.; Garofalo, Roberto P.; Rajarathnam, Krishna

2013-01-01

Proteins that exist in monomer-dimer equilibrium can be found in all organisms ranging from bacteria to humans; this facilitates fine-tuning of activities from signaling to catalysis. However, studying the structural basis of monomer function that naturally exists in monomer-dimer equilibrium is challenging, and most studies to date on designing monomers have focused on disrupting packing or electrostatic interactions that stabilize the dimer interface. In this study, we show that disrupting backbone H-bonding interactions by substituting dimer interface β-strand residues with proline (Pro) results in fully folded and functional monomers, by exploiting proline’s unique feature, the lack of a backbone amide proton. In interleukin-8, we substituted Pro for each of the three residues that form H-bonds across the dimer interface β-strands. We characterized the structures, dynamics, stability, dimerization state, and activity using NMR, molecular dynamics simulations, fluorescence, and functional assays. Our studies show that a single Pro substitution at the middle of the dimer interface β-strand is sufficient to generate a fully functional monomer. Interestingly, double Pro substitutions, compared to single Pro substitution, resulted in higher stability without compromising native monomer fold or function. We propose that Pro substitution of interface β-strand residues is a viable strategy for generating functional monomers of dimeric, and potentially tetrameric and higher-order oligomeric proteins. PMID:24048001

Structure of the complex between teicoplanin and a bacterial cell-wall peptide: use of a carrier-protein approach

DOE Office of Scientific and Technical Information (OSTI.GOV)

Economou, Nicoleta J.; Zentner, Isaac J.; Lazo, Edwin

2013-04-01

Using a carrier-protein strategy, the structure of teicoplanin bound to its bacterial cell-wall target has been determined. The structure reveals the molecular determinants of target recognition, flexibility in the antibiotic backbone and intrinsic radiation sensitivity of teicoplanin. Multidrug-resistant bacterial infections are commonly treated with glycopeptide antibiotics such as teicoplanin. This drug inhibits bacterial cell-wall biosynthesis by binding and sequestering a cell-wall precursor: a d-alanine-containing peptide. A carrier-protein strategy was used to crystallize the complex of teicoplanin and its target peptide by fusing the cell-wall peptide to either MBP or ubiquitin via native chemical ligation and subsequently crystallizing the protein–peptide–antibiotic complex.more » The 2.05 Å resolution MBP–peptide–teicoplanin structure shows that teicoplanin recognizes its ligand through a combination of five hydrogen bonds and multiple van der Waals interactions. Comparison of this teicoplanin structure with that of unliganded teicoplanin reveals a flexibility in the antibiotic peptide backbone that has significant implications for ligand recognition. Diffraction experiments revealed an X-ray-induced dechlorination of the sixth amino acid of the antibiotic; it is shown that teicoplanin is significantly more radiation-sensitive than other similar antibiotics and that ligand binding increases radiosensitivity. Insights derived from this new teicoplanin structure may contribute to the development of next-generation antibacterials designed to overcome bacterial resistance.« less

Structural variability of E. coli thioredoxin captured in the crystal structures of single-point mutants

NASA Astrophysics Data System (ADS)

Noguera, Martín E.; Vazquez, Diego S.; Ferrer-Sueta, Gerardo; Agudelo, William A.; Howard, Eduardo; Rasia, Rodolfo M.; Manta, Bruno; Cousido-Siah, Alexandra; Mitschler, André; Podjarny, Alberto; Santos, Javier

2017-02-01

Thioredoxin is a ubiquitous small protein that catalyzes redox reactions of protein thiols. Additionally, thioredoxin from E. coli (EcTRX) is a widely-used model for structure-function studies. In a previous paper, we characterized several single-point mutants of the C-terminal helix (CTH) that alter global stability of EcTRX. However, spectroscopic signatures and enzymatic activity for some of these mutants were found essentially unaffected. A comprehensive structural characterization at the atomic level of these near-invariant mutants can provide detailed information about structural variability of EcTRX. We address this point through the determination of the crystal structures of four point-mutants, whose mutations occurs within or near the CTH, namely L94A, E101G, N106A and L107A. These structures are mostly unaffected compared with the wild-type variant. Notably, the E101G mutant presents a large region with two alternative traces for the backbone of the same chain. It represents a significant shift in backbone positions. Enzymatic activity measurements and conformational dynamics studies monitored by NMR and molecular dynamic simulations show that E101G mutation results in a small effect in the structural features of the protein. We hypothesize that these alternative conformations represent samples of the native-state ensemble of EcTRX, specifically the magnitude and location of conformational heterogeneity.

Hairy and Slippery Polyoxazoline-Based Copolymers on Model and Cartilage Surfaces.

PubMed

Morgese, Giulia; Ramakrishna, Shivaprakash N; Simic, Rok; Zenobi-Wong, Marcy; Benetti, Edmondo M

2018-02-12

Comb-like polymers presenting a hydroxybenzaldehyde (HBA)-functionalized poly(glutamic acid) (PGA) backbone and poly(2-methyl-2-oxazoline) (PMOXA) side chains chemisorb on aminolized substrates, including cartilage surfaces, forming layers that reduce protein contamination and provide lubrication. The structure, physicochemical, biopassive, and tribological properties of PGA-PMOXA-HBA films are finely determined by the copolymer architecture, its reactivity toward the surface, i.e. PMOXA side-chain crowding and HBA density, and by the copolymer solution concentration during assembly. Highly reactive species with low PMOXA content form inhomogeneous layers due to the limited possibility of surface rearrangements by strongly anchored copolymers, just partially protecting the functionalized surface from protein contamination and providing a relatively weak lubrication on cartilage. Biopassivity and lubrication can be improved by increasing copolymer concentration during assembly, leading to a progressive saturation of surface defects across the films. In a different way, less reactive copolymers presenting high PMOXA side-chain densities form uniform, biopassive, and lubricious films, both on model aminolized silicon oxide surfaces, as well as on cartilage substrates. When assembled at low concentrations these copolymers adopt a "lying down" conformation, i.e. adhering via their backbones onto the substrates, while at high concentrations they undergo a conformational transition, assuming a more densely packed, "standing up" structure, where they stretch perpendicularly from the substrate. This specific arrangement reduces protein contamination and improves lubrication both on model as well as on cartilage surfaces.

