Contributions of NH...O and CH...O hydrogen bonds to the stability of beta-sheets in proteins

Ab initio quantum calculations are applied to both the parallel and the antiparallel arrangements of the beta-sheets of proteins. The energies of the NH...O and CH...O hydrogen bonds present in the beta-sheet are evaluated separately from one another by appropriate modifications of the model systems. The bond energies of these two sorts of hydrogen bonds are found to be very nearly equal in the parallel beta-sheet. The NH...O bonds are stronger than CH...O in the antiparallel geometry but only by a relatively small margin. Moreover, the former NH...O bonds are weakened when placed next to one another, as occurs in the antiparallel beta-sheet. As a result, there is little energetic distinction between the NH...O and CH...O bonds in the full antiparallel beta-sheet, just as in the parallel structure.     

Aromatic-aromatic interactions in proteins: beyond the dimer

Aromatic residues are key widespread elements of protein structures and have been shown to be important for structure stability, folding, protein-protein recognition, and ligand binding. The interactions of pairs of aromatic residues (aromatic dimers) have been extensively studied in protein structures. Isolated aromatic molecules tend to form higher order clusters, like trimers, tetramers, and pentamers, that adopt particular well-defined structures. Taking this into account, we have surveyed protein structures deposited in the Protein Data Bank in order to find clusters of aromatic residues in proteins larger than dimers and characterized them. Our results show that larger clusters are found in one of every two unique proteins crystallized so far, that the clusters are built adopting the same trimer motifs found for benzene clusters in vacuum, and that they are clearly nonlocal brining primary structure distant sites together. We extensively analyze the trimers and tetramers conformations and found two main cluster types: a symmetric cluster and an extended ladder. Finally, using calmodulin as a test case, we show aromatic clsuters possible role in folding and protein-protein interactions. All together, our study highlights the relevance of aromatic clusters beyond the dimer in protein function, stability, and ligand recognition.     

Strength of Calpha-H...O=C hydrogen bonds in transmembrane proteins

A large number of Calpha-H...O contacts are present in transmembrane protein structures, but contribution of such interactions to protein stability is still not well understood. According to previous ab initio quantum calculations, the stabilization energy of a Calpha-H...O contact is about 2-3 kcal/mol. However, experimental studies on two different Calpha-H...O hydrogen bonds present in transmembrane proteins lead to conclusions that one contact is only weakly stabilizing and the other is not even stabilizing. We note that most previous computational studies were on optimized geometries of isolated molecules, but the experimental measurements were on those in the structural context of transmembrane proteins. In the present study, 263 Calpha-H...O=C contacts in alpha-helical transmembrane proteins were extracted from X-ray crystal structures, and interaction energies were calculated with quantum mechanical methods. The average stabilization energy of a Calpha-H...O=C interaction was computed to be 1.4 kcal/mol. About 13% of contacts were stabilizing by more than 3 kcal/mol, and about 11% were destabilizing. Analysis of the relationships between energy and structure revealed four interaction patterns: three types of attractive cases in which additional Calpha-H...O or N-H...O contact is present and a type of repulsive case in which repulsion between two carbonyl oxygen atoms occur. Contribution of Calpha-H...O=C contacts to protein stability is roughly estimated to be greater than 5 kcal/mol per helix pair for about 16% of transmembrane helices but for only 3% of soluble protein helices. The contribution would be larger if Calpha-H...O contacts involving side chain oxygen were also considered.     

Block copolymers confined in a nanopore: pathfinding in a curving and frustrating flatland

We have studied structure formation in a confined block copolymer melt by means of dynamic density functional theory. The confinement is two dimensional, and the confined geometry is that of a cylindrical nanopore. Although the results of this study are general, our coarse-grained molecular model is inspired by an experimental lamella-forming polysterene-polybutadiene diblock copolymer system [K. Shin et al., Science 306, 76 (2004)], in which an exotic toroidal structure was observed upon confinement in alumina nanopores. Our computational study shows that a zoo of exotic structures can be formed, although the majority, including the catenoid, helix, and double helix that were also found in Monte Carlo nanopore studies, are metastable states. We introduce a general classification scheme and consider the role of kinetics and elongational pressure on stability and formation pathway of both equilibrium and metastable structures in detail. We find that helicity and threefold connections mediate structural transitions on a larger scale. Moreover, by matching the remaining parameter in our mesoscopic method, the Flory-Huggins parameter chi, to the experimental system, we obtain a structure that resembles the experimental toroidal structure in great detail. Here, the most important factor seems to be the roughness of the pore, i.e., small variations of the pore radius on a scale that is larger than the characteristic size in the system.     

