An overview of protein-DNA and protein-RNA interactions

In this review the fundamental question of how does protein-DNA or protein-RNA interactions affect the structures and dynamics of DNA, RNA, and protein is addressed. Two models of human serum albumin (HSA) bindings to calf-thymus DNA and transfer RNA (tRNA) are presented here. In these models the binding sites, stability and structural aspects of DNA-protein and RNA-protein are discussed. Electrostatic binding of DNA or RNA via backbone phosphate group to the positively charged amino acids on the surface of protein is prevailing. Two binding sites with K₁ = 4.8 × 10⁵ M^?1 and K₂ = 6.1 × 10⁴ M^?1 for protein-DNA and one binding affinity with K = 1.45 × 10⁴ M^?1 for protein-RNA are observed. A partial B to A-DNA transition is observed for protein-DNA complexes, while tRNA remains in A-family structure upon protein interaction.     

A new application of scanning electrochemical microscopy for the label-free interrogation of antibody-antigen interactions

Within this work we present a ‘proof of principle’ study for the use of scanning electrochemical microscopy (SECM) to detect and image biomolecular interactions in a label-free assay as a potential alternative to current fluorescence techniques. Screen-printed carbon electrodes were used as the substrate for the deposition of a dotted array, where the dots consist of biotinylated polyethyleneimine. These were then further derivatised, first with neutravidin and then with a biotinylated antibody to the protein neuron specific enolase (NSE). SECM using a ferrocene carboxylic acid mediator showed clear differences between the array and the surrounding unmodified carbon. Imaging of the arrays before and following exposure to various concentrations of the antigen showed clear evidence for specific binding of the NSE antigen to the antibody derivatised dots. Non-specific binding was quantified. Control experiments with other proteins showed only non-specific binding across the whole of the substrate, thereby confirming that specific binding does occur between the antibody and antigen at the surface of the dots. Binding of the antigen was accompanied by a measured increase in current response, which may be explained in terms of protein electrostatic interaction and hydrophobic interactions to the mediator, thereby increasing the localised mediator flux. A calibration curve was obtained between 500 fg mL⁻¹ to 200 pg mL⁻¹ NSE which demonstrated a logarithmic relationship between the current change upon binding and antigen concentration without the need for any labelling of the substrate.     

A general NMR method for rapid, efficient, and reliable biochemical screening

High-throughput screening is usually the method of drug-lead discovery. It is now well accepted that, for a functional assay, quality is more important than quantity. The ligand-based or protein-based NMR screening methodologies for detecting compounds binding to the macromolecular target of interest are now well established. A novel and sensitive NMR method for rapid, efficient, and reliable biochemical screening is presented. The method named 3-FABS (three fluorine atoms for biochemical screening) requires the labeling of the substrate with a CF(3) moiety and utilizes (19)F NMR spectroscopy for the detection of the starting and enzymatically modified substrates. The method allows for high-quality screening of large compound or natural product extract collections and for measuring their IC(50) values. Applications of this technique to the screening of inhibitors of the Ser/Thr kinase AKT1 and the protease trypsin are presented. In addition, an interesting application of 3-FABS to functional genomics is also presented.     

(alpha/beta+alpha)-peptide antagonists of BH3 domain/Bcl-x(L) recognition: toward general strategies for foldamer-based inhibition of protein-protein interactions

The development of molecules that bind to specific protein surface sites and inhibit protein-protein interactions is a fundamental challenge in molecular recognition. New strategies for approaching this challenge could have important long-term ramifications in biology and medicine. We are exploring the concept that unnatural oligomers with well-defined conformations ("foldamers") can mimic protein secondary structural elements and thereby block specific protein-protein interactions. Here, we describe the identification and analysis of helical peptide-based foldamers that bind to a specific cleft on the anti-apoptotic protein Bcl-xL by mimicking an alpha-helical BH3 domain. Initial studies, employing a fluorescence polarization (FP) competition assay, revealed that among several alpha/beta- and beta-peptide foldamer backbones only alpha/beta-peptides intended to adopt 14/15-helical secondary structure display significant binding to Bcl-xL. The most tightly binding Bcl-xL ligands are chimeric oligomers in which an N-terminal alpha/beta-peptide segment is fused to a C-terminal alpha-peptide segment ((alpha/beta + alpha)-peptides)). Sequence-affinity relationships were probed via standard and nonstandard techniques (alanine scanning and hydrophile scanning, respectively), and the results allowed us to construct a computational model of the ligand/Bcl-xL complex. Analytical ultracentrifugation with a high-affinity (alpha/beta + alpha)-peptide established 1:1 ligand:Bcl-xL stoichiometry under FP assay conditions. Binding selectivity studies with the most potent (alpha/beta + alpha)-peptide, conducted via surface plasmon resonance measurements, revealed that this ligand binds tightly to Bcl-w as well as to Bcl-xL, while binding to Bcl-2 is somewhat weaker. No binding could be detected with Mcl-1. We show that our most potent (alpha/beta + alpha)-peptide can induce cytochrome C release from mitochondria, an early step in apoptosis, in cell lysates, and that this activity is dependent upon inhibition of protein-protein interactions involving Bcl-xL.     

