Contribution Of Side Chains Research Articles

Different contributions of side-chains in β- d-(1 → 3,6)-galactans on intestinal Peyer’s patch-immunomodulation by polysaccharides from Astragalus mongholics Bunge

Thirteen polysaccharides isolated from an extract of the aerial portions of Astragalus mongholics Bunge demonstrated immunomodulating activity against Peyer’s patch immunocompetent cells. Nine of the active polysaccharide fractions were composed of either arabinogalactans, pectic arabinogalactans or pectins. The activities of the arabinogalactans and pectic arabinogalactans were associated with β- d-(1 → 3)-galactan moieties branched with β- d-(1 → 6)-galactooligosaccharide side-chains having degrees of polymerization of 8 or less. Degradation of the β- d-(1 → 3)-galactan or β- d-(1 → 6)-galactosyl side-chains in the arabinogalactans significantly decreased immunomodulating activity. Rhamnogalacturonan I (RG-I) with β- d-(1 → 3,6)-galactosyl side-chains having terminal β- d-GlcA showed activity in the pectin-enriched fractions. Interestingly, the terminal GlcA was not required for activity of the arabinogalactan-enriched fractions, suggesting at least two different immunomodulating structures.

In situ monitoring of backbone thioester exchange by 19F NMR.

backbone thioester exchange; coiled coil; fluorine; NMR spectroscopy; protein foldingBackbone thioester exchange (BTE) is a new technique for assessing the stability of foldingpatterns adopted by polypeptides.[1–5] Implementation of this method requires thereplacement of a single backbone amide group in the polypeptide of interest with a thioester;this replacement is an approximately isostructural substitution.[ 6,7] Combining the backbone-modified polypeptide analogue (a thiodepsipeptide) with a small thiol in pH 7 aqueous bufferinitiates a thiol–thioester exchange reaction.[8] The equilibrium constant for this exchange isstrongly influenced by the stability of the folded conformation adopted by the full-lengththiodepsipeptide. We have shown that BTE enables examination of sequence–stabilityrelationships for a variety of small folding motifs under native conditions including β-hairpins,coiled coils and a small protein, the chicken villin headpiece subdomain.[1–5] BTE analysisis complementary to traditional methods of assessing protein conformational stability, in whichthe folding pattern is disrupted with heat or chemical additives.[ 9] The traditional approachesrequire extrapolation to obtain folding parameters that apply under native conditions, whileBTE enables direct measurement under native conditions. BTE has provided new insights oncommon protein folding motifs[ 1,3–5] and on contributions of unnatural side chains to tertiarystructural stability,[2] but continued development is necessary to maximize the utility of thisnew tool.Here we report that quantitative BTE analysis can be conducted in situ by using

Thermodynamic properties of peptide solutions 19. Partial molar isothermal compressions at T = 298.15 K of some peptides of sequence gly-X-gly in aqueous solution

The partial molar isobaric expansions at infinite dilution and T = 298.15 K, E 2 ∘ { E 2 ∘ = ( ∂ V 2 ∘ / ∂ T ) p } were determined recently for some tripeptides of sequence glycyl-X-glycine, where X is one of the amino acids alanine, valine, leucine, isoleucine, phenylalanine, methionine, proline, serine, threonine, asparagine, glutamine, histidine, and tyrosine. These quantities, along with partial molar heat capacity data available from the literature, were used to convert the partial molar isentropic compressions at infinite dilution determined previously for these tripeptides into the partial molar isothermal compressions at infinite dilution and T = 298.15 K, K T , 2 ∘ { K T , 2 ∘ = - ( ∂ V 2 ∘ / ∂ p ) T } . These K T , 2 ∘ results, along with that for triglycine, were used to estimate the contributions of the amino acid side-chains to the partial molar isothermal compressions of peptides. The side-chain contributions have been rationalized in terms of the likely peptide–water interactions.

