Monitoring protein-small molecule interactions by local pH modulation

We have developed a technique for sensing protein-small molecule and protein-ion interactions in bulk aqueous solution by utilizing a pH sensitive dye, 5-(and-6)-carboxyfluorescein, conjugated to free lysine residues on the surfaces of designated capture proteins. The fluorescein intensity was found to change by about 6% and 15% for small molecule and ion binding, respectively. The assay works by modulating the local electric fields around a pH sensitive dye. This, in turn, alters the dye's apparent pK(A) value. Such changes may result directly from the charge on the analyte, occur through allosteric effects related to the binding process, or result from a combination of both. The assay was used to follow the binding of Ca(2+) to calmodulin (CaM) and thiamine monophosphate (ThMP) to thiamine binding protein A (TbpA). The results demonstrate a binding constant of 1.1μM for the Ca(2+)/CaM pair and 3.2nM for ThMP/TbpA pair, which are in excellent agreement with literature values. These assays demonstrate the generality of this method for observing the interactions of small molecules and ions with capture proteins. In fact, the assay should work as a biosensor platform for most proteins containing a specific ligand binding site, which would be useful as a simple and rapid preliminary screen of protein-ligand interactions.     

Computational design of protein-small molecule interfaces

The computational design of proteins that bind small molecule ligands is one of the unsolved challenges in protein engineering. It is complicated by the relatively small size of the ligand which limits the number of intermolecular interactions. Furthermore, near-perfect geometries between interacting partners are required to achieve high binding affinities. For apolar, rigid small molecules the interactions are dominated by short-range van der Waals forces. As the number of polar groups in the ligand increases, hydrogen bonds, salt bridges, cation–π, and π–π interactions gain importance. These partial covalent interactions are longer ranged, and additionally, their strength depends on the environment (e.g. solvent exposure). To assess the current state of protein-small molecule interface design, we benchmark the popular computer algorithm Rosetta on a diverse set of 43 protein–ligand complexes. On average, we achieve sequence recoveries in the binding site of 59% when the ligand is allowed limited reorientation, and 48% when the ligand is allowed full reorientation. When simulating the redesign of a protein binding site, sequence recovery among residues that contribute most to binding was 52% when slight ligand reorientation was allowed, and 27% when full ligand reorientation was allowed. As expected, sequence recovery correlates with ligand displacement.     

ROSETTALIGAND: protein-small molecule docking with full side-chain flexibility

Protein-small molecule docking algorithms provide a means to model the structure of protein-small molecule complexes in structural detail and play an important role in drug development. In recent years the necessity of simulating protein side-chain flexibility for an accurate prediction of the protein-small molecule interfaces has become apparent, and an increasing number of docking algorithms probe different approaches to include protein flexibility. Here we describe a new method for docking small molecules into protein binding sites employing a Monte Carlo minimization procedure in which the rigid body position and orientation of the small molecule and the protein side-chain conformations are optimized simultaneously. The energy function comprises van der Waals (VDW) interactions, an implicit solvation model, an explicit orientation hydrogen bonding potential, and an electrostatics model. In an evaluation of the scoring function the computed energy correlated with experimental small molecule binding energy with a correlation coefficient of 0.63 across a diverse set of 229 protein- small molecule complexes. The docking method produced lowest energy models with a root mean square deviation (RMSD) smaller than 2 A in 71 out of 100 protein-small molecule crystal structure complexes (self-docking). In cross-docking calculations in which both protein side-chain and small molecule internal degrees of freedom were varied the lowest energy predictions had RMSDs less than 2 A in 14 of 20 test cases.     

In silico approaches illustrate the evolutionary pattern and protein-small molecule interactions of quinolone synthase from Aegle marmelos Correa

Quinolone synthase from Aegle marmelos (AmQNS) is a Rutacean-specific plant type III polyketide synthase that synthesizes quinolone, acridone, and benzalacetone with therapeutic potential. Simple architecture and broad substrate affinity of AmQNS make it as one of the target enzymes to produce novel structural scaffolds. Another unique feature of AmQNS despite its high similarity to acridone forming type III polyketide synthase from Citrus microcarpa is the variation in the product formation. Hence, to explore the characteristic features of AmQNS, an in-depth sequence and structure-based bioinformatics analyses were performed. Our studies indicated that AmQNS and its nearest homologs have evolved by a series of gene duplication events and strong purifying selection pressure constrains them in the evolutionary process. Additionally, some amino acid alterations were identified in the functionally important region(s), which can contribute to the functional divergence of the enzyme. Prediction of favorable amino acid substitutions will be advantageous in the metabolic engineering of AmQNS for the production of novel compounds. Furthermore, comparative modeling and docking studies were utilized to investigate the structural behavior and small molecule interaction pattern of AmQNS. The observations and results reported here are crucial for advancing our understanding of AmQNS’s phylogenetic position, selection pressure, evolvability, interaction pattern and thus providing the foundation for further studies on the structural and reaction mechanism.     