Applications of LC/MS in structure identifications of small molecules and proteins in drug discovery

With advancements in ionization methods and instrumentation, liquid chromatography/mass spectrometry (LC/MS) has become a powerful technology for the characterization of small molecules and proteins. This article will illustrate the role of LC/MS analysis in drug discovery process. Examples will be given on high-throughput analysis, structural analysis of trace level impurities in drug substances, identification of metabolites, and characterization of therapeutic protein products for process improvement. Some unique MS techniques will also be discussed to demonstrate their effectiveness in facilitating structural identifications.     

Luminescent proteins from Aequorea victoria: applications in drug discovery and in high throughput analysis

Recent progress in generating a vast number of drug targets through genomics and large compound libraries through combinatorial chemistry have stimulated advancements in drug discovery through the development of new high throughput screening (HTS) methods. Automation and HTS techniques are also highly desired in fields such as clinical diagnostics. Luminescence-based assays have emerged as an alternative to radiolabel-based assays in HTS as they approach the sensitivity of radioactive detection along with ease of operation, which makes them amenable to miniaturization. Luminescent proteins provide the advantage of reduced reagent and operating costs because they can be produced in unlimited amounts through the use of genetic engineering tools. In that regard, the use of two naturally occurring and recombinantly produced luminescent proteins from the jellyfish Aequorea victoria, namely, aequorin and the green fluorescent protein (GFP), has attracted attention in a number of analytical applications in diverse research areas. Aequorin is naturally bioluminescent and has therefore, virtually no associated background signal, which allows its detection down to attomole levels. GFP has become the reporter of choice in a variety of applications given that it is an autofluorescent protein that does not require addition of any co-factors for fluorescence emission. Furthermore, the generation of various mutants of GFP with differing luminescent and spectral properties has spurred additional interest in this protein. In this review, we focus on the use of aequorin and GFP in the development of highly sensitive assays that find applications in drug discovery and in high throughput analysis.     

Luminescent proteins from Aequorea victoria: applications in drug discovery and in high throughput analysis

Recent progress in generating a vast number of drug targets through genomics and large compound libraries through combinatorial chemistry have stimulated advancements in drug discovery through the development of new high throughput screening (HTS) methods. Automation and HTS techniques are also highly desired in fields such as clinical diagnostics. Luminescence-based assays have emerged as an alternative to radiolabel-based assays in HTS as they approach the sensitivity of radioactive detection along with ease of operation, which makes them amenable to miniaturization. Luminescent proteins provide the advantage of reduced reagent and operating costs because they can be produced in unlimited amounts through the use of genetic engineering tools. In that regard, the use of two naturally occurring and recombinantly produced luminescent proteins from the jellyfish Aequorea victoria, namely, aequorin and the green fluorescent protein (GFP), has attracted attention in a number of analytical applications in diverse research areas. Aequorin is naturally bioluminescent and has therefore, virtually no associated background signal, which allows its detection down to attomole levels. GFP has become the reporter of choice in a variety of applications given that it is an autofluorescent protein that does not require addition of any co-factors for fluorescence emission. Furthermore, the generation of various mutants of GFP with differing luminescent and spectral properties has spurred additional interest in this protein. In this review, we focus on the use of aequorin and GFP in the development of highly sensitive assays that find applications in drug discovery and in high throughput analysis.     

