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PubMed2005

Interpreting dynamically-averaged scalar couplings in proteins.

The experimental determination of scalar three-bond coupling constants represents a powerful method to probe both the structure and dynamics of proteins. The detailed structural interpretation of such coupling constants is usually based on Karplus relationships, which allow the measured couplings to be related to the torsion angles of the molecules. As the measured couplings are sensitive to thermal fluctuations, the parameters in the Karplus relationships are better derived from ensembles representing the distributions of dihedral angles present in solution, rather than from single conformations. We present a method to derive such parameters that uses ensembles of conformations determined through dynamic-ensemble refinement--a method that provides structural ensembles that simultaneously represent both the structure and the associated dynamics of a protein.

Models, Molecular↗

PubMedDate not supplied

Amino Acid Sequence Autocorrelation vectors and ensembles of Bayesian-Regularized Genetic Neural Networks for prediction of conformational stability of human lysozyme mutants.

Development of novel computational approaches for modeling protein properties from their primary structure is a main goal in applied proteomics. In this work, we reported the extension of the autocorrelation vector formalism to amino acid sequences for encoding protein structural information with modeling purposes. Amino Acid Sequence Autocorrelation (AASA) vectors were calculated by measuring the autocorrelations at sequence lags ranging from 1 to 15 on the protein primary structure of 48 amino acid/residue properties selected from the AAindex database. A total of 720 AASA descriptors were tested for building predictive models of the thermal unfolding Gibbs free energy change of human lysozyme mutants. In this sense, ensembles of Bayesian-Regularized Genetic Neural Networks (BRGNNs) were used for obtaining an optimum nonlinear model for the conformational stability. The ensemble predictor described about 88% and 68% variance of the data in training and test sets, respectively. Furthermore, the optimum AASA vector subset was shown not only to successfully model unfolding thermal stability but also to distribute wild-type and mutant lysozymes on a stability Self-organized Map (SOM) when used for unsupervised training of competitive neurons.

Algorithms↗

PubMedDate not supplied

The sampling properties of some distance geometry algorithms applied to unconstrained polypeptide chains: a study of 1830 independently computed conformations.

In this paper we study the statistical geometry of ensembles of poly (L-alanine) conformations computed by several different distance geometry algorithms. Since basic theory only permits us to predict the statistical properties of such ensembles a priori when the distance constraints have a very simple form, the only constraints used for these calculations are those necessary to obtain reasonable bond lengths and angles, together with a lack of short- and long-range atomic overlaps. The geometric properties studied include the squared end-to-end distance and radius of gyration of the computed conformations, in addition to the usual rms coordinate and phi/psi angle deviations among these conformations. The distance geometry algorithms evaluated include several variations of the well-known embed algorithm, together with optimizations of the torsion angles using the ellipsoid and variable target function algorithms. The conclusions may be summarized as follows: First, the distribution with which the trial distances are chosen in most implementations of the embed algorithm is not appropriate when no long-range upper bounds on the distances are present, because it leads to unjustifiably expanded conformations. Second, chosing the trial distances independently of one another leads to a lack of variation in the degree of expansion, which in turn produces a relatively low rms square coordinate difference among the members of the ensemble. Third, when short-range steric constraints are present, torsion angle optimizations that start from conformations obtained by choosing their phi/psi angles randomly with a uniform distribution between -180 degrees and +180 degrees do not converge to conformations whose angles are uniformly distributed over the sterically allowed regions of the phi/psi plane. Finally, in an appendix we show how the sampling obtained with the embed algorithm can be substantially improved upon by the proper application of existing methodology.

Algorithms↗

PubMed2001

Solution structure of native proteins with irregular folds from Raman optical activity.