Calculation of Proteins’ Total Side-Chain Torsional Entropy and Its Influence on Protein–Ligand Interactions

Despite the high density within a typical protein fold, the ensemble of sterically permissible side-chain repackings is vast. Here, we examine the extent of this variability that survives energetic biases due to van der Waals interactions, hydrogen bonding, salt bridges, and solvation. Monte Carlo simulations of an atomistic model exhibit thermal fluctuations among a diverse set of side-chain arrangements, even with the peptide backbone fixed in its crystallographic conformation. We have quantified the torsional entropy of this native-state ensemble, relative to that of a noninteracting reference system, for 12 small proteins. The reduction in entropy per rotatable bond due to each kind of interaction is remarkably consistent across this set of molecules. To assess the biophysical importance of these fluctuations, we have estimated side-chain entropy contributions to the binding affinity of several peptide ligands with calmodulin. Calculations for our fixed-backbone model correlate very well with experimentally determined binding entropies over a range spanning more than 80 kJ/(mol·308 K).

Quantitative structure-activity relationship (QSAR) study of elastase substrates and inhibitors.

One hundred Suc-X-Y-Ala-pNA peptides: (Suc: succinyl, pNA: p-nitroanilide, X, Y: Gly, Ala, Val, Leu, Ile, Phe, Pro, alpha-aminobutyric acid, norvaline, norleucine) were synthesized and their reaction constants with porcine pancreatic elastase (Km, kcat and kcat/Km) were determined. These reaction constants were quantitatively analyzed using the Free-Wilson/Fujita-Ban method. The contribution of amino acid side chains to the reaction constants Km, kcat and kcat/Km, expressed logarithmically, was found to be additive. On the other hand, 19 elastase inhibitors of the general formula CF3CO-X-Y-Ala-pNA (X,Y: ten amino acids) were synthesized, and their inhibition constants were compared with the Michaelis constant for the corresponding substrates and analyzed using free-energy-related substituent constants. In the analysis of amino acid side chains in the Y position, the Ki value of the inhibitor was generally correlated to the Km value of the substrate, which corresponded to the inhibitor, thus confirming the validity of the equation. log(1/Ki) = 1.271 log(1/Km) + 4.831 This study may serve as a prototypical approach to unraveling structure-activity relationships of peptide substrates and inhibitors of medicinal or agricultural importance.

Comparative structure–activity relationship studies of 1-(5-methylsulfonylpyrid-2-yl)-5-alkyl and (hetero)aryl triazoles and pyrazoles in canine COX inhibition

Structure-activity relationship (SAR) studies of novel 5-alkyl and 5-aryl/heteroaryl substituted 1,2,4-triazoles are described. The in vitro activity is compared to the pyrazole class of compounds with analogous side chains to delineate the contribution of the triazole ring nitrogen in binding to the active site. Both series are quite potent and selective in the canine whole blood (CWB) COX-2 assay, suggesting the increased binding contribution of the hydrophobic side chains.

Phenotypic classification of mutants: a tool for understanding ligand binding and activation of muscarinic acetylcholine receptors

GPCRs (G-protein-coupled receptors) such as the M(1) muscarinic receptor have so far proved recalcitrant to direct structure determination. Nevertheless systematic mutagenesis, particularly alanine scanning, has advanced our understanding of their structure-function relationships. GPCRs exhibit multiple conformational states with different affinities for and abilities to activate their cognate G-proteins. Ligand binding alters these conformational equilibria, thus promoting or inhibiting signalling. Alanine-scanning mutagenesis probes the relative contributions of a particular amino acid side chain to the stability of the ground and activated states of the receptor and its complexes. These determine the phenotype of the mutant receptor. Classification of the phenotypes suggests functional roles for particular amino acid side chains, allowing us to group them accordingly. From a rhodopsin-based homology model of the M(1) mAChR, a coherent view emerges of how these clusters of residues function in ligand anchoring, transduction of binding energy, global structural stabilization and selective stabilization of the ground state or the activated state of the receptor. We can identify differences in ligand-binding modes, and suggest inter- and intra-molecular interactions that are weakened or broken, or formed or intensified during acetylcholine-induced activation. In due course, we may be able to extend these insights to activation by unconventional agonists.