A new H/D exchange- and mass spectrometry-based method for thermodynamic analysis of protein-DNA interactions

The application of SUPREX (stability of unpurified proteins from rates of H/D exchange) to the thermodynamic analysis of protein-DNA complexes is described. A series of five model protein-DNA complexes involving two known DNA binding proteins, Arc repressor and CopG, were analyzed in order to determine the accuracy, precision, and generality of the SUPREX technique for quantifying the strength of protein-DNA interactions. For protein-DNA complexes that reversibly unfold in a two-state manner, we demonstrate that reasonably precise Kd values in agreement with those determined by conventional techniques can be determined by SUPREX. In the case of protein-DNA complexes that are not well modeled by a two-state unfolding mechanism, we find that relative binding affinities can be determined in the SUPREX experiment.     

A general approach for the growth of metal oxide nanorod arrays on graphene sheets and their applications

In the fabrication of flexible devices, highly ordered nanoscale texturing, such as semiconductor metal oxide nanorod arrays on flexible substrates, is critical for optimal performance. Use of transparent conducting films, metallic films, and polymer substrates is limited by mechanical brittleness, chemical and thermal instability, or low electrical conductivity, low melting point, and so on. A simple and general nanocrystal-seed-directed hydrothermal route has now been developed for large-scale growth of nanorod arrays of various semiconductor metal oxides (MO), including TiO(2), ZnO, MnO(2), CuO, and ZrO(2) on both sides of flexible graphene (G) sheets to form sandwichlike MO/G/MO heterostructures. The TiO(2)/G/TiO(2) heterostructures have much higher photocatalytic activity than TiO(2) nanorods, with a photocatalytic degradation rate of methylene blue that is four times faster than that of the TiO(2) nanorods, and are thus promising candidates for photocatalytic decontamination.     

Stair motifs at protein-DNA interfaces: nonadditivity of H-bond, stacking, and cation-pi interactions

At the interface between protein and double-stranded DNA, stair motifs simultaneously involve three different types of pairwise interactions: aromatic base stacking, hydrogen bonding, and cation-pi. The relative importance of these interactions is studied in the stair motif occurring in the 1TC3 crystal structure, which involves an arginine and two stacked guanines, by means of Hartree-Fock (HF) and M?ller-Plesset energy and free energy calculations, including vibrational, rotational, translational contributions, both in a vacuum and various solvents. The results obtained show an anti-cooperative tendency of the HF energy and vibrational free energy terms, and the cooperativity of the rotational, translational, and solvation free energies. Hence, the cooperativity of the stair motif interactions, in the context of protein-DNA recognition, can be viewed as arising from the environment.     

A general approach for the generation of orthogonal tRNAs

BACKGROUND: The addition of new amino acids to the genetic code of Escherichia coli requires an orthogonal suppressor tRNA that is uniquely acylated with a desired unnatural amino acid by an orthogonal aminoacyl-tRNA synthetase. A tRNA(Tyr)(CUA)-tyrosyl-tRNA synthetase pair imported from Methanococcus jannaschii can be used to generate such a pair. In vivo selections have been developed for selecting mutant suppressor tRNAs with enhanced orthogonality, which can be used to site-specifically incorporate unnatural amino acids into proteins in E. coli. RESULTS: A library of amber suppressor tRNAs derived from M. jannaschii tRNA(Tyr) was generated. tRNA(Tyr)(CUA)s that are substrates for endogenous E. coli aminoacyl-tRNA synthetases were deleted from the pool by a negative selection based on suppression of amber nonsense mutations in the barnase gene. The remaining tRNA(Tyr)(CUA)s were then selected for their ability to suppress amber nonsense codons in the beta-lactamase gene in the presence of the cognate M. jannaschii tyrosyl-tRNA synthetase (TyrRS). Four mutant suppressor tRNAs were selected that are poorer substrates for E. coli synthetases than M. jannaschii tRNA(Tyr)(CUA), but still can be charged efficiently by M. jannaschii TyrRS. CONCLUSIONS: The mutant suppressor tRNA(Tyr)(CUA) together with the M. jannaschii TyrRS is an excellent orthogonal tRNA-synthetase pair for the in vivo incorporation of unnatural amino acids into proteins. This general approach may be expanded to generate additional orthogonal tRNA-synthetase pairs as well as probe the interactions between tRNAs and their cognate synthetases.     