Raman optical activity (ROA) spectra have been measured for the proteins hen phosvitin, yeast invertase, bovine alpha-casein, soybean Bowman-Birk protease inhibitor, and rabbit Cd(7)-metallothionein, all of which have irregular folds in the native state. The results show that ROA is able to distinguish between two types of disorder. Specifically, invertase, alpha-casein, the Bowman-Birk inhibitor, and metallothionein appear to possess a "static" type of disorder similar to that in disordered states of poly(L-lysine) and poly(L-glutamic acid); whereas phosvitin appears to possess a more "dynamic" type of disorder similar to that in reduced (unfolded) lysozyme and ribonuclease A and also in molten globule protein states. In the delimiting cases, static disorder corresponds to that found in loops and turns within native proteins with well-defined tertiary folds that contain sequences of residues with fixed but nonrepetitive phi,psi angles; and dynamic disorder corresponds to that envisaged for the model random coil in which there is a distribution of Ramachandran phi,psi angles for each amino acid residue, giving rise to an ensemble of interconverting conformers. In both cases there is a propensity for the phi,psi angles to correspond to the alpha, beta and poly(L-proline) II (PPII) regions of the Ramachandran surface, as in native proteins with well-defined tertiary folds. Our results suggest that, with the exception of invertase and metallothionein, an important conformational element present in the polypeptide and protein states supporting the static type of disorder is that of the PPII helix. Long sequences of relatively unconstrained PPII helix, as in alpha-casein, may impart a plastic (rheomorphic) character to the structure.

Animals↗

PubMed1998

Kinetics of lysozyme refolding: structural characterization of a non-specifically collapsed state using time-resolved X-ray scattering.

We report time-resolved small angle X-ray scattering (SAXS) studies of the structural characteristics of the collapsed state of lysozyme from henegg white (HEL) obtained on initiating refolding by rapidly changing solvent conditions from 8 M to 1.1 M urea at pH 2.9. At this reduced pH the lifetime, of about one second, of the non-specifically collapsed ensemble is considerably prolonged relative to its value at pH 5.2. The SAXS studies are combined with time resolved measurements of tryptophan fluorescence and of the rate of formation of native molecules using interrupted refolding experiments. We observe large burst phase changes in intrinsic tryptophan fluorescence and in the radius of gyration (Rg) which is reduced from 22 A in the fully unfolded state to approximately 19 to 20 A. Subsequent decrease of the Rg to the value for native lysozyme (15 A) follows the time course of formation of native molecules. Single exponential fits to the singular value decomposition (SVD) components of the SAXS data allow reconstruction of the SAXS profile at early time points of refolding. The results of this analysis suggest a globular shape of the collapsed state. A similar fit to the forward scattering amplitude, I(0), suggests that the collapsed state has a solvent accessible surface area which is considerably increased relative to that of the native protein. These results show directly that the non-specifically collapsed state formed during the burst phase in lysozyme refolding indeed represents a molecular compaction and a change in shape from a fully denatured random coil state (albeit restricted by disulfide bonds) to an ensemble of globular conformations which, however, have not yet formed a solvent-protected hydrophobic core.

Animals↗

PubMed2000

Structure-activity relationships in a peptidic alpha7 nicotinic acetylcholine receptor antagonist.

alpha-Conotoxins are small disulfide-constrained peptide toxins which act as antagonists at specific subtypes of nicotinic acetylcholine receptors (nACh receptors). In this study, we analyzed the structures and activities of three mutants of alpha-conotoxin ImI, a 12 amino acid peptide active at alpha7 nACh receptors, in order to gain insight into the primary and tertiary structural requirements of neuronal alpha-conotoxin specificity. NMR solution structures were determined for mutants R11E, R7L, and D5N, resulting in representative ensembles of 20 conformers with average pairwise RMSD values of 0.46, 0.52, and 0.62 A from their mean structures, respectively, for the backbone atoms N, C(alpha), and C' of residues 2-11. The R11E mutant was found to have activity near that of wild-type ImI, while R7L and D5N demonstrated activities reduced by at least two orders of magnitude. Comparison of the structures reveals a common two-loop architecture, with variations observed in backbone and side-chain dihedral angles as well as surface electrostatic potentials upon mutation. Correlation of these structures and activities with those from previously published studies emphasizes that existing hypotheses regarding the molecular determinants of alpha-conotoxin specificity are not adequate for explaining peptide activity, and suggests that more subtle features, visualized here at the atomic level, are important for receptor binding. These data, in conjunction with reported characterizations of the acetylcholine binding site, support a model of toxin activity in which a single solvent-accessible toxin side-chain anchors the complex, with supporting weak interactions determining both the efficacy and the subtype specificity of the inhibitory activity.