Sulfated Glycopeptide Nanostructures for Multipotent Protein Activation

PubMed Central

Lee, Sungsoo S.; Fyrner, Timmy; Chen, Feng; Álvarez, Zaida; Sleep, Eduard; Chun, Danielle S.; Weiner, Joseph A.; Cook, Ralph W.; Freshman, Ryan D.; Schallmo, Michael S.; Katchko, Karina M.; Schneider, Andrew D.; Smith, Justin T.; Yun, Chawon; Singh, Gurmit; Hashmi, Sohaib Z.; McClendon, Mark T.; Yu, Zhilin; Stock, Stuart R.; Hsu, Wellington K.; Hsu, Erin L.; Stupp, Samuel I.

2017-01-01

Biological systems have evolved to utilize numerous proteins with capacity to bind polysaccharides for the purpose of optimizing their function. A well-known subset of these proteins with binding domains for the highly diverse sulfated polysaccharides are important growth factors involved in biological development and tissue repair. We report here on supramolecular sulfated glycopeptide nanostructures, which display a trisulfated monosaccharide on their surfaces and bind five critical proteins with very different polysaccharide binding domains. Binding does not disrupt the filamentous shape of the nanostructures or their internal β-sheet backbone, but must involve accessible adaptive configurations to interact with such different proteins. The glycopeptide nanostructures amplified signaling of bone morphogenetic protein 2 significantly more than the natural sulfated polysaccharide heparin, and promoted regeneration of bone in the spine with a protein dose that is 100-fold lower than expected. These super-bioactive nanostructures may enable many therapies in the horizon involving proteins. PMID:28650443

PBxplore: a tool to analyze local protein structure and deformability with Protein Blocks

PubMed Central

Craveur, Pierrick; Joseph, Agnel Praveen; Jallu, Vincent

2017-01-01

This paper describes the development and application of a suite of tools, called PBxplore, to analyze the dynamics and deformability of protein structures using Protein Blocks (PBs). Proteins are highly dynamic macromolecules, and a classical way to analyze their inherent flexibility is to perform molecular dynamics simulations. The advantage of using small structural prototypes such as PBs is to give a good approximation of the local structure of the protein backbone. More importantly, by reducing the conformational complexity of protein structures, PBs allow analysis of local protein deformability which cannot be done with other methods and had been used efficiently in different applications. PBxplore is able to process large amounts of data such as those produced by molecular dynamics simulations. It produces frequencies, entropy and information logo outputs as text and graphics. PBxplore is available at https://github.com/pierrepo/PBxplore and is released under the open-source MIT license. PMID:29177113

Investigation of protein folding by coarse-grained molecular dynamics with the UNRES force field.

PubMed

Maisuradze, Gia G; Senet, Patrick; Czaplewski, Cezary; Liwo, Adam; Scheraga, Harold A

2010-04-08

Coarse-grained molecular dynamics simulations offer a dramatic extension of the time-scale of simulations compared to all-atom approaches. In this article, we describe the use of the physics-based united-residue (UNRES) force field, developed in our laboratory, in protein-structure simulations. We demonstrate that this force field offers about a 4000-times extension of the simulation time scale; this feature arises both from averaging out the fast-moving degrees of freedom and reduction of the cost of energy and force calculations compared to all-atom approaches with explicit solvent. With massively parallel computers, microsecond folding simulation times of proteins containing about 1000 residues can be obtained in days. A straightforward application of canonical UNRES/MD simulations, demonstrated with the example of the N-terminal part of the B-domain of staphylococcal protein A (PDB code: 1BDD, a three-alpha-helix bundle), discerns the folding mechanism and determines kinetic parameters by parallel simulations of several hundred or more trajectories. Use of generalized-ensemble techniques, of which the multiplexed replica exchange method proved to be the most effective, enables us to compute thermodynamics of folding and carry out fully physics-based prediction of protein structure, in which the predicted structure is determined as a mean over the most populated ensemble below the folding-transition temperature. By using principal component analysis of the UNRES folding trajectories of the formin-binding protein WW domain (PDB code: 1E0L; a three-stranded antiparallel beta-sheet) and 1BDD, we identified representative structures along the folding pathways and demonstrated that only a few (low-indexed) principal components can capture the main structural features of a protein-folding trajectory; the potentials of mean force calculated along these essential modes exhibit multiple minima, as opposed to those along the remaining modes that are unimodal. In addition, a comparison between the structures that are representative of the minima in the free-energy profile along the essential collective coordinates of protein folding (computed by principal component analysis) and the free-energy profile projected along the virtual-bond dihedral angles gamma of the backbone revealed the key residues involved in the transitions between the different basins of the folding free-energy profile, in agreement with existing experimental data for 1E0L .

