Disulfide bonds, their stereospecific environment and conservation in protein structures

We studied the specificity of the non-bonded interaction in the environment of 572 disulfide bonds in 247 polypeptide chains selected from the Protein Data Bank. The preferred geometry of interaction of peptide oxygen atoms is along the back of the two covalent bonds at the sulfur atom of half cystine. With aromatic residues the geometries that direct one of the sulfur lone pair of electrons into the aromatic pi-system are avoided; an orientation in which the sulfide plane is normal or inclined to the aromatic plane and on top of its edge is normally preferred. The importance of the S...aromatic interaction is manifested in the high degree of its conservation across members in homologous protein families. These interactions, while providing extra overall stability to the native fold and reducing the accessibility of the disulfide bond and thereby preventing exchange reactions, also set the orientation of the conserved aromatic rings for further interactions and binding to another molecule. The conformational features and the mode of interactions of disulfide bridges should be useful for molecular design and protein engineering experiments.     

Probing the role of aromaticity in the design of dipeptide based nanostructures

Self-assembly of peptide into nanostructures is believed to be stabilized primarily by aromatic interactions. Using a minimalistic approach, we probed the importance of aromatic interactions in the self-assembly of simple model dipeptides. Our results suggest that aromaticity may not be absolutely essential for self-assembly, even though it tends to provide directionality to the assembly. We found that peptides containing cyclic/linear side chain hydrophobic residues were also capable of forming stable self-assemblies that are stabilized by hydrophobic interactions. Our observations will find relevance in the design of small peptide based nanoparticles.     

Synthesis and Quantitative Structure–Activity Relationships of Selective BCRP Inhibitors

The breast cancer resistance protein (BCRP/ABCG2) is a member of the ABC transporter superfamily. This protein has a number of physiological functions, including protection of the human body from xenobiotics. The overexpression of BCRP in certain tumor cell lines causes cross‐resistance against various drugs used in chemotherapeutic treatment. In a previous work we showed that a new class of compounds derived from XR9576 (tariquidar) selectively inhibits BCRP. In this work we synthesized more members of this class, with modification on the second and third aromatic rings. The inhibitory activities against BCRP and P‐gp were assayed using a Hoechst 33342 assay for BCRP and a calcein AM assay for P‐gp. Finally, quantitative structure–activity relationships for both aromatic rings were established. The results obtained show the importance of the electron density on the third aromatic ring, influenced by substituents, pointing to interactions with aromatic residues of the protein binding site. In the second aromatic ring the activity of compounds is influenced by the steric volume of the substituents.     

The predicted 3D structures of the human M1 muscarinic acetylcholine receptor with agonist or antagonist bound

The muscarinic acetylcholine G-protein-coupled receptors are implicated in diseases ranging from cognitive dysfunctions to smooth-muscle disorders. To provide a structural basis for drug design, we used the MembStruk computational method to predict the 3D structure of the human M1 muscarinic receptor. We validated this structure by using the HierDock method to predict the binding sites for three agonists and four antagonists. The intermolecular ligand-receptor contacts at the predicted binding sites agree well with deductions from available mutagenesis experiments, and the calculated relative binding energies correlate with measured binding affinities. The predicted binding site of all four antagonists is located between transmembrane (TM) helices 3, 4, 5, 6, and 7, whereas the three agonists prefer a site involving residues from TM3, TM6, and TM7. We find that Trp 157(4) contributes directly to antagonist binding, whereas Pro 159(4) provides an indirect conformational switch to position Trp 157(4) in the binding site (the number in parentheses indicates the TM helix). This explains the large decrease in ligand binding affinity and signaling efficacy by mutations of Trp 157(4) and Pro 159(4) not previously explained by homology models. We also found that Asp 105(3) and aromatic residues Tyr 381(6), Tyr 404(7), and Tyr 408(7) are critical for binding the quaternary ammonium head group of the ligand through cation-pi interactions. For ligands with a charged tertiary amine head group, we suggest that proton transfer from the ligand to Asp 105(3) occurs upon binding. Furthermore, we found that an extensive aromatic network involving Tyr 106(3), Trp 157(4), Phe 197(5), Trp 378(6), and Tyr 381(6) is important in stabilizing antagonist binding. For antagonists with two terminal phenyl rings, this aromatic network extends to Trp 164(4), Tyr 179(extracellular loop 2), and Phe 390(6) located at the extracellular end of the TMs. We find that Asn 382(6) forms hydrogen bonds with selected antagonists. Tyr381(6) and Ser 109(3) form hydrogen bonds with the ester moiety of acetylcholine, which binds in the gauche conformation.     