Apparent Molar Volumes and Viscosity B-Coefficients of Some Amino Acids in Aqueous Tetramethylammonium Iodide Solutions at 298.15 K

Apparent molar volumes and viscosity B-coefficients of glycine, l-alanine, and l-valine in 0.05, 0.10, and 0.15 mol·dm-3 aqueous tetramethylammonium iodide (TMAI) solutions have been determined at 298.15 K from density and viscosity measurements. The standard partial molar volumes, standard volumes of transfer, and hydration number of the amino acids have been calculated for rationalizing various interactions in the ternary solutions. It has been found that the partial molar volume and viscosity B-coefficient varies linearly with the number of carbon atoms in the alkyl chain of the amino acids, and they were split into contributions from charged end groups (NH3+, COO-) and the CH2− group of amino acids. Furthermore, the side chain contribution to the partial molar volume and viscosity B-coefficient of the amino acids has been determined using a group additivity approach. The results have been discussed in terms of ion−dipolar, hydrophobic−hydrophobic, and hydrophilic−hydrophobic group interactions.

Double Barrel Shotgun Scanning of the Caveolin-1 Scaffolding Domain

In the postgenomic era, a major challenge remains, elucidating the thermodynamic forces governing receptor-ligand specificity and promiscuity. We report a straightforward approach for mapping side-chain contributions to binding for the multipartner interactions characteristic of the human proteome. Double barrel shotgun scanning dissects binding to two or more targets through combinatorial mutagenesis of one protein binding to multiple targets. Examined here, the caveolin-1 scaffolding domain (CSD) binds to and inhibits both endothelial nitric oxide synthase (eNOS) and protein kinase A (PKA). Homolog shotgun scanning of CSD highlights residues responsible for CSD oligomerization and binding to eNOS and PKA. The experiments uncover a general mechanism in which CSD oligomerizes and deoligomerizes to modulate binding affinity to partner proteins. The results provide a detailed look at a multipartner protein interaction, uncovering strategies for one protein binding to multiple partners.

Comparison between computational alanine scanning and per‐residue binding free energy decomposition for protein–protein association using MM‐GBSA: Application to the TCR‐p‐MHC complex

Recognition by the T-cell receptor (TCR) of immunogenic peptides (p) presented by Class I major histocompatibility complexes (MHC) is the key event in the immune response against virus-infected cells or tumor cells. A study of the 2C TCR/SIYR/H-2K(b) system using a computational alanine scanning and a much faster binding free energy decomposition based on the Molecular Mechanics-Generalized Born Surface Area (MM-GBSA) method is presented. The results show that the TCR-p-MHC binding free energy decomposition using this approach and including entropic terms provides a detailed and reliable description of the interactions between the molecules at an atomistic level. Comparison of the decomposition results with experimentally determined activity differences for alanine mutants yields a correlation of 0.67 when the entropy is neglected and 0.72 when the entropy is taken into account. Similarly, comparison of experimental activities with variations in binding free energies determined by computational alanine scanning yields correlations of 0.72 and 0.74 when the entropy is neglected or taken into account, respectively. Some key interactions for the TCR-p-MHC binding are analyzed and some possible side chains replacements are proposed in the context of TCR protein engineering. In addition, a comparison of the two theoretical approaches for estimating the role of each side chain in the complexation is given, and a new ad hoc approach to decompose the vibrational entropy term into atomic contributions, the linear decomposition of the vibrational entropy (LDVE), is introduced. The latter allows the rapid calculation of the entropic contribution of interesting side chains to the binding. This new method is based on the idea that the most important contributions to the vibrational entropy of a molecule originate from residues that contribute most to the vibrational amplitude of the normal modes. The LDVE approach is shown to provide results very similar to those of the exact but highly computationally demanding method.

Backbone Importance for Protein-Protein Binding.

Although a number of studies have focused on the physical and chemical properties of protein-protein interfaces of complexes to determine their unique features, the importance of the backbone hydrogen bonds to protein-protein binding has been neglected due to the difficulty of quantitatively measuring their contribution to the free binding energy. In this study we are presenting a computational approach that allows the estimation of the contribution to the free binding energy of the CO and NH groups of the backbone of various proteic complexes. A correlation between the quantitative calculated free binding energy contribution of the CO and NH backbone groups of the interfacial residues and the qualitative values expected for this kind of interaction was achieved. The contribution of the backbone to the ΔΔGbinding is significant. The average ΔΔGbinding contribution of the intermolecular hydrogen bonds of the backbone is 1.77 kcal/mol, which is very similar to the average contribution of the different side chains to the ΔΔGbinding, with a value of 1.75 kcal/mol. Therefore, the application of this computational approach as well as an alanine scanning mutagenesis study is essential to a more detailed comprehensive knowledge of proteic complex formation.