Exposing hidden alternative backbone conformations in X-ray crystallography using qFit

DOE PAGES

Keedy, Daniel A.; Fraser, James S.; van den Bedem, Henry; ...

2015-10-27

Proteins must move between different conformations of their native ensemble to perform their functions. Crystal structures obtained from high-resolution X-ray diffraction data reflect this heterogeneity as a spatial and temporal conformational average. Although movement between natively populated alternative conformations can be critical for characterizing molecular mechanisms, it is challenging to identify these conformations within electron density maps. Alternative side chain conformations are generally well separated into distinct rotameric conformations, but alternative backbone conformations can overlap at several atomic positions. Our model building program qFit uses mixed integer quadratic programming (MIQP) to evaluate an extremely large number of combinations of sidechainmore » conformers and backbone fragments to locally explain the electron density. Here, we describe two major modeling enhancements to qFit: peptide flips and alternative glycine conformations. We find that peptide flips fall into four stereotypical clusters and are enriched in glycine residues at the n+1 position. The potential for insights uncovered by new peptide flips and glycine conformations is exemplified by HIV protease, where different inhibitors are associated with peptide flips in the “flap” regions adjacent to the inhibitor binding site. Our results paint a picture of peptide flips as conformational switches, often enabled by glycine flexibility, that result in dramatic local rearrangements. Our results furthermore demonstrate the power of large-scale computational analysis to provide new insights into conformational heterogeneity. Furthermore, improved modeling of backbone heterogeneity with high-resolution X-ray data will connect dynamics to the structure-function relationship and help drive new design strategies for inhibitors of biomedically important systems.« less

Stability Mechanisms of a Thermophilic Laccase Probed by Molecular Dynamics

PubMed Central

Christensen, Niels J.; Kepp, Kasper P.

2013-01-01

Laccases are highly stable, industrially important enzymes capable of oxidizing a large range of substrates. Causes for their stability are, as for other proteins, poorly understood. In this work, multiple-seed molecular dynamics (MD) was applied to a Trametes versicolor laccase in response to variable ionic strengths, temperatures, and glycosylation status. Near-physiological conditions provided excellent agreement with the crystal structure (average RMSD ∼0.92 Å) and residual agreement with experimental B-factors. The persistence of backbone hydrogen bonds was identified as a key descriptor of structural response to environment, whereas solvent-accessibility, radius of gyration, and fluctuations were only locally relevant. Backbone hydrogen bonds decreased systematically with temperature in all simulations (∼9 per 50 K), probing structural changes associated with enthalpy-entropy compensation. Approaching T opt (∼350 K) from 300 K, this change correlated with a beginning “unzipping” of critical β-sheets. 0 M ionic strength triggered partial denucleation of the C-terminal (known experimentally to be sensitive) at 400 K, suggesting a general salt stabilization effect. In contrast, F− (but not Cl−) specifically impaired secondary structure by formation of strong hydrogen bonds with backbone NH, providing a mechanism for experimentally observed small anion destabilization, potentially remedied by site-directed mutagenesis at critical intrusion sites. N-glycosylation was found to support structural integrity by increasing persistent backbone hydrogen bonds by ∼4 across simulations, mainly via prevention of F− intrusion. Hydrogen-bond loss in distinct loop regions and ends of critical β-sheets suggest potential strategies for laboratory optimization of these industrially important enzymes. PMID:23658618

A Novel Method for Sampling Alpha-Helical Protein Backbones

DOE R&D Accomplishments Database

Fain, Boris; Levitt, Michael

2001-01-01

We present a novel technique of sampling the configurations of helical proteins. Assuming knowledge of native secondary structure, we employ assembly rules gathered from a database of existing structures to enumerate the geometrically possible 3-D arrangements of the constituent helices. We produce a library of possible folds for 25 helical protein cores. In each case the method finds significant numbers of conformations close to the native structure. In addition we assign coordinates to all atoms for 4 of the 25 proteins. In the context of database driven exhaustive enumeration our method performs extremely well, yielding significant percentages of structures (0.02%--82%) within 6A of the native structure. The method's speed and efficiency make it a valuable contribution towards the goal of predicting protein structure.

Core–Shell Structure and Aggregation Number of Micelles Composed of Amphiphilic Block Copolymers and Amphiphilic Heterografted Polymer Brushes Determined by Small-Angle X-ray Scattering

DOE Office of Scientific and Technical Information (OSTI.GOV)

Szymusiak, Magdalena; Kalkowski, Joseph; Luo, Hanying

2017-08-31

A large group of functional nanomaterials employed in biomedical applications, including targeted drug delivery, relies on amphiphilic polymers to encapsulate therapeutic payloads via self-assembly processes. Knowledge of the micelle structures will provide critical insights into design of polymeric drug delivery systems. Core–shell micelles composed of linear diblock copolymers poly(ethylene glycol)-b-poly(caprolactone) (PEG-b-PCL), poly(ethylene oxide)-b-poly(lactic acid) (PEG-b-PLA), as well as a heterografted brush consisting of a poly(glycidyl methacrylate) backbone with PEG and PLA branches (PGMA-g-PEG/PLA) were characterized by dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS) measurements to gain structural information regarding the particle morphology, core–shell size, and aggregation number. Themore » structural information at this quasi-equilibrium state can also be used as a reference when studying the kinetics of polymer micellization.« less

Core–Shell Structure and Aggregation Number of Micelles Composed of Amphiphilic Block Copolymers and Amphiphilic Heterografted Polymer Brushes Determined by Small-Angle X-ray Scattering

DOE Office of Scientific and Technical Information (OSTI.GOV)