A modular approach for the construction and modification of glyco-SAMs utilizing 1,3-dipolar cycloaddition

We report the synthesis of a broad variety of functionalized molecules for assembly on gold, allowing the formation of biologically relevant SAMs by a modular approach: either utilizing 1,3-dipolar cycloaddition of alkynes and azides in solution or by 'click on SAM'. Extensive studies into the various parameters of SAM formation and stability have been carried out, leading us to deduce reliable conditions under which glyco-decorated self-assembled monolayers can be formed and studied such as in SPR-supported binding assays.     

AMBER: Assisted model building with energy refinement. A general program for modeling molecules and their interactions

We describe a computer program we have been developing to build models of molecules and calculate their interactions using empirical energy approaches. The program is sufficiently flexible and general to allow modeling of small molecules, as well as polymers. As an illustration, we present applications of the program to study the conformation of actinomycin D. In particular, we study the rotational isomerism about the D -Val-, L -Pro, and L -Pro-Sar amide bonds as well as comparing the energy and structure of the Sobell model and the x-ray structure of actinomycin D.     

A general method for the rapid reduction of alkenes and alkynes using sodium borohydride, acetic acid, and palladium

Alkenes and alkynes are rapidly reduced to the corresponding alkanes using sodium borohydride and acetic acid in the presence of a small amount of palladium catalyst. The heterogeneous reaction is conducted in open air at room temperature. Reactions typically afford conversions to the alkane product of 98% or more within 15 min. The best solvent system was determined to be isopropyl alcohol, though reduction also takes place in solvents such as tetrahydrofuran, chloroform and, with some substrates, even in water. The method described is a convenient alternative to hydrogenations that require an external supply of hydrogen gas.     

A general approach to arylated furans,pyrroles, and thiophenes

A general and practical synthetic method for aryl-substituted five-membered heterocycles has been developed. In the presence of KOH (30%), 1,4-diaryl-1,3-butadiynes undergo the cyclocondensation reaction with water, primary amines, and Na₂S·9H₂O in DMSO at 80 °C to afford 2,5-diarylfurans, 1,2,5-trisubstituted pyrroles, and 2,5-diarylthiophenes in good to high yields. Further studies have disclosed that aryl-substituted five-membered heterocycles can be also synthesized by a one-pot, two-step strategy from the terminal alkynes in DMSO firstly catalyzed by CuCl, and then via addition of KOH to promote the cyclocondensation of 1,3-butadiynes generated in situ.     

A high-throughput approach to lanthanide complexes and their rapid screening in the ring opening polymerisation of caprolactone

Libraries of lanthanide complexes supported by nitrogen and oxygen containing ligands have been synthesised using a high-throughput approach. The complexes were employed in the ring-opening polymerisation of epsilon-caprolactone, in some cases giving polycaprolactone of controlled molecular weight and narrow polydispersity. The libraries, based on twenty-one ligands and eight lanthanide reagents, were developed in order to determine the best combination of lanthanide metal and ligand. They were prepared via transamination reactions of [Ln[N(SiMe(3))(2)](3)] complexes with tetradentate dianionic ligands containing oxygen and nitrogen donors. 1H NMR spectroscopy was used to screen polymerisation activity. The steric demand of the ligand has a significant effect on the polymerisation process, as do the type of nitrogen donor and the size of the central Ln(3+) ion. Ligands containing aryl rings with bulky substituents such as tert-pentyl groups afforded species capable of performing controlled polymerisation of caprolactone, whereas less bulky groups such as methyl were not effective. Yttrium and mid-sized lanthanides such as samarium showed increased activity compared with the larger lanthanides, lanthanum and praseodymium, and the smaller lanthanides like ytterbium. X-ray crystal structures of a sterically demanding chelating amine-bis((2-hydroxyaryl)methyl) ligand and a chloride bridged dinuclear gadolinium complex are reported. The centrosymmetric molecule contains gadolinium in distorted capped trigonal prismatic environments bonded to two amine, two phenolate, one THF and two chloride donors.     

A novel methodological approach for the analysis of host-ligand interactions.