Kinetic Isotope Effects Reveal the Presence of Significant Secondary Structure in the Transition State for the Folding of the N-terminal Domain of L9

Our present understanding of the nature of the transition state for protein folding depends predominantly on studies where individual side-chain contributions are mapped out by mutational analysis (ϕ value analysis). This approach, although extremely powerful, does not in general provide direct information about the formation of backbone hydrogen bonds. Here, we report the results of amide H/D isotope effect studies that probe the development of hydrogen bonded interactions in the transition state for the folding of a small α−β protein, the N-terminal domain of L9. Replacement of amide protons by deuterons in a solvent of constant isotopic composition destabilized the domain, decreasing both its T m and Δ G 0 of unfolding. The folding rate also decreased. The parameter Φ H/D, defined as the ratio of the effect of isotopic substitution upon the activation free energy to the equilibrium free energy was determined to be 0.6 in a D 2O background and 0.75 in a H 2O background, indicating that significant intraprotein hydrogen bond interactions are developed in the transition state for the folding of NTL9. The value is in remarkably good agreement with more traditional measures of the position of the transition state, which report on the relative burial of surface area. The results provide a picture of a compact folding transition state containing significant secondary structure. Indirect analysis argues that the bulk of the kinetic isotope effect arises from the β-sheet-rich region of the protein, and suggests that the development of intraprotein hydrogen bonds in this region plays a critical role in the folding of NTL9.

Solvation Free Energy of Amino Acids and Side-Chain Analogues

The solvation free energies of amino acids and their side-chain analogues in water and cyclohexane are calculated by using Monte Carlo simulation. The molecular interactions are described by the OPLS-AA force field for the amino acids and the TIP4P model for water, and the free energies are determined by using the Bennett acceptance method. Results for the side-chain analogues in cyclohexane and in water are used to evaluate the performance of the force field for the van der Waals and the electrostatic interactions, respectively. Comparison of the calculated hydration free energies for the amino acid analogues and the full amino acids allows assessment of the additivity of the side chain contributions on the number of hydrating water molecules. The hydration free energies of neutral amino acids can be reasonably approximated by adding the contributions of their side chains to that of the hydration of glycine. However, significant nonadditivity in the free energy is found for the zwitterionic form of amino acids with polar side chains. In serine and threonine, intramolecular hydrogen bonds are formed between the polar side chains and backbone groups, leading to weaker solvation than for glycine. In contrast, such nonadditivity is not observed in tyrosine, in which the hydroxyl group is farther separated from, and therefore cannot form an intramolecular hydrogen bond with, the backbone. For histidine we find that a water molecule can form a bridge when the intramolecular hydrogen bond between the polar group and the backbone is broken.

Small-angle X-ray scattering from polystyrene polymacromonomers with relatively short main chains

Synchrotron small-angle X-ray scattering measurements were made on polystyrene polymacromonomers F33 and F110 with 33 and 110 styrene residues, respectively, in each side chain in toluene at 298.2 K and cyclohexane at 307.7 K. Two F33 samples with different weight-average degrees of polymerization of main chain Nw of 37 and 17 (F33-13 and F33-14, respectively) and two F110 samples with Nw of 61 and 27 (F110-10 and F110-11, respectively) were used. The z-average mean-square radii of gyration of these polymacromonomers were explained by the wormlike chain with finite diameter when we considered the contribution of side chains near the main chain to the contour length. This model was also applied to calculate the particle scattering function P(θ), giving satisfactory agreements between calculated and experimental k2P(θ) for F33-13 and F110-10 plotted against the magnitude of the scattering vector k = (4π/λ0)sin(θ/2), where λ0 and θ denote the wavelength of the incident X-ray and the scattering angle, respectively. The data for F33-14 and F110-11, which have short main chains relative to the side chains, cannot be explained by the same model. However, these data were closely fitted by the theoretical values for a comb polymer model with semiflexible main and side chains. The stiffness parameter for side chains in toluene was larger than that in cyclohexane, suggesting that the side chains in the former solvent are more stretched than those in cyclohexane.