Szymusiak, Magdalena; Kalkowski, Joseph; Luo, Hanying

2017-08-16

A large group of functional nanomaterials employed in biomedical applications, including targeted drug delivery, relies on amphiphilic polymers to encapsulate therapeutic payloads via self-assembly processes. Knowledge of the micelle structures will provide critical insights into design of polymeric drug delivery systems. Core–shell micelles composed of linear diblock copolymers poly(ethylene glycol)-b-poly(caprolactone) (PEG-b-PCL), poly(ethylene oxide)-b-poly(lactic acid) (PEG-b-PLA), as well as a heterografted brush consisting of a poly(glycidyl methacrylate) backbone with PEG and PLA branches (PGMA-g-PEG/PLA) were characterized by dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS) measurements to gain structural information regarding the particle morphology, core–shell size, and aggregation number. Themore » structural information at this quasi-equilibrium state can also be used as a reference when studying the kinetics of polymer micellization.« less

Ensemble of Transition State Structures for the Cis-Trans Isomerization of N-Methylacetamide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mantz, Yves A.; Branduardi, Davide; Bussi, Giovanni

2009-09-17

The cis-trans isomerization of N-methylacetamide (NMA), a model peptidic fragment, is studied theoretically in vacuo and in explicit water solvent at 300 K using the metadynamics technique. The computed cis-trans free energy difference is very similar for NMA(g) and NMA(aq), in agreement with experimental measurements of population ratios and theoretical studies at 0 K. By exploiting the flexibility in the definition of a pair of recently introduced collective variables (Branduardi, D.; Gervasio, F. L.; Parrinello, M. J. Chem. Phys. 2007, 126, 054103), an ensemble of transition state structures is generated at finite temperature for both NMA(g) and NMA(aq), as verifiedmore » by computing committor distribution functions. Ensemble members of NMA(g) are shown to have correlated values of the backbone dihedral angle and a second dihedral angle involving the amide hydrogen atom. The dynamical character of these structures is preserved in the presence of solvent, whose influence on the committor functions can be modeled using effective friction/noise terms.« less

Hydrogen/deuterium exchange studies of native rabbit MM-CK dynamics.

PubMed

Mazon, Hortense; Marcillat, Olivier; Forest, Eric; Vial, Christian

2004-02-01

Creatine kinase (CK) isoenzymes catalyse the reversible transfer of a phosphoryl group from ATP onto creatine. This reaction plays a very important role in the regulation of intracellular ATP concentrations in excitable tissues. CK isoenzymes are highly resistant to proteases in native conditions. To appreciate localized backbone dynamics, kinetics of amide hydrogen exchange with deuterium was measured by pulse-labeling the dimeric cytosolic muscle CK isoenzyme. Upon exchange, the protein was digested with pepsin, and the deuterium content of the resulting peptides was determined by liquid chromatography coupled to mass spectrometry (MS). The deuteration kinetics of 47 peptides identified by MS/MS and covering 96% of the CK backbone were analyzed. Four deuteration patterns have been recognized: The less deuterated peptides are located in the saddle-shaped core of CK, whereas most of the highly deuterated peptides are close to the surface and located around the entrance to the active site. Their exchange kinetics are discussed by comparison with the known secondary and tertiary structures of CK with the goal to reveal the conformational dynamics of the protein. Some of the observed dynamic motions may be linked to the conformational changes associated with substrate binding and catalytic mechanism.

Biosynthesis and NMR-studies of a double transmembrane domain from the Y4 receptor, a human GPCR.

PubMed

Zou, Chao; Naider, Fred; Zerbe, Oliver

2008-12-01

The human Y4 receptor, a class A G-protein coupled receptor (GPCR) primarily targeted by the pancreatic polypeptide (PP), is involved in a large number of physiologically important functions. This paper investigates a Y4 receptor fragment (N-TM1-TM2) comprising the N-terminal domain, the first two transmembrane (TM) helices and the first extracellular loop followed by a (His)(6) tag, and addresses synthetic problems encountered when recombinantly producing such fragments from GPCRs in Escherichia coli. Rigorous purification and usage of the optimized detergent mixture 28 mM dodecylphosphocholine (DPC)/118 mM% 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LPPG) resulted in high quality TROSY spectra indicating protein conformational homogeneity. Almost complete assignment of the backbone, including all TM residue resonances was obtained. Data on internal backbone dynamics revealed a high secondary structure content for N-TM1-TM2. Secondary chemical shifts and sequential amide proton nuclear Overhauser effects defined the TM helices. Interestingly, the properties of the N-terminal domain of this large fragment are highly similar to those determined on the isolated N-terminal domain in the presence of DPC micelles.

RNA Bricks—a database of RNA 3D motifs and their interactions

PubMed Central

Chojnowski, Grzegorz; Waleń, Tomasz; Bujnicki, Janusz M.

2014-01-01

The RNA Bricks database (http://iimcb.genesilico.pl/rnabricks), stores information about recurrent RNA 3D motifs and their interactions, found in experimentally determined RNA structures and in RNA–protein complexes. In contrast to other similar tools (RNA 3D Motif Atlas, RNA Frabase, Rloom) RNA motifs, i.e. ‘RNA bricks’ are presented in the molecular environment, in which they were determined, including RNA, protein, metal ions, water molecules and ligands. All nucleotide residues in RNA bricks are annotated with structural quality scores that describe real-space correlation coefficients with the electron density data (if available), backbone geometry and possible steric conflicts, which can be used to identify poorly modeled residues. The database is also equipped with an algorithm for 3D motif search and comparison. The algorithm compares spatial positions of backbone atoms of the user-provided query structure and of stored RNA motifs, without relying on sequence or secondary structure information. This enables the identification of local structural similarities among evolutionarily related and unrelated RNA molecules. Besides, the search utility enables searching ‘RNA bricks’ according to sequence similarity, and makes it possible to identify motifs with modified ribonucleotide residues at specific positions. PMID:24220091

NMR 1H,13C, 15N backbone and 13C side chain resonance assignment of the G12C mutant of human K-Ras bound to GDP.