Amino Acid Sequence↗

PubMed2006

A marriage made in torsional space: using GALAHAD models to drive pharmacophore multiplet searches.

Pharmacophore multiplets are useful tools for 3D database searching, with the queries used ordinarily being derived from ensembles of random conformations of active ligands. It seems reasonable to expect that their usefulness can be augmented by instead using queries derived from single ligand conformations obtained from aligned ligands. Comparisons of pharmacophore multiplet searching using random conformations with multiplet searching using single conformations derived from GALAHAD (a genetic algorithm with linear assignment for hypermolecular alignment of datasets) models do indeed show that, while query hypotheses based on random conformations are quite effective, hypotheses based on aligned conformations do a better job of discriminating between active and inactive compounds. In particular, the hypothesis created from a neuraminidase inhibitor model was more similar to half of 18 known actives than all but 0.2% of the compounds in a structurally diverse subset of the World Drug Index. Similarly, a model developed from five angiotensin II antagonists yielded hypotheses that placed 65 known antagonists within the top 0.1-1% of decoy databases. The differences in discriminating power ranged from 2 to 20-fold, depending on the protein target and the type of pharmacophore multiplet used.

Algorithms↗

PubMed1990

Spectroscopic studies on elastin-like synthetic polypeptides.

Spectroscopic studies on synthetic polypeptides containing the unit-X-G-G (X=V or L) are reported. The sequences, constituting either fragments or model of elastin, were shown to adopt type II beta-turns together with an ensemble of unordered conformations. Furthermore, it was found that the stability of the beta-turns was depending on the nature of the X residue, on the hydration of the chain and, in the case of the sequence G-V-G-G-L, was decreasing by increasing the length of the chain.

Amino Acid Sequence↗

PubMed1992

Na+/K(+)-ATPase regulation by neurotransmitters.

A long period of experimental work has led to the conclusion that Na+/K(+)-ATPase is the enzymatic version of the Na+/K+ pump. This enzymatic system is in charge of various important cell functions. Among them cationic equilibrium and recovering of resting membrane potential in neurons is relevant. A tetrameric ensemble of peptides conform the system known as alpha and beta subunits. The alpha subunit is subdivided in alpha 1, alpha 2 and alpha 3, according to different location and properties. Regulatory factors intrinsic to the Na+/K(+)-ATPase system are: ATP, Na+ and Mg2+ concentrations inside the cell, and K+ outside. The enzyme activity is also regulated by extrinsic factors like some hormones (insulin and thyroxine). Induction of gene expression or post-translational modifications of the preexisting pool of the enzyme are the basic mechanisms of regulation proposed. Other extrinsic factors that seem to regulate the enzyme activity are some neurotransmitters. Among them the most extensively studied are catecholamines, mainly norepinephrine (NE) and lately serotonin (5-HT). The mechanism suggested for NE activation of the enzyme seems to involve specific receptors or a non-specific chelating action related to the catechol group that would relieve the inhibition by divalent cations. Another possibility is that NE removes an endogenous inhibitory factor present in the cytoplasm. The Na+/K(+)-ATPase is activated also by 5-HT. In vivo pharmacological and nutriological manipulations of brain 5-HT are accompanied by parallel responses of Na+/K(+)-ATPase activity. Serotonin agonists do activate the enzyme and antagonists neutralize the activation. In vitro there is a different dose dependent activation, according to the brain region. The mechanism involved seems to implicate a specific receptor system. Serotonin-Na+/K(+)-ATPase interaction in the rat brain is probably of functional relevance because it disappears in amygdaloid kindling. Also it seems to influence the ionic regulation of the pigment transport mechanism in crayfish photoreceptors. In relation to other neurotransmitters, a weak response to histamine was observed with acetylcholine, GABA and glutamic acid, the results were negative.