P2X5 Subunit Assembly Requires Scaffolding by the Second Transmembrane Domain and a Conserved Aspartate

Functional homomeric and heteromeric ATP-gated P2X receptor channels have been shown to display a characteristic trimeric architecture. Of the seven different isoforms (designated P2X(1)-P2X(7)), P2X(5) occurs in humans primarily as a non-functional variant lacking the C-terminal end of the ectodomain and the outer half of the second transmembrane domain. We show that this truncated variant, which results from the splice-skipping of exon 10, is prone to subunit aggregation because the residual transmembrane domain 2 is too short to insert into the membrane. Alleviation of the negative hydrophobic mismatch by the addition of a stretch of moderately hydrophobic residues enabled formation of a second membrane-spanning domain and strictly parallel homotrimerization. Systematic mutagenesis identified only one transmembrane domain 2 residue, Asp(355), which supported homotrimerization in a side chain-specific manner. Our results indicate that transmembrane domain 2 formation contributes 2-fold to hP2X(5) homotrimerization by tethering the end of the ectodomain to the membrane, thereby topologically restricting conformational mobility, and by intramembrane positioning of Asp(355). While transmembrane domain 2 appears to favor assembly by enabling productive subunit interactions in the ectodomain, Asp(355) seems to assist by simultaneously driving intramembrane helix interactions. Overall, these results indicate a complex interplay between topology, helix-helix interactions, and oligomerization to achieve a correctly folded structure.

Novel antitumor agent family of 1 H-benzo[ c, d]indol-2-one with flexible basic side chains: Synthesis and biological evaluation

A series of mono-1 H-benzo[ c, d]indol-2-one with different amine side chains and bis-1 H-benzo[ c, d]indol-2-one as novel family of DNA intercalators were designed and synthesized, the contributions of aromatic chromophores and amine side chains for DNA binding properties, for example, intercalation and electrostatic binding, respectively, were evaluated. Among them, A3 tailed with N, N-dimethylamino-ethyl-ethane-1,2-diamine showed selective anti-tumor activities against cell lines A549 and P388 with IC 50 0.428 μm and 1.69 μm.

Interactions of some α-amino acids with tetra- n-alkylammonium bromides in aqueous medium at different temperatures

The densities and viscosities were measured for binary mixtures of 0.01 M aqueous tetramethylammonium bromide (TMAB) and tetraethylammonium bromide (TEAB) and ternary mixtures of glycine, dl-alanine and dl-valine (0.01 to 0.05 M) in 0.01 M-aqueous tetra- n-alkylammonium bromides at T = (298.15, 303.15, 308.15, and 313.15) K and at atmospheric pressure. The apparent molar volume, ϕ v, was computed using the density data. The limiting apparent molar volume, ϕ v ∘ of amino acids and the slope, S v ∗ were obtained using the Masson’s equation. The transfer volume, ϕ v ∘ ( tr ) of the amino acids and their side-chain contribution, ϕ v ∘ ( R ) have also been determined. The viscosity data have been analyzed on the basis of the Jones–Dole equation, the viscosity A and B-coefficients and free energy of activation of viscous flow, per mole of solvent, Δ μ 1 ∘ # and solute, Δ μ 2 ∘ # have been evaluated. Both ϕ v ∘ and Δ μ 2 ∘ # vary linearly with increase in number of C atoms in the alkyl chain of the amino acids. This linearity is used to find the contribution of the zwitterionic end groups, ( NH 4 + , COO - ) and those of CH 2 groups of side-chain to the ϕ v ∘ and Δ μ 2 ∘ # . The variations of these parameters with concentration and temperature clearly suggests the role of alkyl cation on the solute–solvent interactions in aqueous medium. The structural influence of the large cations of quaternary ammonium salts upon solvent is also taken into consideration.