PubMed

Sharma, Alok K; Lee, Seung-Joo; Rigby, Alan C; Townson, Sharon A

2018-05-02

K-Ras is a key driver of oncogenesis, accounting for approximately 80% of Ras-driven human cancers. The small GTPase cycles between an inactive, GDP-bound and an active, GTP-bound state, regulated by guanine nucleotide exchange factors and GTPase activating proteins, respectively. Activated K-Ras regulates cell proliferation, differentiation and survival by signaling through several effector pathways, including Raf-MAPK. Oncogenic mutations that impair the GTPase activity of K-Ras result in a hyperactivated state, leading to uncontrolled cellular proliferation and tumorogenesis. A cysteine mutation at glycine 12 is commonly found in K-Ras associated cancers, and has become a recent focus for therapeutic intervention. We report here 1 H N, 15 N, and 13 C resonance assignments for the 19.3 kDa (aa 1-169) human K-Ras protein harboring an oncogenic G12C mutation in the GDP-bound form (K-RAS G12C-GDP ), using heteronuclear, multidimensional NMR spectroscopy. Backbone 1 H- 15 N correlations have been assigned for all non-proline residues, except for the first methionine residue.

Frequent side chain methyl carbon-oxygen hydrogen bonding in proteins revealed by computational and stereochemical analysis of neutron structures.

PubMed

Yesselman, Joseph D; Horowitz, Scott; Brooks, Charles L; Trievel, Raymond C

2015-03-01

The propensity of backbone Cα atoms to engage in carbon-oxygen (CH · · · O) hydrogen bonding is well-appreciated in protein structure, but side chain CH · · · O hydrogen bonding remains largely uncharacterized. The extent to which side chain methyl groups in proteins participate in CH · · · O hydrogen bonding is examined through a survey of neutron crystal structures, quantum chemistry calculations, and molecular dynamics simulations. Using these approaches, methyl groups were observed to form stabilizing CH · · · O hydrogen bonds within protein structure that are maintained through protein dynamics and participate in correlated motion. Collectively, these findings illustrate that side chain methyl CH · · · O hydrogen bonding contributes to the energetics of protein structure and folding. © 2014 Wiley Periodicals, Inc.

1H, 13C, and 15N resonance assignments for the protein coded by gene locus BB0938 of Bordetella bronchiseptica

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rossi, Paolo; Ramelot, Theresa A.; Xiao, Rong

2005-11-01

The product of gene locus BB0938 from Bordetella bronchiseptica (Swiss-Prot ID: Q7WNU7-BORBR; NESG target ID: BoR11; Wunderlich et al., 2004; Pfam ID: PF03476) is a 128-residue protein of unknown function. This broadly conserved protein family is found in eubacteria and eukaryotes. Using triple resonance NMR techniques, we have determined 98% of backbone and 94% of side chain 1H, 13C, and 15N resonance assignments. The chemical shift and 3J(HN?Ha) scalar coupling data reveal a b topology with a seven-residue helical insert, ??????????. BMRB deposit with accession number 6693. Reference: Wunderlich et al. (2004) Proteins, 56, 181?187.

A Global Protein Kinase and Phosphatase Interaction Network in Yeast

PubMed Central

Breitkreutz, Ashton; Choi, Hyungwon; Sharom, Jeffrey R.; Boucher, Lorrie; Neduva, Victor; Larsen, Brett; Lin, Zhen-Yuan; Breitkreutz, Bobby-Joe; Stark, Chris; Liu, Guomin; Ahn, Jessica; Dewar-Darch, Danielle; Reguly, Teresa; Tang, Xiaojing; Almeida, Ricardo; Qin, Zhaohui Steve; Pawson, Tony; Gingras, Anne-Claude; Nesvizhskii, Alexey I.; Tyers, Mike

2011-01-01

The interactions of protein kinases and phosphatases with their regulatory subunits and substrates underpin cellular regulation. We identified a kinase and phosphatase interaction (KPI) network of 1844 interactions in budding yeast by mass spectrometric analysis of protein complexes. The KPI network contained many dense local regions of interactions that suggested new functions. Notably, the cell cycle phosphatase Cdc14 associated with multiple kinases that revealed roles for Cdc14 in mitogen-activated protein kinase signaling, the DNA damage response, and metabolism, whereas interactions of the target of rapamycin complex 1 (TORC1) uncovered new effector kinases in nitrogen and carbon metabolism. An extensive backbone of kinase-kinase interactions cross-connects the proteome and may serve to coordinate diverse cellular responses. PMID:20489023

1H, 13C, and 15N resonance assignments for Escherichia coli ytfP, a member of the broadly conserved UPF0131 protein domain family

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aramini, James M.; Swapna, G.V.T.; Huang, Yuanpeng

2005-11-01

Protein ytfP from Escherichia coli (Swiss-Prot ID: YTFP-ECOLI; NESG target ID: ER111; Wunderlich et al., 2004) is a 113-residue member of the UPF0131 protein family (Pfam ID: PF03674) of unknown function. This domain family is found in organisms from all three kingdoms, archaea, eubacteria and eukaryotes. Using triple resonance NMR techniques, we have determined 97% of backbone and 91% of side chain 1H, 13C, and 15N resonance assignments. The chemical shift and 3J(HN?Ha) scalar coupling data reveal a mixed a/b topology,????????. BMRB deposit with Accession No. 6448. Reference: Wunderlich et al. (2004) Proteins, 56, 181?187.