Experimental measurement of noncovalent interactions between halogens and aromatic rings

Chemical double mutant cycles have been used to quantify the interactions of halogens with the faces of aromatic rings in chloroform. The halogens are forced over the face of an aromatic ring by an array of hydrogen-bonding interactions that lock the complexes in a single, well-defined conformation. These interactions can also be engineered into the crystal structures of simpler model compounds, but experiments in solution show that the halogen-aromatic interactions observed in the solid state are all unfavourable, regardless of whether the aromatic rings contain electron-withdrawing or electron-donating substituents. The halogen-aromatic interactions are repulsive by 1-3 kJ mol(-1). The interactions with fluorine are slightly less favourable than with chlorine and bromine.     

High-resolution X-ray analysis reveals binding of arginine to aromatic residues of lysozyme surface: implication of suppression of protein aggregation by arginine

While biotechnological applications of arginine (Arg) as a solution additive that prevents protein aggregation are increasing, the molecular mechanism of its effects remains unclear. In this study, we investigated the Arg-lysozyme complex by high-resolution crystallographic analysis. Three Arg molecules were observed to be in close proximity to aromatic amino acid residues of the protein surface, and their occupancies gradually increased with increasing Arg concentration. These interactions were mediated by electrostatic, hydrophobic and cation-π interactions with the surface residues. The binding of Arg decreased the accessible surface area of aromatic residues by 40%, but increased that of charged residues by 10%. These changes might prevent intermolecular hydrophobic interactions by shielding hydrophobic regions of the lysozyme surface, resulting in an increase in protein solubility.     

Chemically prepared hevein domains: effect of C-terminal truncation and the mutagenesis of aromatic residues on the affinity for chitin

Chemically prepared hevein domains (HDs), N-terminal domainof an antifungal protein from Nicotiana tabacum (CBP20-N) andan antimicrobial peptide from Amaranthus caudatus (Ac-AMP2),were examined for their affinity for chitin, a ß-1,4-linkedpolymer of N-acetylglucosamine. An intact binding domain, CBP20-N,showed a higher affinity than a C-terminal truncated domain,Ac-AMP2. The formation of a pyroglutamate residue from N-terminalGln of CBP20-N increased the affinity. The single replacementof any aromatic residue of Ac-AMP2 with Ala resulted in a significantreduction in affinity, suggesting the importance of the completeset of three aromatic residues in the ligand binding site. Themutations of Phe18 of Ac-AMP2 to the residues with larger aromaticrings, i.e. Trp, ß-(1-naphthyl)alanine or ß-(2-naphthyl)alanine,enhanced the affinity, whereas the mutation of Tyr20 to Trpreduced the affinity. The affinity of an HD for chitin mightbe improved by adjusting the size and substituent group of stackingaromatic rings.     

Effects of volatile-char interactions on the evolution of char structure during the gasification of Victorian brown coal in steam

The purpose of this study is to investigate the effects of volatile-char interactions on the evolution of char structure during the gasification of Victorian brown coal in steam. A novel one-stage fluidised-bed/fixed-bed quartz reactor was employed to carry out the experiments in the presence and absence of volatile-char interactions. The effects of thermal annealing on char structure were also investigated under similar conditions. The structural features of char were evaluated using FT-Raman spectroscopy. The results indicate that the char structural features were considerably affected by volatile-char interactions, which was shown from the Raman band area or the ratios between the band areas. H radicals from the thermal cracking/reforming of volatiles are believed to play a vital role in the changes in char structure due to the volatile-char interactions. H radicals could penetrate into char matrix and favour the condensation of aromatic rings, which was the main reason for the decrease in the ratio of small (less than 6 fused rings) to large aromatic rings during the volatile-char interactions. The volatile-char interactions also greatly affected the concentrations of O-containing groups in char and thus significantly altered the observed Raman intensity of the char.     