Traditional analysis of drug-binding data relies upon the Scatchard formalism. These methods rely upon the fitting of a linear equation providing intercept and gradient data that relate to physical properties, such as the binding constant, cooperativity coefficients and number of binding sites. However, the existence of different binding modes with different binding constants makes the implementation of these models difficult. This article describes a novel approach to the binding model of host-ligand interactions by using a derived analytical function describing the observed signal. The benefit of this method is that physically significant parameters, that is, binding constants and number of binding sites, are automatically derived by the use of a minimisation routine. This methodology was utilised to analyse the interactions between a novel antitumour agent and DNA. An optical spectroscopy study confirms that the pentacyclic acridine derivative (DH208) binds to nucleic acids. Two binding modes can be identified: a stronger one that involves intercalation and a weaker one that involves oriented outer-sphere binding. In both cases the plane of the bound acridine ring is parallel to the nucleic acid bases, orthogonal to the phosphate backbone. Ultraviolet (UV) and circular dichroism (CD) data were fitted using the proposed model. The binding constants and the number of binding sites derived from the model remained consistent across the different techniques used. The different wavelengths at which the measurements were made maintained the coherence of the results.     

A thorough benchmark of density functional methods for general main group thermochemistry, kinetics, and noncovalent interactions

A thorough energy benchmark study of various density functionals (DFs) is carried out with the new GMTKN30 database for general main group thermochemistry, kinetics and noncovalent interactions [Goerigk and Grimme, J. Chem. Theor. Comput., 2010, 6, 107; Goerigk and Grimme, J. Chem. Theor. Comput., 2011, 7, 291]. In total, 47 DFs are investigated: two LDAs, 14 GGAs, three meta-GGAs, 23 hybrids and five double-hybrids. Besides the double-hybrids, also other modern approaches, i.e., the M05 and M06 classes of functionals and range-separated hybrids, are tested. For almost all functionals, the new DFT-D3 correction is applied in order to consistently test the performance also for important noncovalent interactions; the parameters are taken from previous works or determined for the present study. Basis set and quadrature grid issues are also considered. The general aim of the study is to work out which functionals are generally well applicable and robust to describe the energies of molecules. In summary, we recommend on the GGA level the B97-D3 and revPBE-D3 functionals. The best meta-GGA is oTPSS-D3 although meta-GGAs represent in general no clear improvement compared to numerically simpler GGAs. Notably, the widely used B3LYP functional performs worse than the average of all tested hybrids and is also very sensitive to the application of dispersion corrections. We discourage its usage as a standard method without closer inspection of the results, as it still seems to be often done nowadays. Surprisingly, long-range corrected exchange functionals do in general not perform better than the corresponding standard hybrids. However, the ωB97X-D functional seems to be a promising method. The most robust hybrid is Zhao and Truhlar's PW6B95 functional in combination with DFT-D3. If higher accuracy is required, double-hybrids should be applied. The corresponding DSD-BLYP-D3 and PWPB95-D3 variants are the most accurate and robust functionals of the entire study. Additional calculations with MP2 and and its spin-scaled variants SCS-MP2, S2-MP2 and SOS-MP2 revealed that double-hybrids in general outperform those. Only SCS-MP2 can be recommended, particularly for reaction energies. We suggest its usage when a large self-interaction error is expected that prohibits usage of double-hybrids. Perdews' metaphoric picture of Jacob's Ladder for the classification of density functionals' performance could unbiasedly be confirmed with GMTKN30. We also show that there is no statistical correlation between a functional's accuracy for atomization energies and the performance for chemically more relevant reaction energies.     

The development and characterization of protein-based stationary phases for studying drug-protein and protein-protein interactions

Protein-based liquid chromatography stationary phases are used in bioaffinity chromatography for studying drug-protein interactions, the determination of binding affinities, competitive and allosteric interactions, as well as for studying protein-protein interactions. This review addresses the development and characterization of protein-based stationary phase, and the application of these phases using frontal and zonal chromatography techniques. The approach will be illustrated using immobilized heat shock protein 90α and the immobilized estrogen related receptor stationary phases. In addition, the review discusses the use of the protein-coated magnetic beads for ligand and protein fishing as well as for the identification of unknown ligands from cellular or botanical extracts.     

A rapid and general method for the asymmetric synthesis of 2-substituted pyrrolidines using tert-butanesulfinamide

A new method for the asymmetric synthesis of 2-substituted pyrrolidines in three steps from commercially available starting materials is described. Addition of the Grignard reagent prepared from 2-(2-bromoethyl)-1,3-dioxane to N-tert-butanesulfinyl aldimines proceeds in high yields and with good diastereoselectivities. The sulfinamide products are then cleanly converted into pyrrolidines in one step.