[Preparation of anti-hCG single domain antibody by antibody grafting technique using an antigen-binding peptide].

PubMed

Peng, Jing; Wang, Qiong; Cheng, Xiaoling; Liu, Mengwen; Wang, Mei; Xin, Huawei

2018-04-25

We used the antibody grafting technology to prepare anti-hCG single-domain antibodies on the basis of antigen-binding peptide to simplify the single-domain antibody preparation process and improving the biochemical stability of peptide. By using a universal single-domain antibody backbone (cAbBCII10), CDR1 or CDR3 was replaced by the hCG-binding peptide, and the grafted antibody gene sequences were synthesized and cloned into the prokaryotic expression vector pET30a(+) in fusion with a C-terminal sfGFP gene, i.e. pET30a-(His6)-cAbBCII10-CDR1/hCGBP1-sfGFP and pET30a-(His6)-cAbBCII10-CDR3/hCGBP3-sfGFP. The recombinant plasmids were transformed into E. coli BL21(DE3), and the fusion proteins were induced by IPTG. Highly soluble recombinant fusion proteins were obtained and purified by Ni-NTA affinity column. SDS-PAGE confirmed the purified protein as the target protein. The antigen-antibody binding assay showed that both the CDR1 and CDR3 grafted antibodies have hCG-binding activities. While the titers of the two grafted antibodies were similar, the binding affinity of CDR3 grafted antibody was higher than that of CDR1 grafted protein (about 2-3 times). The grafted antibodies retained the relatively high biochemical stability of the single-domain antibody backbone and were relatively thermostable and alkaline tolerant. The obtained antibodies also had a relatively high antigen-binding specificity to hCG. This study provided a reliable experimental basis for further optimization of anti-hCG single domain antibody by antibody grafting technology using antigen-binding peptide.

Conformational switching between protein substates studied with 2D IR vibrational echo spectroscopy and molecular dynamics simulations.

PubMed

Bagchi, Sayan; Thorpe, Dayton G; Thorpe, Ian F; Voth, Gregory A; Fayer, M D

2010-12-30

Myoglobin is an important protein for the study of structure and dynamics. Three conformational substates have been identified for the carbonmonoxy form of myoglobin (MbCO). These are manifested as distinct peaks in the IR absorption spectrum of the CO stretching mode. Ultrafast 2D IR vibrational echo chemical exchange experiments are used to observed switching between two of these substates, A(1) and A(3), on a time scale of <100 ps for two mutants of wild-type Mb. The two mutants are a single mutation of Mb, L29I, and a double mutation, T67R/S92D. Molecular dynamics (MD) simulations are used to model the structural differences between the substates of the two MbCO mutants. The MD simulations are also employed to examine the substate switching in the two mutants as a test of the ability of MD simulations to predict protein dynamics correctly for a system in which there is a well-defined transition over a significant potential barrier between two substates. For one mutant, L29I, the simulations show that translation of the His64 backbone may differentiate the two substates. The simulations accurately reproduce the experimentally observed interconversion time for the L29I mutant. However, MD simulations exploring the same His64 backbone coordinate fail to display substate interconversion for the other mutant, T67R/S92D, thus pointing to the likely complexity of the underlying protein interactions. We anticipate that understanding conformational dynamics in MbCO via ultrafast 2D IR vibrational echo chemical exchange experiments can help to elucidate fast conformational switching processes in other proteins.

Integrating NOE and RDC using sum-of-squares relaxation for protein structure determination.

PubMed

Khoo, Y; Singer, A; Cowburn, D

2017-07-01

We revisit the problem of protein structure determination from geometrical restraints from NMR, using convex optimization. It is well-known that the NP-hard distance geometry problem of determining atomic positions from pairwise distance restraints can be relaxed into a convex semidefinite program (SDP). However, often the NOE distance restraints are too imprecise and sparse for accurate structure determination. Residual dipolar coupling (RDC) measurements provide additional geometric information on the angles between atom-pair directions and axes of the principal-axis-frame. The optimization problem involving RDC is highly non-convex and requires a good initialization even within the simulated annealing framework. In this paper, we model the protein backbone as an articulated structure composed of rigid units. Determining the rotation of each rigid unit gives the full protein structure. We propose solving the non-convex optimization problems using the sum-of-squares (SOS) hierarchy, a hierarchy of convex relaxations with increasing complexity and approximation power. Unlike classical global optimization approaches, SOS optimization returns a certificate of optimality if the global optimum is found. Based on the SOS method, we proposed two algorithms-RDC-SOS and RDC-NOE-SOS, that have polynomial time complexity in the number of amino-acid residues and run efficiently on a standard desktop. In many instances, the proposed methods exactly recover the solution to the original non-convex optimization problem. To the best of our knowledge this is the first time SOS relaxation is introduced to solve non-convex optimization problems in structural biology. We further introduce a statistical tool, the Cramér-Rao bound (CRB), to provide an information theoretic bound on the highest resolution one can hope to achieve when determining protein structure from noisy measurements using any unbiased estimator. Our simulation results show that when the RDC measurements are corrupted by Gaussian noise of realistic variance, both SOS based algorithms attain the CRB. We successfully apply our method in a divide-and-conquer fashion to determine the structure of ubiquitin from experimental NOE and RDC measurements obtained in two alignment media, achieving more accurate and faster reconstructions compared to the current state of the art.