Anion binding involving pi-acidic heteroaromatic rings

Anions are essential species in biological systems and, particularly, in enzyme-substrate recognition. Therefore, the design and preparation of anion receptors is a topical field of supramolecular chemistry. Most host-guest systems successfully developed are based on noncovalent (ionic and hydrogen-bonded) interactions between anions and ammonium-type functionalities or Lewis acid groups. However, since the past 5 years, an alternative route toward the synthesis of efficient anion hosts has emerged, namely, the use of "anion-pi" interactions involving nitrogen-containing electron-deficient aromatic rings, as the result of several favorable theoretical investigations. In this Account, the state of the (new) art in this growing area of anion-binding research is presented and several selected examples from our work and that of other groups will be discussed.     

Influence of cation-π interactions in protein-DNA complexes

Protein-DNA recognition plays a crucial role in gene expression and regulation. In this work, we have analyzed the influence of cation-π interactions to the stability of 62 protein-DNA complexes. A new criterion has been formulated to delineate the cation-π interactions based on (i) the distribution of atoms in the π system (5 and 6-member rings) of DNA bases around the positive charged atoms of Lys and Arg and (ii) the energetic contribution of contacting atoms from electrostatic and van der Waals interactions. Our method shows the presence of cation-π interactions in 92% of the complexes. The side chain of Arg is more likely than that of Lys to be in cation-π interactions. In both Lys and Arg, the cationic groups have stronger cation-π interaction energy than the atoms with effective positive charge. The aromatic chains of purines (A and G) are exhibiting more cation-π interactions than pyrimidines (C and T). The Arg-G pair has the strongest interaction energy of −4.3 kcal/mol among all the possible pairs of amino acids and bases. The interaction energy is always positive for T and we observed few favorable interactions with C. Further, we found that the cation-π interactions due to 5-member rings of A and G are stronger than that with the atoms in 6-member rings. The distribution of base atoms around the charged atoms shows that the N7 in the 5-member ring of G is making significant number of close contacts with NZ of Arg, which is important to establish dominant cation-π interactions.     

Self-assembly of peptide nanotubes and amyloid-like structures by charged-termini-capped diphenylalanine peptide analogues

We had recently demonstrated that the diphenylalanine peptide, the core recognition motif of the Alzheimer's ß-amyloid polypeptide, self-assembles into a novel class of peptide nanotubes. The formation of well-ordered supramolecular structures at the nanoscale by such a simple peptide was consistent with our suggestion that aromatic interactions may provide order and directionality needed for the formation of fibrillar peptide structures. Yet, we could not rule out a contribution of the charged amine and carboxyl moieties at the termini of the short peptide. In order to explore the potential role of electrostatic interaction in the assembly process we have studied a modified non-charged peptide analogue, Ac-Phe–Phe-NH₂, in which the N-terminal amine was acetylated and the C-terminal carboxyl was amidated. Scanning and transmission electron microscopy analyses demonstrated that this peptide analogue self-assembles into highly-ordered tubular structures, as observed with the NH₂-Phe–Phe-COOH. Also, infrared spectroscopy revealed an amide I absorbance pattern that is very similar to that of the non-modified peptide. Furthermore, an amidated NH₂-Phe–Phe-NH₂ peptide, which has a net positive charge, also self-assembled into ordered tubular structures. On the other hand, the amine-modified analogues Boc-Phe–Phe-COOH, Z-Phe–Phe-COOH, and Fmoc-Phe–Phe-COOH peptides formed amyloid-like structures that had a significantly smaller diameter. Taken together, the current study further supports our hypothesis regarding the role of aromatic interactions in the self-assembly of amyloid fibrils and amyloid-associated nanostructures that can be modulated by simple chemical modifications.     