Animals↗

PubMed2004

The role of sequence and structure in protein folding kinetics; the diffusion-collision model applied to proteins L and G.

The diffusion-collision model (DCM) is applied to the folding kinetics of protein L and protein G. In the DCM, the two proteins are treated as consisting of two beta-hairpins and one alpha-helix, so that they are isomorphous with the three-helix bundle DCM model. In the absence of sequence dependent factors, both proteins would fold in the same way in the DCM, with the coalescence of the N-terminal hairpin and the helix slightly favored over the C-terminal hairpin and the helix because the former are closer together than the latter. However, sequence dependent factors make the N-terminal hairpin of protein L and the C-terminal hairpin of protein G more stable in the ensemble of unfolded conformations. This difference in the stabilities gives rise to the difference in the calculated folding behavior, in agreement with experiment.

Amino Acid Sequence↗

PubMed2004

Computational methods for generating models of denatured and partially folded proteins.

Partially folded and denatured proteins can give important insights into protein folding, misfolding, and aggregation. Such non-native states of proteins are however very difficult to characterise in detail as they are dynamic, heterogeneous systems comprising of ensembles of interconverting conformers. This article describes methods that produce models for non-native proteins in atomic detail. A variety of molecular dynamics based protocols are discussed together with some recent procedures that include restraints from experimental data. These models provide an important framework for interpreting experimental data from studies of non-native states using nuclear magnetic resonance spectroscopy, fluorescence, circular dichroism, and small angle scattering techniques.

Animals↗

PubMed2001

A semiflexible polymer model applied to loop formation in DNA hairpins.

A statistical mechanical "zipper" model is applied to describe the equilibrium melting of short DNA hairpins with poly(dT) loops ranging from 4 to 12 bases in the loop. The free energy of loop formation is expressed in terms of the persistence length of the chain. This method provides a new measurement of the persistence length of single-stranded DNA, which is found to be approximately 1.4 nm for poly(dT) strands in 100 mM NaCl. The free energy of the hairpin relative to the random coil state is found to scale with the loop size with an apparent exponent of > or = 7, much larger than the exponent of approximately 1.5-1.8 expected from considerations of loop entropy alone. This result indicates a strong dependence of the excess stability of the hairpins, from stacking interactions of the bases within the loop, on the size of the loop. We interpret this excess stability as arising from favorable hydrophobic interactions among the bases in tight loops and which diminish as the loops get larger. Free energy profiles along a generalized reaction coordinate are calculated from the equilibrium zipper model. The transition state for hairpin formation is identified as an ensemble of looped conformations with one basepair closing the loop, and with a lower enthalpy than the random coil state. The equilibrium model predicts apparent activation energy of approximately -11 kcal/mol for the hairpin closing step, in remarkable agreement with the value obtained from kinetics measurements.

Base Composition↗

PubMed1998

Monomeric bovine beta-lactoglobulin adopts a beta-barrel fold at pH 2.

We have determined a crude structure of the apo form of bovine beta-lactoglobulin, a protein of 162 amino acid residues with a molecular mass of 18 kDa, at a low pH on the basis of data collected using only homonuclear 1H NMR spectroscopy. An ensemble of protein conformations was calculated with the distance-geometry algorithm for NMR applications (DYANA). The monomeric protein at low pH adopts a beta-barrel fold, well-superimposable on the structure determined by X-ray crystallography for the dimer at physiological pH. NMR evidence suggests the presence of disordered loop regions and terminal segments. Structural differences between the monomer at pH 2 and the dimer at pH 7, obtained by X-ray crystallography, are discussed, paying particular attention to surface electrostatic properties, in view of the high charge state of the protein at low pH.

Algorithms↗

PubMed2002

Structure of a beta-alanine-linked polyamide bound to a full helical turn of purine tract DNA in the 1:1 motif.