Relationship between the bond lengths in N-H...N,O-H...O,F-H...F,and Cl-H...Cl hydrogen bridges

The applicability of the equation $ e^{ - ((r_1 - r_0 )/b)^{5/3} } + e^{ - ((r_2 - r_0 )/b)^{5/3} } = 1 $ e^{ - ((r_1 - r_0 )/b)^{5/3} } + e^{ - ((r_2 - r_0 )/b)^{5/3} } = 1 has been studied. The equation defines the relationship between the experimental values of the covalent (r ₁) and hydrogen (r ₂) bond lengths in O-H...O bridges for describing the relation between the experimental interatomic distances in N-H...N bridges and the parameters of X-H...X fragments (X = O, N, F, Cl) calculated by the density functional method (B3LYP/6-31++G(d,p)) for neutral, positive, and negative molecular complexes. Here r ₀ is the mean value of the X-H bond length in free molecules; r _sym is the X...H distance in the symmetrical bridge; and b is the coefficient defined by the equation b = (r _sym − r ₀)/(ln2)^3/5. This equation allows us to adequately describe the relationships between bond lengths in nearly linear hydrogen bridges formed by oxygen, nitrogen, fluorine, and chlorine atoms. It is thus universal and can be used in studies of a wide range of substances.     

Computational discovery of putative quorum sensing inhibitors against LasR and RhlR receptor proteins of Pseudomonas aeruginosa

Drugs have been discovered in the past mainly either by identification of active components from traditional remedies or by unpredicted discovery. A key motivation for the study of structure based virtual screening is the exploitation of such information to design targeted drugs. In this study, structure based virtual screening was used in search for putative quorum sensing inhibitors (QSI) of Pseudomonas aeruginosa. The virtual screening programme Glide version 5.5 was applied to screen 1,920 natural compounds/drugs against LasR and RhlR receptor proteins of P. aeruginosa. Based on the results of in silico docking analysis, five top ranking compounds namely rosmarinic acid, naringin, chlorogenic acid, morin and mangiferin were subjected to in vitro bioassays against laboratory strain PAO1 and two more antibiotic resistant clinical isolates, P. aeruginosa AS1 (GU447237) and P. aeruginosa AS2 (GU447238). Among the five compounds studied, except mangiferin other four compounds showed significant inhibition in the production of protease, elastase and hemolysin. Further, all the five compounds potentially inhibited the biofilm related behaviours. This interaction study provided promising ligands to inhibit the quorum sensing (QS) mediated virulence factors production in P. aeruginosa.     

Interplay of phenyl-perfluorophenyl stacking,C-H...F,C-F...pi and F...F interactions in some crystalline aromatic azines

Analysis of phenyl-perfluorophenyl stacking synthon, C-H...F, C-F...pi interactions, and F...F tetramer in three closely related azine crystal structures shows the dominance of Ar-ArF synthon while other interactions are turned on/off depending on the H/F stoichiometry in the molecule.     

Weak C-H...O and C-H...F-C hydrogen bonds in the oxirane-trifluoromethane dimer

The oxirane-trifluoromethane dimer generated in a supersonic expansion has been characterized by Fourier transform microwave spectroscopy. The rotational spectra of the parent species and of its two (13)C isotopomers in combination with ab initio calculations have been used to establish a C(s)() geometry for the dimer with the two monomers bound by one C-H.O and two C-H.F-C hydrogen bonds. An overall bonding energy of about 6.7 kJ/mol has been derived from the centrifugal distortion analysis. The lengths of the C-H.O and C-H.F hydrogen bonds, r(O.H) and r(F.H), are 2.37 and 2.68 A, respectively. The C-H.F-C interactions give rise to the HCF(3) internal rotation motion barrier of 0.55(1) kJ/mol, which causes the A-E splittings observed in the rotational spectra. The analysis of the structural and energetic features of the C-H.O and C-H.F-C interactions allows us to classify them as weak hydrogen bonds. Ab initio calculations predict these weak interactions to produce blue shifts in the C-H vibrational frequencies and shortenings of the C-H lengths.     