The chemical and physical structure of hydrogenation residues of maceral concentrates

The chemical structure of the residues from hydrogenation of maceral concentrates of an Australian bituminous coal have been investigated using nuclear magnetic resonance and infrared spectroscopic techniques and microscopy. It is shown that even before coal morphology is modified, aromatic substitution reactions result in loss of ether and/or phenolic groups from the coal. The results also demonstrate that the structure of residues depends markedly on the hydrogen source available for reaction. Tetralin, 1-methylnaphthalene and molecular hydrogen provide hydrogen to cap radicals, the order of efficiency being tetralin > molecular hydrogen > 1-methylnaphthalene. Although molecular hydrogen and 1-methylnaphthalene are insufficiently reactive to cause liquefaction, they reduce the degree of cross-linking of aromatic rings, thereby enhancing the plasticity of the residues.     

Analysis of cation-π interactions to the structural stability of RNA binding proteins

Cation-π interactions play an important role to the stability of protein structures. In this work, we have analyzed the influence of cation-π interactions in RNA binding proteins. We observed cation-π interactions in 32 out of 51 RNA binding proteins and there is a strong correlation between the number of amino acid residues and number of cation-π interactions. The analysis on the influence of short (<±3 residues), medium (±3 or ±4 residues) and long range contacts (>±4 residues) showed that the cation-π interactions are mainly formed by long-range contacts. The cation-π interaction energy for Arg-Trp is found to be the strongest among all interacting pairs. Analysis on the preferred secondary structural conformation of the residues involved in cation-π interaction indicates that the cationic Lys and Arg prefer to be in α-helices and β-strands, respectively, whereas the aromatic residues prefer to be in strand and coil regions. Most of the cation-π interactions forming residues in RNA binding proteins are conserved among homologous sequences. Further, the cation-π interactions have distinct roles to the stability of RNA binding proteins in addition to other conventional non-covalent interactions. The results observed in the present study will be useful in understanding the contribution of cation-π interactions to the stability of RNA binding proteins.     

Bimodal character of polyester–solvent interactions. I. Evaluation of the solubility parameters of the aromatic and the aliphatic ester residues of poly(ethylene terephthalate)

The Hildebrand and the Hansen solubility parameters of the aromatic and the aliphatic ester residues of poly(ethylene terephthalate), PET, are evaluated and compared to those determined experimentally. The interactions of nonaqueous solvents with the aromatic and aliphatic ester residues of PET are also described in terms of their relative basicity and acidity in the Lewis sense, where the aromatic residue may be taken as a Lewis acid and the aliphatic ester residue may be taken as a Lewis base.     

π-Stacking of rhodamine B onto water-soluble polymers containing aromatic groups

Evidences of the π-stacking of rhodamine B onto poly(sodium 4-styrenesulfonate) are given by ¹H NMR spectroscopy. As a consequence of these π-π interactions, changes on the diafiltration and UV-vis absorbance and fluorescence patterns are shown comparing water-soluble polymers containing aromatic rings as poly(sodium 4-styrenesulfonate) and poly(N-methacryloyl-5-aminosalicylic acid) with other polyanions that do not contain aromatic rings as poly(sodium vinylsulfonate) and poly(acrylic acid).     

Reactions and Intermediates at the Metal-Polymer Interface as Observed by XPS and NEXAFS Spectroscopy