Modeling protein-small molecule interactions: structure and thermodynamics of noble gases binding in a cavity in mutant phage T4 lysozyme L99A

The complexes of phage T4 lysozyme L99A with noble gases have been studied by molecular dynamics simulation. In a long simulation of the complex with one Xe atom, the structure was found to undergo global conformation change involving a reversible opening and closing of the entrance to the substrate-binding site, during which the conformations of the N and C-terminal domains varied little. The distributions of Xe positions sampled in dynamics simulations were refined in terms of anisotropic Gaussian distributions via least-squares minimization of the difference between Fourier transforms. In addition, molecular transformation simulations have been applied in order to calculate the binding free energies of Xe, Kr and Ar relative to a standard state at a pressure of 1 bar. A single bound Xe is found to assume an equilibrium distribution over three adjacent preferred sites, while in a two-Xe complex, the two Xe atoms preferentially occupy two of these. The positions of the three sites agree closely with the positions of bound Xe determined in the refined crystal structure of a complex formed at a pressure of 8 bar Xe, and the calculated affinities agree well with the observed partial occupancies. At a pressure of 8 bar, a mixture of one-Xe and two-Xe complexes is present, and similarly for complexes with Kr and Ar, with single occupancy relatively more prevalent with Kr and Ar. (Binding of a third Xe atom is found to be quite unfavorable.) A comparison with simulation results for the binding of benzene to the same site leads to the conclusion that binding of Xe within cavities in proteins is common because of several favorable factors: (1) Xe has a large atomic polarizability; (2) Xe can be applied at a relatively high pressure, i.e. high chemical potential; (3) an unfavorable entropic term related to the need to orient the ligand in the binding site is absent. Finally, it is found that the model's binding energy of a water molecule in the cavity is insufficient to overcome the unfavorable binding entropy.     

A dialysis cell for nuclear magnetic resonance spectroscopic measurement of protein-small molecule binding

A dialysis cell is described for use in an NMR spectrometer, to make spectroscopic determinations of protein-small molecule binding. The protein solution is contained within a cylindrical dialysis tube which is concentrically suspended in an NMR tube containing a protein-free dialysis buffer. Simultaneous determinations of the equilibrium transmembrane distribution of the small molecule and the chemical shifts in both compartments are made spectroscopically, providing estimates of the dissociation constant and the chemical shift of the bound species. The cell is used for 31P NMR spectroscopic measurement of the degree of binding of 2,3-diphosphoglycerate to hemoglobin in a 2.8 mM carboxyhemoglobin solution at pH 6.9 and 21 degrees C. The Kd is found to be 2.4 x 10(-3) M.     

PathoPlant: a database on plant-pathogen interactions

Pathogen recognition and signal transduction during plant pathogenesis is essential for the activation of plant defense mechanisms. To facilitate easy access to published data and to permit comparative studies of different pathogen response pathways, a database is indispensable to give a broad overview of the components and reactions so far known. PathoPlant has been developed as a relational database to display relevant components and reactions involved in signal transduction related to plant-pathogen interactions. On the organism level, the tables 'plant', 'pathogen' and 'interaction' are used to describe incompatible interactions between plants and pathogens or diseases. On the molecular level, plant pathogenesis related information is organized in PathoPlant's main tables 'molecule', 'reaction' and 'location'. Signal transduction pathways are modeled as consecutive sequences of known molecules and corresponding reactions. PathoPlant entries are linked to associated internal records as well as to entries in external databases such as SWISS-PROT, GenBank, PubMed, and TRANSFAC. PathoPlant is available as a web-based service at http://www.pathoplant.de.     

NPIDB: a database of nucleic acids-protein interactions

The database NPIDB (Nucleic Acids-Protein Interaction DataBase) contains information derived from structures of DNA-protein and RNA-protein complexes extracted from PDB (1834 complexes in July 2007). It is organized as a collection of files in PDB format and is equipped with a web-interface and a set of tools for extracting biologically meaningful characteristics of complexes. The content of the database is weekly updated. AVAILABILITY: http://monkey.belozersky.msu.ru/NPIDB/     

Relibase: design and development of a database for comprehensive analysis of protein-ligand interactions

Knowledge discovery from the exponentially growing body of structurally characterised protein-ligand complexes as a source of information in structure-based drug design is a major challenge in contemporary drug research. Given the need for powerful data retrieval, integration and analysis tools, Relibase was developed as a database system particularly designed to handle protein-ligand related problems and tasks. Here, we describe the design and functionality of the Relibase core database system. Features of Relibase include, e.g. the detailed analysis of superimposed ligand binding sites, ligand similarity and substructure searches, and 3D searches for protein-ligand and protein-protein interaction patterns. The broad range of functions provided in Relibase and its high level of data integration, along with its flexible and intuitive interface, makes Relibase an invaluable data mining tool which can significantly enhance the drug development process. An example, illustrating a 3D query for quarternary ligand nitrogen atoms interacting with aromatic ring systems in proteins, a pattern found in pharmaceutically relevant target proteins such as, e.g. acetylcholine-esterase, is discussed.     