Thermodynamic and transport properties of l-serine and l-threonine in aqueous sodium acetate and magnesium acetate solutions at T = 298.15 K

The apparent molar volumes, V 2, ϕ , apparent molar adiabatic compressibilities, κ S,2, ϕ , and relative viscosities, η r, of l-serine and l-threonine in different concentrations of aqueous sodium acetate and magnesium acetate solutions at T = 298.15 K have been obtained from the measurements of densities, speed of sound, and flow time, respectively. These data are used to derive the partial molar volumes, V 2 ∘ , partial molar adiabatic compressibilities, κ S , 2 ∘ (at infinite dilution), and viscosity B-coefficients. The corresponding quantities of transfer, (Δ t V 0, Δ t κ S , 2 ∘ , and Δ t B), have been obtained. The activation free energy, Δ μ 2 ∘ ≠ , for viscous flow has been calculated for l-serine and l-threonine in aqueous solutions. The hydration numbers, n H, side chain contributions, and interaction coefficients have also been calculated. The values of V 2 ∘ , κ S , 2 ∘ , viscosity B-coefficient, and Δ μ 2 ∘ ≠ for viscous flow increase with the concentration of sodium acetate and magnesium acetate solutions. The influence of magnesium acetate on the transfer properties is greater than sodium acetate in aqueous solutions. A comparison of Δ t V 0, Δ κ S , 2 ∘ , and Δ t B values for l-serine and l-threonine with the reported data for dl-α-alanine and dl-α-amino- n-butyric acid in aqueous sodium acetate and magnesium acetate solutions shows that the values are greater for l-serine and l-threonine at the same concentration of sodium acetate and magnesium acetate.

Simulation of FT-IR spectra and 2D-COS analysis for the H/D exchange of two related ligands

Two-dimensional IR correlation spectroscopy (2D-COS) is a relatively novel method that provides the analysis of infrared spectra with the capacity to differentiate overlapping peaks and to distinguish between in-phase and out-of-phase spectral responses. The technique is becoming popular for protein dynamic studies, like H/D exchange attenuated total reflectance Fourier transform-infrared (ATR-FT-IR) spectroscopy. The use of this technique provides the capability to resolve the dynamic molecular events that occur upon H → D exchange of accessible amide protons and exchangeable protons within side chain (Arg, Tyr) that are commonly overlapped. The value of the use of simulated spectra to generate the 2D-COS plots is that it allows for confirmation of the existence of the auto-peaks and its correlations. Because of this, we have employed the use of simulated spectra originated from backbone vibrational contributions such as amide I, I′, II and II′ bands for a case study presented herein. Intensity, bandwidth, and band position were the parameters used to study their variation in the correlation plots during a typical H/D exchange ATR-FT-IR experiment. These simulations are compared with experimental spectral data from ATR-FT-IR analysis in order to separate the backbone contribution from the highly overlapped side chain contributions (mainly Arg and Tyr).

Application of Near-Infrared and Fourier Transform Infrared Spectroscopy in the Characterization of Ligand-Induced Conformation Changes in Folate Binding Protein Purified from Bovine Milk: Influence of Buffer Type and pH

Fourier transform infrared (FT-IR) and near-infrared (NIR) spectroscopy have been applied to detect structural alterations in folate binding protein (FBP) induced by ligation in different buffer types. The amide I region pointed to a beta-sheet to alpha-helix transition upon ligation in acetate and phosphate buffers, and the formation of intermolecular beta-sheet was indicated at pH 5.0, in agreement with a dimerization of FBP taking place at this pH. The ligand-induced changes in the 2100-2300 nm NIR region were significant for FBP in acetate and phosphate buffers of pH 5.0, and the variations were interpreted as secondary structure changes, based on previous assignments of secondary structures to the combination bands in the NIR region. In the case of acetate buffer, variations in the amide combination bands agreed with the amide I analysis, but for the other buffer types some discrepancies were found and explained by side-chain contributions to the NIR, which could reflect the tertiary and quaternary structure differences. NIR spectra of FBP at pH 7.4 and 5.0 revealed contradictory effects on the side chains, reflecting different polymerization events at the two pH values, whereas the amide I region indicated similar changes at the two pH values. Therefore, we suggest that FT-IR and NIR spectroscopy may complement each other, such that the two techniques in combination may give information on all three types of protein conformational changes. While the secondary structure changes are revealed by FT-IR, the tertiary and quaternary structure changes are reflected in the NIR spectra, although the general influence of the latter changes on the NIR spectra remains to be confirmed.