Polyamides composed of N-methylpyrrole (Py), N-methylimidazole (Im) and N-methylhydroxypyrrole (Hp) amino acids linked by beta-alanine (beta) bind the minor groove of DNA in 1:1 and 2:1 ligand to DNA stoichiometries. Although the energetics and structure of the 2:1 complex has been explored extensively, there is remarkably less understood about 1:1 recognition beyond the initial studies on netropsin and distamycin. We present here the 1:1 solution structure of ImPy-beta-Im-beta-ImPy-beta-Dp bound in a single orientation to its match site within the DNA duplex 5'-CCAAAGAGAAGCG-3'.5'-CGCTTCTCTTTGG-3' (match site in bold), as determined by 2D (1)H NMR methods. The representative ensemble of 12 conformers has no distance constraint violations greater than 0.13 A and a pairwise RMSD over the binding site of 0.80 A. Intermolecular NOEs place the polyamide deep inside the minor groove, and oriented N-C with the 3'-5' direction of the purine-rich strand. Analysis of the high-resolution structure reveals the ligand bound 1:1 completely within the minor groove for a full turn of the DNA helix. The DNA is B-form (average rise=3.3 A, twist=38 degrees ) with a narrow minor groove closing down to 3.0-4.5 A in the binding site. The ligand and DNA are aligned in register, with each polyamide NH group forming bifurcated hydrogen bonds of similar length to purine N3 and pyrimidine O2 atoms on the floor of the minor groove. Each imidazole group is hydrogen bonded via its N3 atom to its proximal guanine's exocyclic amino group. The important roles of beta-alanine and imidazole for 1:1 binding are discussed.

Base Pairing↗

PubMed1995

Comparison of NMR solution structures of the receptor binding domains of Pseudomonas aeruginosa pili strains PAO, KB7, and PAK: implications for receptor binding and synthetic vaccine design.

The solution structures of peptide antigens from the receptor binding domains of Pseudomonas aeruginosa strains PAO and KB7 have been determined using two-dimensional 1H NMR techniques. Ensembles of solution conformations for the trans forms of these 17-residue disulfide-bridged peptides have been generated using a simulated annealing procedure in conjunction with distance and torsion angle restraints derived from NMR data. Comparison of the NMR-derived solution structures of the PAO and KB7 peptides, with that previously determined (McInnes et al., 1993) and herein refined for the PAK peptide reveals a common structural motif. All three peptide structures contain a type I beta-turn in the conserved sequence Asp134-X-X-Phe137 and a type II beta-turn in the conserved sequence Pro139-X-Gly-Cys142. However, the overall folds of the three peptides differ as well as the disposition of the side chains comprising the hydrophobic pockets. The similarities and differences between the structures of the three strains which bind to a common cell surface receptor are discussed in light of their contributions to synthetic vaccine design.

Amino Acid Sequence↗

PubMed1994

High-resolution solution structure of reduced parsley plastocyanin.

A high-resolution three-dimensional solution structure of parsley plastocyanin has been determined using 1H-NMR-derived data. An ensemble of 30 conformers has been calculated, exhibiting an atomic root mean square distribution about the mean coordinate positions of 0.37 +/- 0.03 A for backbone atoms and 0.75 +/- 0.04 A for all heavy atoms. (These values exclude residues 8-10 which are disordered.) The global fold of parsley plastocyanin is closely similar to those of other plastocyanins which have been structurally characterized by X-ray diffraction and NMR methods. However, deletion of residues at positions 57 and 58 of the consensus plastocyanin sequence causes elimination of a turn found in most higher plant plastocyanins. This turn is located in an acidic patch binding site, which consists of two clusters of acidic residues at positions 42-45 and 59-61. These residues surround the side chain of Tyr 83, which has been shown to be involved in binding of and electron transfer from cytochrome f, one of plastocyanin's physiological partners. The acidic recognition site is further disrupted in parsley plastocyanin by nonconservative substitution of two charged residues at positions 59 and 60. The NMR-derived structures show that E53, E85, and E95 compensate for these substitutions and give parsley plastocyanin an acidic recognition site of similar extent to that of other higher plant plastocyanins.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed2001

Characterization of carotenoid and chlorophyll photooxidation in photosystem II.