Biomimetic oligosaccharide and peptide surfactant polymers designed for cardiovascular biomaterials

NASA Astrophysics Data System (ADS)

Ruegsegger, Mark Andrew

A common problem associated with cardiovascular devices is surface induced thrombosis initiated by the rapid, non-specific adsorption of plasma proteins onto the biomaterial surface. Control of the initial protein adsorption is crucial to achieve the desired longevity of the implanted biomaterial. The cell membrane glycocalyx acts as a non-thrombogenic interface through passive (dense oligosaccharide structures) and active (ligand/receptor interactions) mechanisms. This thesis is designed to investigate biomimicry of the cell glycocalyx to minimize non-specific protein adsorption and promote specific ligand/receptor interactions. Biomimetic macromolecules were designed through the molecular-scale engineering of polymer surfactants, utilizing a poly(vinyl amine) (PVAm) backbone to which hydrophilic (dextran, maltose, peptide) and hydrophobic alkyl (hexanoyl or hexanal) chains are simultaneously attached. The structure was controlled through the molar feed ratio of hydrophobic-to-hydrophilic groups, which also provided control of the solution and surface-active properties. To mimic passive properties, a series of oligomaltose surfactants were synthesized with increasing saccharide length (n = 2, 7, 15 where n is number of glucose units) to investigate the effect of coating height on protein adsorption. The surfactants were characterized by infra red (IR) and nuclear magnetic resonance (NMR) spectroscopies for structural properties and atomic force microscopy (AFM) and contact angle goniometry for surface activity. Protein adsorption under dynamic flow (5 dyn/cm2) was reduced by 85%--95% over the bare hydrophobic substrate; platelet adhesion dropped by ˜80% compared to glass. Peptide ligands were incorporated into the oligosaccharide surfactant to promote functional activity of the passive coating. The surfactants were synthesized to contain 0%, 25%, 50%, 75%, and 100% peptide ligand density and were stable on hydrophobic surfaces. The peptide surface density was calculated to be 0.86 ligands/nm2 for PVAm(Pep)(100%), as determined by total internal reflection fluorescence (TIRF) spectroscopy. Similar cell growth was observed on the 100% peptide surfactant as for the fibronectin control, and no cell growth was seen on the 0% peptide. Increasing cell viability was observed for the surfaces with increasing peptide density. These results indicate much promise for surfactant polymers in surface modification and the capability to mimic the passive and active properties of the cell glycocalyx.

A Bayesian Approach for Determining Protein Side-Chain Rotamer Conformations Using Unassigned NOE Data

PubMed Central

Zeng, Jianyang; Roberts, Kyle E.; Zhou, Pei

2011-01-01

Abstract A major bottleneck in protein structure determination via nuclear magnetic resonance (NMR) is the lengthy and laborious process of assigning resonances and nuclear Overhauser effect (NOE) cross peaks. Recent studies have shown that accurate backbone folds can be determined using sparse NMR data, such as residual dipolar couplings (RDCs) or backbone chemical shifts. This opens a question of whether we can also determine the accurate protein side-chain conformations using sparse or unassigned NMR data. We attack this question by using unassigned nuclear Overhauser effect spectroscopy (NOESY) data, which records the through-space dipolar interactions between protons nearby in three-dimensional (3D) space. We propose a Bayesian approach with a Markov random field (MRF) model to integrate the likelihood function derived from observed experimental data, with prior information (i.e., empirical molecular mechanics energies) about the protein structures. We unify the side-chain structure prediction problem with the side-chain structure determination problem using unassigned NMR data, and apply the deterministic dead-end elimination (DEE) and A* search algorithms to provably find the global optimum solution that maximizes the posterior probability. We employ a Hausdorff-based measure to derive the likelihood of a rotamer or a pairwise rotamer interaction from unassigned NOESY data. In addition, we apply a systematic and rigorous approach to estimate the experimental noise in NMR data, which also determines the weighting factor of the data term in the scoring function derived from the Bayesian framework. We tested our approach on real NMR data of three proteins: the FF Domain 2 of human transcription elongation factor CA150 (FF2), the B1 domain of Protein G (GB1), and human ubiquitin. The promising results indicate that our algorithm can be applied in high-resolution protein structure determination. Since our approach does not require any NOE assignment, it can accelerate the NMR structure determination process. PMID:21970619

A Protein Structure Initiative Approach to Expression, Purification, and In Situ Delivery of Human Cytochrome b5 to Membrane Vesicles†

PubMed Central

Sobrado, Pablo; Goren, Michael A.; James, Declan; Amundson, Carissa K.; Fox, Brian G.

2008-01-01

A specialized vector backbone from the Protein Structure Initiative was used to express full-length human cytochrome b5 as a C-terminal fusion to His8-maltose binding protein in Escherichia coli. The fusion protein could be completely cleaved by tobacco etch virus protease, and a yield of ~18 mg of purified full-length human cytochrome b5 per liter of culture medium was obtained (2.3 mg per]of wet weight bacterial cells). In situ proteolysis of the fusion protein in the presence of chemically defined synthetic liposomes allowed facile spontaneous delivery of the functional peripheral membrane protein into a defined membrane environment without prior exposure to detergents or other lipids. The utility of this approach as a delivery method for production and incorporation of monotopic (peripheral) membrane proteins is discussed. PMID:18226920

A Protein Structure Initiative approach to expression, purification, and in situ delivery of human cytochrome b5 to membrane vesicles.