NCIR: a database of non-canonical interactions in known RNA structures

The secondary and tertiary structure of an RNA molecule typically includes a number of non-canonical base–base interactions. The known occurrences of these interactions are tabulated in the NCIR database, which can be accessed from http://prion.bchs.uh.edu/bp_type/. The number of examples is now over 1400, which is an increase of >700% since the database was first published. This dramatic increase reflects the addition of data from the recently published crystal structures of the 50S (2.4 Å) and 30S (3.0 Å) ribosomal subunits. In addition, non-canonical interactions observed in published crystal and NMR structures of tRNAs, group I introns, ribozymes, RNA aptamers and synthetic oligonucleotides are included. Properties associated with these interactions, such as sequence context, sugar pucker conformation, glycosidic angle conformation, melting temperature, chemical shift and free energy, are also reported when available. Out of the 29 anticipated pairs with at least two hydrogen bonds, 28 have been observed to date. In addition, several novel examples, not generally predicted, have also been encountered, bringing the total of such pairs to 36. Added to this list are a variety of single, bifurcated, triple and quadruple interactions. The most common non-canonical pairs are the sheared GA, GA imino, AU reverse Hoogsteen, and the GU and AC wobble pairs. The most frequent triple interaction connects N3 of an A with the amino of a G that is also involved in a standard Watson–Crick pair.     

GiSAO.db: a database for ageing research

Background

Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that causes acute respiratory disease in chickens worldwide. To date, only one complete genomic sequence of ILTV has been reported. This sequence was generated by concatenating partial sequences from six different ILTV strains. Thus, the full genomic sequence of a single (individual) strain of ILTV has not been determined previously. This study aimed to use high throughput sequencing technology to determine the complete genomic sequence of a live attenuated vaccine strain of ILTV.

Results

The complete genomic sequence of the Serva vaccine strain of ILTV was determined, annotated and compared to the concatenated ILTV reference sequence. The genome size of the Serva strain was 152,628 bp, with a G + C content of 48%. A total of 80 predicted open reading frames were identified. The Serva strain had 96.5% DNA sequence identity with the concatenated ILTV sequence. Notably, the concatenated ILTV sequence was found to lack four large regions of sequence, including 528 bp and 594 bp of sequence in the UL29 and UL36 genes, respectively, and two copies of a 1,563 bp sequence in the repeat regions. Considerable differences in the size of the predicted translation products of 4 other genes (UL54, UL30, UL37 and UL38) were also identified. More than 530 single-nucleotide polymorphisms (SNPs) were identified. Most SNPs were located within three genomic regions, corresponding to sequence from the SA-2 ILTV vaccine strain in the concatenated ILTV sequence.

Conclusions

This is the first complete genomic sequence of an individual ILTV strain. This sequence will facilitate future comparative genomic studies of ILTV by providing an appropriate reference sequence for the sequence analysis of other ILTV strains.     

POINT: a database for the prediction of protein-protein interactions based on the orthologous interactome

One possible path towards understanding the biological function of a target protein is through the discovery of how it interfaces within protein-protein interaction networks. The goal of this study was to create a virtual protein-protein interaction model using the concepts of orthologous conservation (or interologs) to elucidate the interacting networks of a particular target protein. POINT (the prediction of interactome database) is a functional database for the prediction of the human protein-protein interactome based on available orthologous interactome datasets. POINT integrates several publicly accessible databases, with emphasis placed on the extraction of a large quantity of mouse, fruit fly, worm and yeast protein-protein interactions datasets from the Database of Interacting Proteins (DIP), followed by conversion of them into a predicted human interactome. In addition, protein-protein interactions require both temporal synchronicity and precise spatial proximity. POINT therefore also incorporates correlated mRNA expression clusters obtained from cell cycle microarray databases and subcellular localization from Gene Ontology to further pinpoint the likelihood of biological relevance of each predicted interacting sets of protein partners.     

NRSub: a non-redundant database for Bacillus subtilis.

In the context of the international project aimed at sequencing the whole genome of Bacillus subtilis we have developed a non-redundant, fully annotated database of sequences from this organism. Starting from the B.subtilis sequences available in the EMBL, GenBank and DDBJ collections we have removed all encountered duplications and then added extra annotations to the sequences (e.g. accession numbers for the genes, locations on the genetic map, codon usage, etc.) We have also added cross-references to the EMBL, MEDLINE, SWISS-PROT and ENZYME data banks. The present system results from merging of the NRSub and SubtiList databases and the sequence contigs used in the two systems are identical. NRSub is distributed as a flatfile in EMBL format (which is supported by most sequence analysis software packages) and as an ACNUC database, while SubtiList is distributed as a relational database under 4th Dimension. It is possible to access the data through two dedicated World Wide Web servers located in France and Japan.     

Macromolecule--small molecule interactions: analytical and graphical reexamination

An analysis of multiple binding of small molecules by a macromolecule has been made in terms of probabilistic considerations instead of equilibrium ones. This leads to algebraic expressions which suggest alternative nonlinear graphical representations of binding data. One of these, a double-logarithmic presentation of moles bound versus free ligand has been analyzed in detail to show how the number of binding sites and the intrinsic affinity constant can be obtained from the graph.