Photosystem II (PSII) contains two accessory chlorophylls (Chl(Z), ligated to D1-His118, and Chl(D), ligated to D2-His117), carotenoid (Car), and heme (cytochrome b(559)) cofactors that function as alternate electron donors under conditions in which the primary electron-donation pathway from the O(2)-evolving complex to P680(+) is inhibited. The photooxidation of the redox-active accessory chlorophylls and Car has been characterized by near-infrared (near-IR) absorbance, shifted-excitation Raman difference spectroscopy (SERDS), and electron paramagnetic resonance (EPR) spectroscopy over a range of cryogenic temperatures from 6 to 120 K in both Synechocystis PSII core complexes and spinach PSII membranes. The following key observations were made: (1) only one Chl(+) near-IR band is observed at 814 nm in Synechocystis PSII core complexes, which is assigned to Chl(Z)(+) based on previous spectroscopic studies of the D1-H118Q and D2-H117Q mutants [Stewart, D. H., Cua, A., Chisholm, D. A., Diner, B. A., Bocian, D. F., and Brudvig, G. W. (1998) Biochemistry 37, 10040-10046]; (2) two Chl(+) near-IR bands are observed at 817 and 850 nm in spinach PSII membranes which are formed with variable relative yields depending on the illumination temperature and are assigned to Chl(Z)(+), and Chl(D)(+), respectively; (3) the Chl and Car cation radicals have significantly different stabilities at reduced temperatures with Car(+) decaying much faster; (4) in Synechocystis PSII core complexes, Car(+) decays by recombination with Q(A)(-) and not by Chl(Z)/Chl(D) oxidation, with multiphasic kinetics that are attributed to an ensemble of protein conformers that are trapped as the protein is frozen; and (5) in spinach PSII membranes, Car(+) decays mainly by recombination with Q(A)(-), but also partly by formation of the 850 nm Chl cation radical. The greater stability of Chl(Z)(+) at low temperatures enabled us to confirm that resonance Raman bands previously assigned to Chl(Z)(+) are correctly assigned. In addition, the formation and decay of these cations provide insight into the alternate electron-donation pathways to P680(+).

Carotenoids↗

PubMed2001

Monomeric solution structure of the prototypical 'C' chemokine lymphotactin.

Lymphotactin, the sole identified member of the C class of chemokines, specifically attracts T lymphocytes and natural killer cells. This 93-residue protein lacks 2 of the 4 conserved cysteine residues characteristic of the other 3 classes of chemokines and possesses an extended carboxyl terminus, which is required for chemotactic activity. We have determined the three-dimensional solution structure of recombinant human lymphotactin by NMR spectroscopy. Under the conditions used for the structure determination, lymphotactin was predominantly monomeric; however, pulsed field gradient NMR self-diffusion measurements and analytical ultracentrifugation revealed evidence of dimer formation. Sequence-specific chemical shift assignments were determined through analysis of two- and three-dimensional NMR spectra of (15)N- and (13)C/(15)N-enriched protein samples. Input for the torsion angle dynamics calculations used in determining the structure included 1258 unique NOE-derived distance constraints and 60 dihedral angle constraints obtained from chemical-shift-based searching of a protein conformational database. The ensemble of 20 structures chosen to represent the structure had backbone and heavy atom rms deviations of 0.46 +/- 0.11 and 1.02 +/- 0.14 A, respectively. The results revealed that human lymphotactin adopts the conserved chemokine fold, which is characterized by a three-stranded antiparallel beta-sheet and a C-terminal alpha-helix. Two regions are dynamically disordered as evidenced by (1)H and (13)C chemical shifts and [(15)N]-(1)H NOEs: residues 1-9 of the amino terminus and residues 69-93 of the C-terminal extension. A functional role for the C-terminal extension, which is unique to lymphotactin, remains to be elucidated.

Amino Acid Sequence↗