PubMed

Sobrado, Pablo; Goren, Michael A; James, Declan; Amundson, Carissa K; Fox, Brian G

2008-04-01

A specialized vector backbone from the Protein Structure Initiative was used to express full-length human cytochrome b5 as a C-terminal fusion to His8-maltose binding protein in Escherichia coli. The fusion protein could be completely cleaved by tobacco etch virus protease, and a yield of approximately 18 mg of purified full-length human cytochrome b5 per liter of culture medium was obtained (2.3mg per g of wet weight bacterial cells). In situ proteolysis of the fusion protein in the presence of chemically defined synthetic liposomes allowed facile spontaneous delivery of the functional peripheral membrane protein into a defined membrane environment without prior exposure to detergents or other lipids. The utility of this approach as a delivery method for production and incorporation of monotopic (peripheral) membrane proteins is discussed.

Sphingolipid hydroxylation in mammals, yeast and plants - An integrated view.

PubMed

Marquês, Joaquim Trigo; Susana Marinho, H; de Almeida, Rodrigo Freire Martins

2018-05-07

This review is focused on sphingolipid backbone hydroxylation, a small but widespread structural feature, with profound impact on membrane biophysical properties. We start by summarizing sphingolipid metabolism in mammalian cells, yeast and plants, focusing on how distinct hydroxylation patterns emerge in different eukaryotic kingdoms. Then, a comparison of the biophysical properties in membrane model systems and cellular membranes from diverse organisms is made. From an integrative perspective, these results can be rationalized considering that superficial hydroxyl groups in the backbone of sphingolipids (by intervening in the H-bond network) alter the balance of favorable interactions between membrane lipids. They may strengthen the bonding or compete with other hydroxyl groups, in particular the one of membrane sterols. Different sphingolipid hydroxylation patterns can stabilize/disrupt specific membrane domains or change whole plasma membrane properties, and therefore be important in the control of protein distribution, function and lateral diffusion and in the formation and overtime stability of signaling platforms. The recent examples explored throughout this review unveil a potentially key role for sphingolipid backbone hydroxylation in both physiological and pathological situations, as they can be of extreme importance for the proper organization of cell membranes in mammalian cells, yeast and, most likely, also in plants. Copyright © 2017. Published by Elsevier Ltd.

Aggregation of flexible polyelectrolytes: Phase diagram and dynamics.

PubMed

Tom, Anvy Moly; Rajesh, R; Vemparala, Satyavani

2017-10-14

Similarly charged polymers in solution, known as polyelectrolytes, are known to form aggregated structures in the presence of oppositely charged counterions. Understanding the dependence of the equilibrium phases and the dynamics of the process of aggregation on parameters such as backbone flexibility and charge density of such polymers is crucial for insights into various biological processes which involve biological polyelectrolytes such as protein, DNA, etc. Here, we use large-scale coarse-grained molecular dynamics simulations to obtain the phase diagram of the aggregated structures of flexible charged polymers and characterize the morphology of the aggregates as well as the aggregation dynamics, in the presence of trivalent counterions. Three different phases are observed depending on the charge density: no aggregation, a finite bundle phase where multiple small aggregates coexist with a large aggregate and a fully phase separated phase. We show that the flexibility of the polymer backbone causes strong entanglement between charged polymers leading to additional time scales in the aggregation process. Such slowing down of the aggregation dynamics results in the exponent, characterizing the power law decay of the number of aggregates with time, to be dependent on the charge density of the polymers. These results are contrary to those obtained for rigid polyelectrolytes, emphasizing the role of backbone flexibility.

Phi ({Phi}) and psi ({Psi}) angles involved in malarial peptide bonds determine sterile protective immunity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Patarroyo, Manuel E., E-mail: mepatarr@gmail.com; Universidad Nacional de Colombia, Bogota; Moreno-Vranich, Armando

Highlights: Black-Right-Pointing-Pointer Phi ({Phi}) and psi ({Psi}) angles determine sterile protective immunity. Black-Right-Pointing-Pointer Modified peptide's tendency to assume a regular conformation related to a PPII{sub L}. Black-Right-Pointing-Pointer Structural modifications in mHABPs induce Ab and protective immunity. Black-Right-Pointing-Pointer mHABP backbone atom's interaction with HLA-DR{beta}1{sup Asterisk-Operator} is stabilised by H-bonds. -- Abstract: Modified HABP (mHABP) regions interacting with HLA-DR{beta}1{sup Asterisk-Operator} molecules have a more restricted conformation and/or sequence than other mHABPs which do not fit perfectly into their peptide binding regions (PBR) and do not induce an acceptable immune response due to the critical role of their {Phi} and {Psi} torsion angles.more » These angle's critical role was determined in such highly immunogenic, protection-inducing response against experimental malaria using the conformers (mHABPs) obtained by {sup 1}H-NMR and superimposed into HLA-DR{beta}1{sup Asterisk-Operator }-like Aotus monkey molecules; their phi ({Phi}) and psi ({Psi}) angles were measured and the H-bond formation between these molecules was evaluated. The aforementioned mHABP propensity to assume a regular conformation similar to a left-handed polyproline type II helix (PPII{sub L}) led to suggesting that favouring these conformations according to their amino acid sequence would lead to high antibody titre production and sterile protective immunity induction against malaria, thereby adding new principles or rules for vaccine development, malaria being one of them.« less