Assessment of metals bound to marine plankton proteins and to dissolved proteins in seawater

Studies based on laser ablation–inductively coupled plasma-mass spectrometry (LA–ICP-MS) have been performed to assess metal bound to dissolved proteins and proteins from marine plankton after two-dimensional polyacrylamide gel electrophoresis (2D PAGE). Dissolved proteins were pre-concentrated from surface seawater (60 L) by tangential ultrafiltration with 10 kDa molecular weight cut-off (MWCO) membranes and further centrifugal ultrafiltration (10 kDa) before proteins isolation by methanol/chloroform/water precipitation. Proteins isolation from plankton was assessed after different trichloroacetic acid (TCA)/acetone and methanol washing stages, and further proteins extraction with a phenol solution. LA–ICP-MS analysis of the electrophoretic profiles obtained for dissolved proteins shows the presence of Cd, Cr, Cu, and Zn in five spots analyzed. These proteins exhibit quite similar molecular weights (within the 10–14 kDa range) and pIs (from 5.8 to 7.3). Cd, Cr, Cu, and Zn have also been found to be associated to proteins isolated from plankton samples. In this case, Cd has been found to be bound to proteins of quite different molecular weight (9, 13 and 22 kDa) and pIs (4.5, 5.2, 5.5, and 10). However, trace elements such as Cr, Cu and Zn appear to be mainly bound to plankton proteins of low molecular weight and variable pI.     

Modern drug discovery technologies: opportunities and challenges in lead discovery

The identification of promising hits and the generation of high quality leads are crucial steps in the early stages of drug discovery projects. The definition and assessment of both chemical and biological space have revitalized the screening process model and emphasized the importance of exploring the intrinsic complementary nature of classical and modern methods in drug research. In this context, the widespread use of combinatorial chemistry and sophisticated screening methods for the discovery of lead compounds has created a large demand for small organic molecules that act on specific drug targets. Modern drug discovery involves the employment of a wide variety of technologies and expertise in multidisciplinary research teams. The synergistic effects between experimental and computational approaches on the selection and optimization of bioactive compounds emphasize the importance of the integration of advanced technologies in drug discovery programs. These technologies (VS, HTS, SBDD, LBDD, QSAR, and so on) are complementary in the sense that they have mutual goals, thereby the combination of both empirical and in silico efforts is feasible at many different levels of lead optimization and new chemical entity (NCE) discovery. This paper provides a brief perspective on the evolution and use of key drug design technologies, highlighting opportunities and challenges.     

Citrinin revisited: from monomers to dimers and beyond

Detailed chemical analysis of the solid phase fermentation of an Australian Penicillium citrinum isolate has returned the known compounds citrinin (1), phenol A acid (6), dihydrocitrinone (7) and dihydrocitrinin (8), together with a novel cytotoxic dimer, dicitrinin A (5). Dicitrinin A (5) was determined to be a dimerised artefact of the major co-metabolite citrinin, and its structure solved by spectroscopic analysis and chemical modification. Analysis of the products encountered during the controlled decomposition of citrinin led to the discovery of additional citrinin dimers and delineated a plausible mechanistic pathway linking all monomeric and dimeric citrinin degradation products.     

Quinolactacins revisited: from lactams to imide and beyond

Chemical analysis of a solid phase fermentation of an Australian Penicillium citrinum strain has returned all known examples of a rare class of N-methyl quinolone lactams, quinolactacins A2 (1), B2 (2), C2 (3) and A1 (4), together with the new quinolactacins B1 (5), C1 (6), D1 (7) and D2 (8), and the novel derivatives quinolonimide (9) and quinolonic acid (10). Complete stereostructures were assigned to all these compounds by detailed spectroscopic analysis and chemical interconversion. Carefully controlled and monitored decomposition studies have confirmed that quinolactacins readily undergo C-3 epimerization and oxidation, and under appropriate conditions convert to quinolonimide and quinolonic acid. Mechanisms for key transformations are proposed. The decomposition studies suggested that only quinolactacins A2 (1) and B2 (2) are genuine natural products, with all other isolated compounds being decomposition artefacts. Quinolactacins C1 (6), C2 (3), and the racemic mixture of quinolactacins D1/D2 (8/7) all displayed notable cytotoxic activity.