Potassium or chromium were evaporated by means of a Knudsen effusion cell under ultra-high vacuum conditions onto a number of common polymers, prepared as stretched foils and spin-coated films. The metal-polymer interface was studied by X-ray Absorption and X-ray Photoelectron Spectroscopy. Evaporated samples were analyzed without exposure to the atmosphere. Different general types of reactions of the metal atoms with the polymers were observed. With deposited K and Cr a redox process including the transfer of substrate oxygen atoms across the interface was found. The formation of π-electron complexes and covalent metal-carbon bonds were obtained exclusively with chromium. Aromatic rings, carbonyl groups and, to a lesser extent, ether linkages are scissioned by metal-polymer interactions. The fourfold substitution of aromatic rings and the exclusive existence of C ─ O ─ C structures within polyphenylene ether (PPE) make this polymer most stable toward reactions with chromium. In contrast, bisphenol-A polycarbonate is susceptible to redox reactions with the carbonate group and to forming Cr complexes with aromatic rings using the chromium 3d orbitals. The result is a succession of complex formation, followed by Cr substitution onto aromatic rings and destruction of rings with the formation of chromium carbides.     

Reactions and Intermediates at the Metal-Polymer Interface as Observed by XPS and NEXAFS Spectroscopy

Potassium or chromium were evaporated by means of a Knudsen effusion cell under ultra-high vacuum conditions onto a number of common polymers, prepared as stretched foils and spin-coated films. The metal-polymer interface was studied by X-ray Absorption and X-ray Photoelectron Spectroscopy. Evaporated samples were analyzed without exposure to the atmosphere. Different general types of reactions of the metal atoms with the polymers were observed. With deposited K and Cr a redox process including the transfer of substrate oxygen atoms across the interface was found. The formation of π-electron complexes and covalent metal-carbon bonds were obtained exclusively with chromium. Aromatic rings, carbonyl groups and, to a lesser extent, ether linkages are scissioned by metal-polymer interactions. The fourfold substitution of aromatic rings and the exclusive existence of C ─ O ─ C structures within polyphenylene ether (PPE) make this polymer most stable toward reactions with chromium. In contrast, bisphenol-A polycarbonate is susceptible to redox reactions with the carbonate group and to forming Cr complexes with aromatic rings using the chromium 3d orbitals. The result is a succession of complex formation, followed by Cr substitution onto aromatic rings and destruction of rings with the formation of chromium carbides.     

Targeting EphA2‐Sam and Its Interactome: Design and Evaluation of Helical Peptides Enriched in Charged Residues

The EphA2 receptor controls diverse physiological and pathological conditions and its levels are often upregulated in cancer. Targeting receptor overexpression, through modulation of endocytosis and consequent degradation, appears to be an appealing strategy for attacking tumor malignancy. In this scenario, the Sam domain of EphA2 plays a pivotal role because it is the site where protein regulators of endocytosis and stability are recruited by means of heterotypic Sam–Sam interactions. Because EphA2‐Sam heterotypic complexes are largely based on electrostatic contacts, we have investigated the possibility of attacking these interactions with helical peptides enriched in charged residues. Several peptide sequences with high predicted helical propensities were designed, and detailed conformational analyses were conducted by diverse techniques including NMR, CD, and molecular dynamics (MD) simulations. Interaction studies were also performed by NMR, surface plasmon resonance (SPR), and microscale thermophoresis (MST) and led to the identification of two peptides capable of binding to the first Sam domain of Odin. These molecules represent early candidates for the generation of efficient Sam domain binders and antagonists of Sam–Sam interactions involving EphA2.     

Chemical transformations during pyrolysis of Rundle oil shale

Rundle shale (Queensland, Australia) was pyrolysed at 12.5 K min⁻¹ to 350–500 °C for 10–240 min. The structures of the liquid products and pyrolysis residues were investigated by a number of n.m.r. spectroscopic techniques including cross-polarization and dipolar dephasing. N.m.r. provided a simple method for detecting nitrile carbon and measuring terminal and internal olefinic hydrogen in shale oil. It was found that the ratio of terminal olefinic hydrogen to internal olefinic hydrogen in shale oil increases by a factor of three over the range 350–500 °C. Moreover, the results suggest that aromatic rings in Rundle shale residues are not highly substituted and hence that aromatic ring condensation reactions are not important during pyrolysis. From elemental, yield and n.m.r. data, the conversion of aliphatic carbon to aromatic carbon during pyrolysis was found to be as high as 25% at 500 °C.